Cultural categorization of febrile illnesses in

Journal of Ethnopharmacology 85 (2003) 179–185

Cultural categorization of febrile illnesses in correlation with herbal remedies used for treatment in Southwestern Nigeria E.O. Ajaiyeoba a,∗ , O. Oladepo b , O.I. Fawole c , O.M. Bolaji d , D.O. Akinboye e , O.A.T. Ogundahunsi f , C.O. Falade f , G.O. Gbotosho f , O.A. Itiola g , T.C. Happi e , O.O. Ebong h , I.M. Ononiwu h , O.S. Osowole b , O.O. Oduola f , J.S. Ashidi a , A.M.J. Oduola d a Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria Department of Public Health and Health Education, University of Ibadan, Ibadan, Nigeria Department of Epidemiology, Medical Statistics and Environmental Health, University of Ibadan, Ibadan, Nigeria d Postgraduate Institute of Medical Research and Training, University of Ibadan, Ibadan, Nigeria e Department of Zoology, University of Ibadan, Ibadan, Nigeria f Department of Pharmacology & Therapeutics, University of Ibadan, Ibadan, Nigeria g Department of Pharmaceutics and Industrial Pharmacy, University of Ibadan, Ibadan, Nigeria h Department of Pharmacology, University of Port-Harcourt, Port-Harcourt, Nigeria b

c

Received 9 March 2002; received in revised form 8 November 2002; accepted 8 November 2002

Abstract The ethnographic study was conducted in two communities in Oyo State in Southwestern Nigeria. The study sites consisted of a rural and an urban local government area located in the tropical rain forest zone of Nigeria. The study was designed to obtain information on febrile illnesses and herbal remedies for treatment with the aim of identifying potential antimalarial drugs. The study revealed that fever is a general term for describing illnesses associated with elevated body temperature. The indigenous Yoruba ethnic population has categorized fever based on symptoms and causes. The present communication is the result of focus group discussion and semi-structured questionnaire administered to traditional healers, herb sellers, elders and mothers. This was on types of fevers, symptoms and causes of febrile illnesses. The investigation also included use of traditional herbs in the prevention and treatment of the illnesses in the two communities. A total of 514 respondents were interviewed. This was made up of 266 (51.8%) from Atiba local government area (LGA), an urban centre while 248 (48.2%) respondents were interviewed from Itesiwaju LGA, a rural community. The LGAs are located in Oyo State of Nigeria. The respondents proffered 12 types of febrile illnesses in a multiple response answering system in Yoruba language. The most common ones (direct translation into English) were: yellow fever (39.1%), typhoid (34.8%), ordinary (28.8%), rainy season (20.8%) and headache (10.5%) fevers, respectively. Perceived causes of each of the febrile illnesses included stress, mosquito bites, unclean water, rains and over exposure to the sun. Methods of fever prevention were mainly with the use of herbal decoctions, powdered herbs, orthodox medications and maintenance of proper hygiene. Of a total of 112 different herbal remedies used in the treatment of the febrile illnesses compiled from the study, 25 recipes are presented. Recipes consisted of 2–7 ingredients. Oral decoctions (84%), oral powders (63%), use as soaps and creams (40%) in a multiple response system, were the most prevalent routes of administration of prepared herbs used in the treatment of the fevers. Boiling in water or alcohol was the most common method used in the preparation of the remedies. The four most frequently mentioned (multiple response system) plants in the Southwest ethnobotany for fevers were Azadirachta indica (87.5%), Mangifera indica (75.0%), Morinda lucida (68.8%) and Citrus medica (68.8%). © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fevers; Phytomedicine; Herbal recipes; Malaria; Southwest Nigeria

1. Introduction

∗ Corresponding author. Present address: Department of Chemistry, Virginia Polytechnic Institute & State University, Hahn Hall 3012, Blacksburg, VA 24061, USA. Tel.: +1-540-231-4929; fax: +1-540-231-7702. E-mail address: [email protected] (E.O. Ajaiyeoba).

In the last 20 years, drug discovery utilizing ethnopharmacology and traditional uses of herbal remedies have received a lot of attention. Plant products such as morphine, quinine and tubocuraraine have been used as templates for the design of new therapeutic compounds (Phillipson and Wright, 1991; Phillipson, 1994). From one of such plants in the Chinese

0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(02)00357-4

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E.O. Ajaiyeoba et al. / Journal of Ethnopharmacology 85 (2003) 179–185

ethnomedicine, Artemisinin was isolated from Artemisia annua and various derivatives of artemisinin are being used for the treatment of malaria infections (Lugt, 2000). In African ethnomedicines, it is well known that traditional healers make use of a large variety of herbs in the treatment of parasitic diseases including malaria and a wide proportion of herbal remedies dispensed by traditional healers are widely believed by their clients to be effective (Kirby, 1997; Warrell, 1997). In another study (Etkin, 1997), antimalarial plant medicines used by the Hausa in Northern was compiled. The morbidity and mortality of falciparum malaria are seen in children under the age of 5 years. In indigenous African communities, only 8–25% of people with malaria visit health services (Brinkmann and Brinkmann, 1991), this has necessitated the investigation into African ethnobotany and ethnomedicine. For the first time, the categorization, causes and corresponding herbal recipes used for their treatment of each mentioned febrile illness in Southwest Nigeria, are presented from the Nigeria. The objective of the present study was to obtain information on characterization of febrile illnesses and the utilization of phytomedicines for treatment of fevers in Southwestern Nigeria. In the indigenous communities, fever (Iba), is a general term for describing all diseases with elated body temperatures including malaria. The practical control of malaria relies mainly on early diagnosis and treatment, so it became necessary to survey the ethnobotany in Nigeria and determine how malaria is diagnosed and differentiated from other febrile illnesses before herbal treatment. The overall contribution is to enhance the development of antimalarial drugs from the Nigerian phytomedicine in continuation of our studies (Ajaiyeoba et al., 1999), of identification and clinical evaluation of potential antimalarial components from the Nigerian flora.

2. Materials and methods 2.1. Study areas The study areas consisted of rural and urban communities, Itesiwaju and Atiba LGAs, respectively. Itesiwaju local government is a totally rural community. The residents are mostly farmers. The town lacks the usual social amenities and has a low-density population. Atiba LGA on the other hand, a community of civil servants and traders, is densely populated. It is in the Oyo town and links Western Nigeria to the Northern Nigeria through Kwara state. The town has social amenities such as electricity supply and pipe borne water. The residents of both areas belong to the Yoruba ethnic group.

the study to them and obtain their cooperation. The health workers at the primary health care department of the communities were visited. Familiarization visits were also made to community leaders and opinion leaders in the community to solicit their support in the study. 2.3. Training of interviewers and pretest Research assistants did the pre-testing of instruments. They were trained over a period of 1 week by the investigators. After they had satisfactorily performed, they were allowed to commence with the pretest. The pretest was carried out in Akinyele and Egbeda local government areas thereafter, amendments were made to the instruments before the main study. 2.4. Informed consent The objectives of the study were explained to the intending participants. Informed consent was obtained from each of the participants. An approval for the study was obtained from the traditional healers’ association of the respective communities. 2.5. Development of study instruments 2.5.1. Translation of instruments into Yoruba language The study instruments were prepared in English language and were translated into Yoruba language before administration of the instruments in both communities. 2.5.2. Focus group discussion A total of eight focus group discussions (FGDs) were held. Four of the FGDs (two among men and women who were less than 45 years and two among men and women who were older than 45 years) were conducted in Atiba LGA. Four other groups with similar composition were conduced in Itesiwaju LGA, targeted at the general populace. The respondents were traditional healers, herb sellers, elders and mothers. The discussions were recorded on audiotapes for ease of reference. Important issues were documented. 2.5.3. General questionnaire Following the FGD, a semi-structured questionnaire was administered to mothers, community leaders, traditional healers, herbalists, herb sellers and adults in the community. A total of 514 respondents were interviewed. In addition to demographic characteristics of the respondents, the questions addressed include:

2.2. Advocacy visits Advocacy visits were made to the traditional healers association in the study communities to explain the purpose of

(i) Types of fever, causes and people at risk. (ii) Treatment of fevers. (iii) Herbal preparations and methods of administration.


3. Results 3.1. Focus group discussion (FGD) Analysis of the focus group discussions indicated fever, stomach pain, cough, convulsion, cholera, diarrhoea, body-ache, dizziness, typhoid fever, diabetes, ulcer, tetanus, shivers and teeth eruption as common illnesses among children. Illnesses reported among adults were fever, joint pain, headache, body-ache, stroke, cholera, back pain, toothache, chills and catarrh. Other illnesses listed were dermatitis, hernia, dizziness and allergy. The participants further mentioned “internal heat”, oedema, vomiting, worm infestation, malaria, yellow fever, typhoid fever, bleeding, cough, weakness and numbness as the common illnesses among pregnant women. The participants categorized fever as ordinary fever, typhoid fever, yellow fever, tiredness fever and rain fever. Other categories were blood-sucking fever, fever associated with elevated body temperature alone and body-ache fever. Most of the participants mentioned mosquito bite as the cause of fever, others mentioned were drinking unclean water and eating unhygienic food, staying in the rain for a long time, over exposure to sun, physical exertion, dust, too much water, unclean environments, spicy food, anaemia and housefly. The use of herbal remedies at home was identified, as a major treatment while attendance of orthodox health care centres, such as hospitals was a follow up where herbal remedies did not produce relief. On drugs commonly used in treating fevers, participants mentioned analgesics, chloroquine, multivitamin preparation. All these drugs were said to be effective but itching was an undesirable effect resulting from chloroquine use. No dosage was mentioned. Many plants were mentioned by respondents for the treatment of febrile illnesses, these included Dongoyaro (Neem), Mango, Citrus, Paw-paw, among others. Dosage for herbs is a “handful” for children and “one cup” for adults depending on the method of preparation. For the improvement of treatment, participants suggested research into traditional medicine, co-operation between traditional healers and orthodox doctors, availability of traditional medicine, provision of drugs and personnel to health facilities. 3.2. Cultural characterization of febrile illnesses

Table 1 The types of fever commonly mentioned Percentagesa

Types of fever Local nameb

English translation

Iba ponju Iba taifod Iba lasan Ibajo Iba orififo

Yellow Typhoid Ordinary Rainy season Headache

a b

39.1 34.8 28.8 20.8 10.5

Multiple response. Local name in Yoruba.

One hundred and ninety-six respondents (38.1%), had no formal education, 140 (27.2%) had primary school education and 117 (22.7%) had secondary education. Seventy-five percent of the respondents were married. 3.2.2. Types of fever, perception of causes and people at risk Almost all the respondents 495 (96.3%) had heard of fever and 12 different types of fever were described in the indigenous language. The five frequently mentioned fevers are listed in Table 1. The other mentioned fevers were fever due to exhaustion (orere), blood sucking fever, fever associated with elevated body temperature alone, dry season fever, dizziness fever, severe fever and body-ache fever. Specifically, the strongest notions of causes of fever are listed in Table 2. For blood sucking fever, 20% of the respondents mentioned mosquito bite as the major cause of fever while 33% of the respondents mentioned rain. Forty-seven percent of the respondents associated bad water with typhoid fever and 11% associated over-exposure to sun to fever characterized by elevated temperatures. Forty-five percent of the respondents associated stress with yellow fever. Other causes of fever mentioned are dust, exhaustion, supernatural cause, bangs on the head, strange and bad food, catarrh, blood dysfunction, lack of hygiene, premature birth and hereditary. The duration of these fevers ranged from 1 to 90 days. Respondents claimed that everyone was at risk of contracting any of the types of fever. However, specific groups like the light complexioned people, pregnant women, farmers, people who do not eat balanced diet and people living in dirty areas were mentioned as being at risk of rainy season

Table 2 Types of febrile illness and perceived causes Types of fever

3.2.1. Respondent’s demographic The respondents included mothers, fathers, adult members of the community leaders/elders, herbs sellers and traditional healers. The ages of the respondents ranged between 14 and 95 years with a mean age of 40 ± 16.7 years. Most of the respondents 333 (64.8%) were Moslems, 160 (31.1%) Christians, 16 (3.1%) traditional religion practitioners while 5 (0.9%) did not respond to the question on religion.

181

Yellow Typhoid Ordinary Rainy season Headache a

Percentagesa Stress

Mosquito bites

Bad water

44.5 4.1 17.2 10.0 2.3

13.4 5.4 36.1 10.0 4.3

11.6 47.3 1.6 2.5

Multiple response.

Rains

2.0 2.5 33.8

Too much sun 16.9 0.7 22.1 11.3

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Table 3 Types of febrile illness and perceived symptoms Types of fever

Yellow Typhoid Ordinary Rainy season Headache a

Percentagesa Headache

High body temperature

Chills

Body pain

Loss of appetite

Yellow eyes/urine

Weakness

Dizziness

7.2 11.4 26.6 13.8 50.9

11.5 7.4 28.1 23.8 11.8

9.1 2.3 23.0 58.8 7.8

12.0 8.6 18.5 16.3 9.8

6.3 5.7 5.2 1.3 2.0

58.2 13.1 10.4 5.0 3.9

15.4 10.3 25.2 12.8 13.7

1.9 2.9 4.4 2.5 2.0

Multiple response.

fever, yellow fever, ordinary fever, typhoid fever, headache fever and fever due to tiredness. 3.2.3. Fevers and associated symptoms The frequently mentioned symptoms associated with the most commonly fevers are enumerated in Table 3. Elevated body temperature was reported as the major symptom for rainy season fever, ordinary fever, yellow fever and typhoid fever. Other symptoms not listed in the table were insomnia, vomiting, stomach pain, catarrh, thirst, blood dysfunction, dark stool and yawning. 3.2.4. Seasonality and occurrence Most of the fevers mentioned occur all year round. However, yellow fever, ordinary fever, typhoid fever, and headache fever were reported to be more common during the dry season while rainy season fever is during the rainy season. 3.2.5. Methods of preventing different types of fevers The preventive measures mentioned for different types of fever varied (Table 4). Aqueous or alcoholic extract of herbs (22.6%), powdered herb (22.6%) were frequently mentioned for the prevention of yellow fever. The use of orthodox drugs (20.1%) and aqueous or alcoholic extract of herbs (13.0%) were mentioned for prevention of typhoid fever. In addition, the use of orthodox drugs (25.0 or 26.3%) and aqueous or alcoholic extract of herbs (26.6 or 34.7%) were frequently mentioned for the prevention of ordinary or rain fever. Other preventive measures are cleanliness, eating balance diet, medicinal herbal scarification, use of herbal soaps and reduction in physical exertion. The use

Table 4 Methods of prevention of fever Types of fever

Yellow Typhoid Ordinary Rainy season a

Table 5 Recipes used for the treatment of febrile illnesses in Southwestern Nigeria Fever

Recipes

Yellow

1. M. lucida (b), C. medica (j), M. indica (l), Carica papaya (wl) and Cymbopogon citratus (l)a 2. A. indica (l), C. medica (l) and C. citratus (l)a 3. A. indica (b, l), Anarcadium occidentalis (l) and A. occidentalis (b)a 4. A. indica (l), C. medica (l), C. citratus (l) and C. papaya (l)a 5. M. indica (l), Khaya grandifoliola (l), M. lucida (l) and A. occidentalis (l)a

Typhoid

1. A. indica (b), M. indica (b), Solanum erianthum (b), C. papaya (wl) and C. citratus (l)a 2. C. medica (j), Gossypium barbadense (l), M. lucida (l), A. indica (b) and M. indica (b)a 3. Cassia saemia (l), Chromolena odoratum (l), C. medica (l), C. citratus (l), C. papaya (wl), M. indica (l, b), and A. indica (l)a 4. Vernonia amygdalina (l) and C. medica (j)b 5. P. guajava (l), M. indica (b, l) and Bambusa vulgaris (l)a

Ordinary

1. C. citratus (l), Newboldia laevis (l), Lawsonia inermis (l), Citrus sinensis (f) and Zea mays (ws)a 2. A. indica (l), C. citratus (l) and M. indicaa 3. N. laevis (l) and Spritea 4. L. inermis (l), C. medica (f) and Z. mays (ws)a

Rainy season 1. C. medica (l), Capsium annum (f) and M. lucida (l)c 2. A. indica (l), C. odoratum (l), L. inermis (l), C. citratus and C. papaya (l)a 3. M. lucida (l) and O. gratissimum (l)c 4. A. indica (l), C. medica (j), C. papaya (l) and C. citratus (l)a 5. Mormodica charantia (l), C. medica (j), C. saemia (l), M. indica (l) and A. indica (l)a Headache

Percentagesa Herbal liquid

Powder herb

Orthodox drug

Cleanliness

22.6 13.0 25.0 26.3

22.6 1.3 3.1 3.2

39.5 20.1 26.6 34.7

13.7 9.1 17.2 6.3

Multiple response.

of insecticide, avoiding direct sun rays, use of mosquito bed/window nets, and having adequate rest were also proffered. Some respondents, however, believed no adequate preventive measure that could be taken against fever.

1. M. lucida (l) and C. medica (j)c 2. A. occidentalis (l, b), M. indica (l) and C. medica (j)d 3. O. gratissimum (l), C. medica (j) and C. sinensis (j)c 4. C. papaya (x)d 5. V. amygdalina (l), C. citrus (l), M. indica (l) and C. medica (f)a

Plant parts: l, leaves; wl, withered leaves; j, juice; b, bark; f, fruit; ws, water from fermented starch and x, non-fruiting stem. Method of crude drug preparation: a boiling (in water); b steeping (in alcohol); c squeezing; d pounding.


Different complications were associated with the types of fever mentioned. Death was a major complication cited. Others include: weight loss, insomnia, pile, convulsion, weakness, anaemia, illness, asthma, stroke, stomach ache, chills, yellow eyes, psychosis, sterility and vomiting. Some respondents said there are no complications associated with fever. 3.2.6. Treatment of fevers Most respondents affirmed that traditional herbs were effective in treating the different types of fever mentioned. Different herbal recipes were mentioned in the treatment of the different types of fevers. 3.2.7. Herbal remedies, their preparation and methods of administration A total of 112 herbal recipes used in the treatment of different febrile illnesses in Southwestern Nigeria were compiled (Ajaiyeoba et al., 2000). Twenty-five of these remedies are presented in Table 5. For each of the five febrile illnesses, five recipes are outlined. From the results in Table 5, boiling was the most common form of preparation. Soaking the plant materials in water for specific number of days was next. The third most common was crushing the herbs to extract the fluid in them. Oral administration was the most common route of administration followed by topical application (bathing/rubbing), inhalation and scarification. In most cases, the herbs were the sole treatment modality. It was not common to combine use of herbs with orthodox medicine. Majority of the respondents do not experience problems with the use of the herbs, however, a few of the respondents mentioned dizziness, anaemia, diarrhoea, stomach pain, vomiting, additional illness and itching.

4. Discussion and conclusion Most of the febrile illnesses mentioned occurred all the year round. Yellow fever was the most commonly mentioned fever (39.1%), in the multiple response questionnaires. Other relevant ones mentioned were typhoid fever, ordinary season fever, rain fever and headache fever (see Table 1 for details). In these communities, ordinary fever suggests that no other appellation could be given to the febrile illness, meaning it could actually be due to malaria infection. Others were typhoid fever, yellow fever, high temperature fever and headache fevers, were reported to be more common during the dry season. Rain fever was reported to be more common during the raining season. One can infer that rain fever period coincided with the period of highest malaria transmission season and so this fever could be due to malaria infection. The results of the perceived causes of the febrile illnesses are presented in Table 2. The proffered causes of respective febrile illnesses, among respondents were observed to be dependent on time of occurrence, seasonality and symptoms. Of these causes, stress (44.5%) was the most significant

183

for yellow fever, while bad water (47.3%) was the major cause of typhoid fever mentioned. However, for ordinary fever, mosquito (36.1%) was the major cause. This goes a step further to infer that ordinary fever could be due to malaria infection. The second highest cause was exposure to too much sun (22.1%). In the case of rainy season fever, the perceived cause was rains (33.8%). No significant cause could be perceived as causing headache fever as presented in Table 2. Symptoms of the febrile illnesses are presented in Table 3. Yellow eyes and urine were the predominantly mentioned symptoms for yellow (58.4%) and typhoid (13.1%) fevers, respectively. Pyrexia (28.1%), headache (26.6%) and weakness (25.2%) were the perceived symptoms of ordinary fever. Rainy season fever symptoms were mainly chills (58.8%). Other symptoms were pyrexia and headache. Lastly, for headache fever, headache (50.9%) was the most mentioned symptom. Generally, the proffered symptoms in Table 3 suggest that symptoms associated with ordinary fever and rain fever are compactable with malaria. From literature, no other categorization, symptoms and causes of febrile illnesses have been reported hitherto in Southwestern Nigeria or from any other ethnobotany in Nigeria, or Africa, for comparative analysis. Different methods of prevention were proffered for each of the febrile illnesses mentioned as presented in Table 4. These preventive measures were found to be linked with respondents’ perceived causes. The most important preventive method mentioned was Agbo, an aqueous or alcoholic extract of herbs, prepared by boiling (a few hours) or steeping (12–72 h). Pounding of dry plant material in a recipe with a wooden mortar and pestle gives the powdered herbs (Agunmu) usually taken in soups or maize pap, was another preventive method mentioned. The use of orthodox drugs and clean surroundings were other prophylactic measures perceived by the respondents. Dosages were dependent on the method of preparation. It ranged from a glass full (300 ml) when obtained from boiling or steeping in water/alcohol to 100 ml. When extract is obtained by squeezing the plant material to obtain exudates, then dosage would be between 5 and 20 ml. Powdered plant materials are given as a handful and eaten in maize pap or soups. Death was a major complication associated with the types of fevers mentioned. Other complications were headache, body-ache, loss of appetite, weight loss, insomnia, convulsion, vomiting and so on. Some respondents indicated that there were no complications. Most of the respondents confirmed the efficacy of traditional herbs mentioned and Table 5 gives details of recipes (five each) proffered for each fever. Remedies consisted of 1–7 ingredients, which were mainly prepared by boiling or steeping in water or alcohol. The plant ingredients consisted of aerial plant parts, roots, fruits and juice. Others included non-fruiting stem and aqueous extract of fermented maize starch as presented

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Table 6 List of plants commonly mentioned in the treatment of febrile illnesses in Southwest Nigeria Plant in Yoruba

Botanical name

Plant part(s)

Frequency (%)

Eke Mangoro Oruwo Osan wewe Ibepe Kasia Ogano Koko Oyinbo Kaju Sepeleba Owu Efinrin Omi edun agbado Opo Oyinbo Girepi Otili Alubosa Goafa Emile Ehin olebe

A. indica (Meliaceae) M. indica (Anarcadiaceae) M. lucida (Rubiaceae) C. medica (Rutaceae) C. papaya (Caricaeae) Cassia sp. (Caesalpinaceae) Khaya sp. (Meliaceae) C. citratus (Graminea) A. occidentalis (Anacardiaceae) Tithonia diversiflora (Asteraceae) Gossypium sp. (Malvaceae) Ocimum gratissimum (Labiatea) Z. mays (Graminea) Ananas cosmos (Bromeliaceae) Citrus decumana (Rutaceae) Cajanus cajan (Papilionaceae) Allium cepa (Alliaceae) Psidium guajava (Rutaceae) Euphorbia hirta (Euphorbiaceae) Phyllantus amarus (Euphorbiaceae)

Bark and leaves Bark and leaves Leaves Leaves and juice Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Extracta Fruit Juice Bark Leaves Bark Whole Leaves

87.5 75.0 68.8 68.8 56.3 43.8 43.8 37.5 31.3 31.3 25.0 25.0 18.8 18.8 18.8 12.5 12.5 12.5 6.3 6.3

a

Aqueous extract from fermented starch.

Fig. 1. Methods of administration of herbal remedied used for treatment of fevers.

in Table 6. The most commonly mentioned plant was Neem (75%, leaves and bark). Others were Mango leaves and bark (23%). Remedies consisted of 1–7 ingredients. From the results presented in Table 6, a great majority (96%) of the recipes contained the fruit or juice of a citrus species. The inference here could be addition of Vitamin C to the remedy to aid healing, similar to what obtains in orthodox medical practice. Fig. 1 gives a representation of the methods of administration of the remedies. The commonest route of administration was oral, followed by topical (bathing/rubbing), inhalation and scarification. Combining the use of herbs with orthodox medicine was not common. Most of the respondents did not report experiencing problems with the use of herbs although a few specified dizziness, anaemia, diarrhoea, stomach pain, vomiting, itching and some other illnesses, which were not specified. From the results of the ethnobotanical surveys in this study, one can conclude that, in Southwestern Nigeria, herbal

preparations are used for the prevention and treatment of the febrile illnesses. Respondents were knowledgeable about the causes of mentioned febrile illnesses, partly intermingled with some wrong notions. The causes of febrile illnesses proffered were affected by the belief of the traditional healer or caregiver. In categorizing febrile illnesses, alongside the causes and symptoms, ordinary fever and rainy season fever are the two illnesses compactable with malaria infection. The highest malaria transmission season is during the rainy season (March–September). Observation of the efficacy of the herbal remedies and microscopic clearance of Plasmodium falciparum has been done in a further study and the results will be presented elsewhere. Furthermore, based on the results of this study, the herbal ingredients identified from the Southwest Nigerian phytomedicine, are being investigated pharmacologically in our laboratories (Malaria Research Group, in IAMRAT, University of Ibadan), to confirm efficacy of the plants for antiplasmodial/antimalarial properties.


Acknowledgements This research received financial support from WHO/TDR/ MIM Africa research capability strengthening Grant ID 980046. We are grateful to all the respondents, the Traditional Healers Associations in Atiba and Otu Local Government Areas of Oyo State, Southwestern Nigeria for consenting to participate in the Studies. References Ajaiyeoba, E.O., Oladepo, O., Ogundahunsi, O.A.T., Ebong, O.O., Fawole, O.I, Gbotosho, G.O., Bolaji, O.M., Itiola, O.A., Akinboye, D.O., Happi, T.C., Falade, C.O., Oduola, A.M.J., 1999. Identifying antimalarial moieties from the phytomedicine compendium of Nigeria. Poster presented at the MIM African Malaria Conference, Durban, March, C10 p. Ajaiyeoba, E.O., Ebong, O.O., Akinboye, D.O., Bolaji, O.M., Gbotosho, G.O., Fawole, O.I., Falade, C.O., Itiola, A.O., Oladepo, O., Osowole,

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O.S., Oduola, O.O., Ashidi, J.S., Falade, M.O., Ononiwu, I.M., Ezeiruakwu, N.P., 2000. Identification and clinical evaluation of potential antimalarial components from the Nigerian phytomedicine compendium. Second Year Progress Report submitted to WHO/TDR/MIM, Geneva, pp. 32–35. Brinkmann, U., Brinkmann, A., 1991. Malaria and health in Africa: the present situation and epidemiological trends. Tropical Medicine and Parasitology 42, 204–213. Etkin, N.L., 1997. Antimalarial plants used by the Hausas in Northern Nigeria. Tropical Doctor 27 (Suppl. 1), 12–16. Kirby, G.C., 1997. Plants as sources of antimalarial components. Tropical Doctor 27 (Suppl. 1), 7–11. Lugt, C.B., 2000. Dutch registration for artemotil injections. TDR News, No. 63, October. Phillipson, J.D., 1994. Natural products as drugs. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 17–19. Phillipson, J.D., Wright, C.W., 1991. Can ethnopharmacology contribute to the development of antimalarial agents? Journal of Ethnopharmacology 32 (1–3), 155–171. Warrell, D.A., 1997. Herbal remedies for malaria. Tropical Doctor 27 (Suppl. 1), 5–6.


Anti-inflammatory activity of ‘TAF’ an active fraction from the plant Barleria prionitis Linn. B. Singh∗ , S. Bani, D.K. Gupta, B.K. Chandan, A. Kaul Departments of Pharmacology and Natural Products Chemistry, Regional Research Laboratory, Canal Road, Jammu 180016, India Received 8 July 2002; received in revised form 8 November 2002; accepted 8 November 2002

Abstract ‘TAF’ fraction from the methanol–water extract of Barleria prionitis Linn. was evaluated for anti-inflammatory and anti-arthritic activities against different acute and chronic animal test models. It exhibited significant anti-inflammatory activity against different inflammagens like carrageenan, histamine and dextran. The anti-inflammatory activity in adrenalectomised rats was maintained showing that the effect of fraction ‘TAF’ is not activated by the pituitary–adrenal axis. Significant anti-arthritic activity was observed in adjuvant-induced polyarthritis test in rats. ‘TAF’ also showed inhibition of vascular permeability and leucocytes migration in vivo into the site of inflammatory insult. Ibuprofen (Cadilla India Ltd., Mumbai) was used as a standard reference drug. The oral (p.o.) LD50 was more than 3000 mg/kg, with no signs of abnormalities or any mortality observed for 15 days after single-dose drug administration. The intraperitoneal (i.p.) LD50 was found to be 2530 mg/kg (±87 mg/kg S.E.) [Proceedings of Society of Experimental Biological Medicine 57 (1944) 261]. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Barleria prionitis; Leucocyte migration; Phlogistic; Inflammation; Carrageenan; Vascular permeability; Polyarthritis

1. Introduction

2.2. Preparation of extract and fractionation

Barleria prionitis (Family Acanthaceae; Hindi— Katsareya; Sanskrit—Karunta), is an annual shrub, 1–3 feet high, found throughout tropical Asia (India and Sri Lanka) and in South Africa. The white flower variety is bitter and in indigenous system of medicine in India the leaves are used in fever, toothache and liver ailments, aerial parts are used in inflammation, bark in cough, the whole plant and especially the roots are used as tonic and diuretic (Chopra et al., 1956, 1958; Kiritkar and Basu, 2000; Nadkarni, 1994).

Shade dried coarsely powdered whole plant (7.5 kg) was extracted with alcohol and water (80:20) by percolation (15 times) to obtain aqueous alcoholic extract (873 g, yield: 11.64%). The extract so obtained was dissolved in 10% methanol in water and extracted successively with hexane, chloroform and n-butanol (eight times each) to obtain hexane fraction (65.47 g, yield: 7.5%); chloroform fraction (46.70 g, yield 5.35%); n-butanol fraction (25.49 g, yield: 2.92%) and aqueous marc (732.44 g, yield: 83.90% of the total alcoholic extract). Hexane, chloroform and n-butanol fractions were dried at low temperature (40–50 ◦ C) under vacuum whereas the aqueous marc was lyophilised by freeze dryer. All the fractions were subjected to anti-inflammatory activity against carrageenan-induced oedema in rats which led us to establish that n-butanol and aqueous fraction has significant anti-inflammatory activity (Table 1). Keeping in view the yield and activity, the aqueous fraction (TAF) was taken up for detailed anti-inflammatory activity. On chemical analysis, this aqueous fraction was found to be a mixture of iridoid glucosides, which on TLC (silica gel) gives three spots of rf values 0.84, 0.60 and 0.34 solvent system chloroform–methanol (80:20). One major spot of rf value 0.34 was further confirmed by Co TLC with a reference standard shanzhiside methyl ester (iridoid glucoside).

2. Materials and methods 2.1. Plant material The B. prionitis was collected from the Bahu fort area of Jammu (Jammu & Kashmir, India) and authenticated by T.N. Srivastava, taxonomist at Janki Ammal Herbarium of the Institute (Regional Research Laboratory, Jammu, India) where voucher specimen has been deposited.

∗ Corresponding

author. Fax: +91-191-543829. E-mail address: [email protected] (B. Singh).


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Table 1 Effects of oral administration of B. prionitis Linn (different fractions) and Ibu on anti-inflammatory response to carrageenan-induced oedema in rats Treatment

Dose (mg/kg)

Oedema (ml) (normal rats)a

Control Hexane fractions Chloroform fractions n-Butanol fractions Aqueous marc (TAF) fractions Ibu (reference drug)

– 100.00 100.00 100.00 100.00 50.00

0.92 0.78 0.82 0.55 0.48 0.46

± ± ± ± ± ±

0.03 0.03 ns 0.02 ns 0.03∗∗ 0.02∗∗ 0.02∗∗

Inhibition (%)b – 15.02 10.26 40.22 47.83 50.02

± ± ± ± ±

3.04 2.76 2.08 2.04 1.98

ns: not significant (P > 0.05) in relation to respective control values (Dunnett’s t-test). a Values represent the increase in paw volume (mean ± S.E.) of six animals in each group. b Values represent the percent (mean ± S.E.) inhibition of inflammation by the drug treatment. ∗∗ P < 0.01.

Further chemical profile showed mainly three iridoid glucosides, i.e. acetyl barlerin (6,8-di-o-acetylshanzhiside methyl ester), barlein (8-o-acetylshanzhiside methyl ester) and shanshiside methyl ester of formula 1, 2 and 3.

2.4. Treatment Different doses of test drug, standard and the vehicle were given orally (p.o.). Freshly prepared solution of ‘TAF’ (1%, w/v) in normal saline was used in all the experiments except for the toxicity study where 10% solution was used. The control animals were given the vehicle only. Ibuprofen (Ibu) was used as standard for authenticity of the experiments. The oedema was measured using plethysmographic recordings of paw volume (volume differential meter, Model 7101, Ugo Basile, Italy) of the experimental rats. 2.5. Carrageenan-induced oedema in rats

Standardisation of the active fraction is obtained using the above iridoid glucoside as the markers. 2.3. Animals Charles Foster rats (150–180 g) and Swiss albino mice (24–30 g) of either sex, bred in the Institute’s animal house, were used in the study. Animals were housed under standard conditions (23 ± 2 ◦ C, 60–70% relative humidity and 12-h photo period) and were maintained on standard rodent pellet diet (Lipton India Ltd., Mumbai) and water ad libitum.

Acute oedema (Winter et al., 1962) in left hind paws of the rat was induced by the subplantar injection of 0.1 ml of freshly prepared (1%, w/v) carrageenan (Marine Colloids, USA) suspension in normal saline 1 h after the drug administration. The paw volume was measured before and 4 h after the carrageenan injection (Table 2). 2.6. Histamine-induced oedema in rats The method of Horakova and Moratova (1964) was followed. Oedema in one of the hind paws of the rat was induced by the subplantar injection of 0.1 ml freshly prepared 0.1% (w/v) histamine (Sigma Chemical Co., St. Louis, MO,

Table 2 Effects of oral administration of B. prionitis Linn. ‘TAF’ and Ibu on anti-inflammatory response to carrageenan-induced oedema in normal and adrenalectomised rats Treatment

Dose (mg/kg)

Oedema (ml)a (normal rats)

Control TAF TAF TAF TAF Ibu

– 12.50 25.00 50.00 100.00 50.00

0.98 0.83 0.67 0.58 0.48 0.48

± ± ± ± ± ±

0.03 0.02∗∗ 0.04∗∗ 0.02∗∗ 0.02∗∗ 0.02∗∗

Inhibitionb (%)

Oedema (ml)a (adrenalectomised rats)

– 15.30 31.63 40.62 51.02 51.02

0.95 0.83 0.78 0.62 0.52 0.43

± ± ± ± ± ±

Values represent the increase in paw volume (mean ± S.E.) of six animals in each group. Values represent the percent (mean ± S.E.) inhibition of inflammation by the drug treatment. ∗∗ P < 0.01 in relation to respective control values (Dunnett’s t-test). a

b

0.05 0.03∗∗ 0.02∗∗ 0.02∗∗ 0.02∗∗ 0.02∗∗

Inhibitionb (%) – 11.93 17.89 33.86 44.56 54.03

± ± ± ± ±

3.23 2.17 2.58 2.23 2.51

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Table 3 Effects of oral administration of B. prionitis Linn. ‘TAF’ and Ibu on anti-inflammatory response to histamine and dextran-induced oedema in rats Treatment

Dose (mg/kg)

Histamine oedema (ml)a

Control TAF TAF TAF TAF Ibu

– 12.50 25.00 50.00 100.00 50.00

1.11 0.97 0.89 0.81 0.70 0.74

± ± ± ± ± ±

0.07 0.02 ns 0.02∗ 0.04∗∗ 0.04∗∗ 0.03∗∗

Inhibitionb (%)

Dextran oedema (ml)a

– 12.16 19.07 27.04 36.14 31.83

1.16 1.04 0.94 0.85 0.76 0.68

± ± ± ± ±

2.67 2.46 3.67 4.14 3.39

± ± ± ± ± ±

0.04 0.03 ns 0.04∗∗ 0.04∗∗ 0.03∗∗ 0.02∗∗

Inhibitionb (%) – 12.35 19.10 26.58 34.05 41.23

± ± ± ± ±

1.91 3.48 3.74 3.31 2.55

ns: not significant (P > 0.05) in relation to respective control values (Dunnett’s t-test). a Data shows oedema in ml (mean ± S.E.) in paw volume of six animals in each group. b Values represent the percent (mean ± S.E.) inhibition of oedema by the drug treatment. ∗ P < 0.05. ∗∗ P < 0.01.

USA) in normal saline. The oedema was quantitated before and 1 h after the histamine injection (Table 3). 2.7. Dextran-induced oedema in rats Acute oedema (Winter, 1964) in one of the hind paws of the rat was induced by the subplantar injection of 0.1 ml freshly prepared 6% (w/v) dextran (Sigma) in normal saline. The paw volume was measured before and 1 h after the phlogistic injection. Different doses of the test and reference drug dissolved in normal saline was given p.o. 1 h before the dextran injection (Table 3). 2.8. Adjuvant-induced polyarthritis in rats Adjuvant arthritis (Newbould, 1963) was induced by the subplantar injection of 0.05 ml freshly prepared suspension (5.0 mg/ml) of steam killed Mycobacterium tuberculosis (Difco, USA) in liquid paraffin. The volume of the injected (“primary response”; Arrigoni-Martelli and Brahm, 1975) and un-injected (“secondary” or immune mediated responses; Arrigoni-Martelli et al., 1976), hinds paws were quantitated before and on alternate days from Days 1 to 21 after the adjuvant injection. Different doses of the test drug dissolved in normal saline and the vehicle (normal saline) were given p.o. 1 h before the injection and then once daily for 21 days. Erythrocyte sedimentation rate (ESR) of all the animals were monitored on Day 21 by the Wintrobe method (Raphael, 1976), (Table 4). 2.9. Carrageenan-induced oedema in adrenalectomised rats The adrenal glands of the animals were removed surgically (Ingle and Griffith, 1949) and saline was provided to these rats instead of water for drinking. Two days later the carrageenan acute oedema test (Winter et al., 1962) was carried out as described above (Table 2). 2.10. Leucocytes migration (in vivo) in rats Following the procedure of Meacock and Kitchen (1979) pleurisy was induced in the rats by injecting 0.5 ml of

carrageenan (1%, w/v in sterilised normal saline) into the pleural cavity of the rats. Different doses of the test and reference drug were given p.o. 1 h before and 6 h after the carrageenan injection. After 24 h of carrageenan injection, rats were anesthetised, the pleural exudates volume measured and the total leucocyte count of the exudate done by the method of Bauer (1980) (Table 4). 2.11. Acetic acid-induced vascular permeability in mice Using the method of Whittle (1964) mice were injected with 0.2% solution of Evans blue dye (0.25%, w/v in normal saline) intravenously after 30 min of oral administration of the drug. Fifteen minutes later the mice were injected intraperitoneally (1 ml/100 g of body weight) with freshly prepared 0.6% of acetic acid (v/v) in normal saline. After 30 min of acetic acid injection mice of all the groups were sacrificed, their peritoneal cavities washed with 10 ml of haparinised sterile normal saline and centrifuged (3000 × g) for 10 min. Absorbance of the supernatant was measured at 610 nm using a spectrophotometer (Uvikon-810, Kontron Instruments, Switzerland) (Table 4). Anti-inflammatory activity (A) was calculated by the following equation: TS − V A= 1− × 100 VS − V where V is the vehicle, TS and VS are the drug + phlogistic agent and vehicle + phlogistic agent-treated groups of animals, respectively. 2.12. Acute toxicity studies Different doses (100–3000 mg/kg, p.o. and i.p.) of ‘TAF’ were given to the group of 10 mice for each dose, while one group with same number of mice served as control. The animals were observed continuously for 1 h and then at half-hourly intervals for 4 h for any gross behavioural change, fecal and urine output and feeding behaviour etc. Acute LD50 values p.o. and i.p. in mice were calculated by the method of Miller and Tainter (1944).

ns: not significant (P > 0.05) in relation to respective control values (Dunnett’s t-test) (N = 6 per group). a Data shows increase of leucocytes count and exudate volume (mean ± S.E.) in carrageenan-induced pleurisy in the rats (in vivo) and, within parentheses, the percent (mean ± S.E.) reduction of leucocytes count and exudate volume by drug treatment. b Data shows increase in concentration of dye due to excessive vascular permeability (mean ± S.E.) in mice, and within parentheses, the percent (mean ± S.E.) reduction of dye concentration by drug treatment. c Data shows increase of ESR (mean ± S.E.) in polyarthritis rats, and within parentheses, the percent (mean ± S.E.) inhibition of ESR by TAF treatment. ∗ P < 0.05. ∗∗ P < 0.01.

± ± ± ± ± ± 14.22 14.05 11.86 10.41 10.00 12.47 0.02 0.02 ns (10.35 ± 4.79) 0.02∗∗ (19.75 ± 3.53) 0.03∗∗ (33.06 ± 5.46) 0.02∗∗ (39.19 ± 2.46) 0.01∗∗ (33.01 ± 1.69) ± ± ± ± ± ± 0.62 0.55 0.49 0.41 0.37 0.41 0.34 0.20 ns (8.95 ± 4.90) 0.23 ns (16.50 ± 6.74) 0.34 ns (22.62 ± 8.78) 0.16∗ (32.48 ± 4.34) 0.39 ns (22.11 ± 10.22) ± ± ± ± ± ± 3.89 3.54 3.25 3.01 2.62 3.03 1.91 2.54 ns (14.96 ± 4.53) 2.40∗∗ (19.77 ± 4.27) 2.53∗∗ (26.46 ± 4.51) 2.53∗∗ (32.00 ± 4.51) 1.85∗∗ (28.69 ± 3.30) ± ± ± ± ± ± 56.20 47.78 45.08 41.32 38.22 40.00 – 12.5 25.0 50.0 100 50 Control TAF TAF TAF TAF Ibu

Dye concentration (␮g/25 g mouse)b Exudate volumea TLC (mm ×10−3 )a Dose (mg/kg) Treatment

Table 4 Effects of B. prionitis Linn. ‘TAF’ and Ibu on (in vivo) leucocytes migration and vascular permeability

0.56 0.79 ns (1.19 ± 5.56) 1.02 ns (10.33 ± 5.58) 0.67 ns (16.60 ± 7.22) 0.68∗∗ (26.79 ± 4.77) 0.65∗∗ (12.28 ± 4.59)

B. Singh et al. / Journal of Ethnopharmacology 85 (2003) 187–193 ESR (mm) (Day 21, polyarthritis)c

190

2.13. Statistical analysis The data obtained was subjected to statistical analysis using ANOVA (Armitage and Berry, 1987) for comparing different groups and Dunnett’s t-test for control and multiple test groups (Dunnett, 1964). The regression coefficient (slope b) correlation coefficient (r) with its P value and ED50 with 95% confidence limit (CL) were determined by regression analysis using log dose and percent effect of antiinflammatory activity (Swinscow, 1980; Ghosh, 1984). The P value of 3000 mg/kg, p.o. and 2530±87 (S.E.) mg/kg, i.p. by the graphic method (Miller and Tainter, 1944). Its apparent safe-over long-term administration in the chronic studies is encouraging enough to warrant further studies to explore its possible therapeutic role in modern clinical practice.

4. Discussion The therapy of arthritis usually employs NSAIDs, steroids or disease modifying drugs. The long-term use of these, however, may not limit the disease progression leading to joint deformity. All of these drugs, however, have side effects and the search for a noval anti-arthritic drug continues. The present investigations indicate that the active fraction ‘TAF’ of the plant B. prionitis possess marked dose related anti-inflammatory and anti-arthritic activity as evidenced by several acute and chronic test models. ‘TAF’ showed significant anti-inflammatory activity against the acute inflammation induced by carrageenan, histamine and dextran in rats (Tables 2 and 3) and does not seem to act through the activation of adrenal–pituitary axis as it significantly inhibited the carageenan-induced oedema in adrenalectomised

Acknowledgements The authors wish to thank Miss. Reeta Bhat, Mr. Benjamin and Mr. Karan Singh for their technical assistance in experimental work.

References Armitage, P., Berry, G., 1987. In: Statistical methods in medical research. Blackwell Scientific Publication, Oxford, pp. 186–196. Arrigoni-Martelli, E., Brahm, E., 1975. Investigations in the influence of cyclophosphamide, gold sodium thiomalate and d-penicillamine on nystatin oedema and adjuvant arthritis. Agents and Actions 5, 264–267.

B. Singh et al. / Journal of Ethnopharmacology 85 (2003) 187–193 Arrigoni-Martelli, E., Brahm, E., Huskisson, E.C., Willoghby, D.A., Dieppe, P.A., 1976. Pertussis vaccine oedema: an experimental model for the action of penicillamine-like drugs. Agents Actions 6, 613–617. Bauer, J.D., 1980. Numerical evaluation of formed elements of blood. In: Sonnenwirth, A.C., Jarett, L. (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, vol. 1, 8th ed. The C.V. Mosby Company, 11830 Westline Industrial Drive, St. Louis, Missouri, USA, pp. 785–808. Billingham, M.E.J., Davies, G.E., 1979. Experimental models of arthritis in animals as screening tests for drugs to treat arthritis in man. Handbook of Experimental Pharmacology 50 (2), 108–144. Bowen, D.L., Fauci, A.S., 1988. Adrenal corticosteroids. In: Gallin, J.I., Goldstein, I.M., Synderman, R. (Eds.), Inflammation Basic Principles and Clinical Correlates. Raven Press, New York, pp. 877–896. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Barleria prionitis Linn. In: Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research Publication, New Delhi, India, pp. 33–34. Chopra, R.N., Chopra, I.C., Handa, K.L., Kapur, L.D., 1958. Barleria prionitis Linn. In: Chopra’s Indigenous Drugs of India, 2nd ed. U.N. Dhar and Sons Pvt. Ltd., Calcutta, India, p. 595. Dunnett, C.W., 1964. New tables for multiple comparisons with control. Biometrics 20, 482–494. Ferreira, S.H., Vane, J.R., 1979. Mode of action of anti-inflammatory agents which are prostaglandin synthetesa inhibitors. Anti-inflammatory Drugs 50, 348–392. Ghosh, M.N., 1984. In: Fundamentals of Experimental Pharmacology, 2nd ed. Scientific Book Agency, Calcutta, India, pp. 177–190. Glenn, E.M., Bowman, B.J., Lyster, S.C., Rohloff, N.A., 1971. Aggregation of red cells and effects on non-steroidal anti-inflammatory drugs. Proceedings of Society of Experimental Biological Medicine 138, 235– 239. Gokhale, A.B., Damre, A.S., Kulkarni, K.R., Saraf, M.N., 2002. Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine 9, 433–437. Horakova, Z., Moratova, J., 1964. Means of influencing the oedematous component of inflammation. In: International Symposium on Non-steroidal Anti-inflammatory Drugs. M.N. Excerpta, Amsterdam, pp. 237–244. Ingle, D.J., Griffith, J.O., 1949. In: Farris, E.J., Griffith Jr., J.G. (Eds.), The Rats in Laboratory Investigation. Hafner Publishing Co., Inc., New York, pp. 444–490. Kiritkar, K.R., Basu, B.D., 2000. Barleria prionitis Linn. In: Caicus, E.J.F., Mhaskar, K.S. (Eds.), Indian Medicinal Plants, Vol. III, Revised and Enlarged, 3rd ed. Sri Satguru Publications, Indian Book Centre, Delhi, India, pp. 2587–2590. Meacock, S.C.R., Kitchen, B.A., 1979. Effects of the non-steroidal anti-inflammatory drug benoxaprofen on leucocyte migration. Journal of Pharmacology 31, 366–370.

193

Miller, L.C., Tainter, M.L., 1944. Evaluation of ED50 and its error by means of logarithmic Probit graph papers. Proceedings of Society of Experimental Biological Medicine 57, 261–264. Mujumdar, A.M., Naik, D.G., Dange, C.N., Puntambekar, H.M., 2000. Antiinflammatory activity of Curcuma amada Roxb in albino rats. Indian Journal of Pharmacology 32, 375–377. Nadkarni, A.K., 1994. Barleria prionitis Linn. In: Dr. K.M. Nadkarni’s Indian Materia Medica, 3rd ed. Reprint vol. 1, Popular Book Depot, Mumbai, India. Newbould, B.B., 1963. Chemotherapy of rat arthritis induced in rats by injection of mycobacterium adjuvant. British Journal of Pharmacology 21, 127–136. Otterness, I.G., Bliven, M.L., 1985. In: Lombardino, J.G. (Ed.), Non Steroidal Antiinflammatory Drugs. Wiley, New York, pp. 111–252. Raphael, S.S., 1976. Basic hematologic techniques. In: Lynch’s Medical Laboratory Technology, 3rd Asian ed. W.B. Saunders Company, IGAKU SHOIN Ltd., Tokyo, pp. 1073–1130. Rippe, B., Kaniya, A., Folkon, B., 1979. Transcapillary passage of albumin, effects of tissue cooling and of increase in filteration and plasma colloid osmotic pressure. Acta Physiologica Scandinavica 105, 171– 175. Simionesen, N., Simionescn, M., Palede, G.E., 1978. Open junction in endothelium of the post capillary venules of the diaphram. Journal of Cell Biology 79, 27–29. Swingle, K.F., 1974. Evaluation for anti-inflammatory activity. In: Scherrer, R.A., Whitehouse, M.W. (Eds.), Antiinflammatory Agents. Chemistry and Pharmacology, vol. 11. Academic Press, New York, pp. 33–112. Swingle, K.F., Jaques, L.W., Kvam, D.C., 1969. Differences in the severity of adjuvant arthritis in four strains of rats. Proceedings of Society of Experimental Biology (NY) 132, 608–612. Swinscow, T.D.V., 1980. In: Statistics at Square One, 6th ed. British Medical Association, London, pp. 62–70. Ward, J.R., Cloud, R.S., Kranitt, E.L., Jones, R.S., 1964. Studies on adjuvant induced polyartheritis in rats. The effect of immunosuppressive agents on arthritis and tuberculin hypersensitivity. Arthritis Rheumatic 1, 644–661. Whittle, B.A., 1964. The use of changes in capillary permeability in mice to distinguish between narcotic and non-narcotic analgesics. British Journal of Pharmacology 22, 246–253. Winter, C.A., 1964. Anti-inflammatory testing methods: comparative evaluation of indomethacin and other agents. In: International Symposium on Nonsteroidal Anti-inflammatory Drugs. M.N. Excerpta, Amsterdem, pp. 190–202. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan induced oedema in hind paw of the rats as an assay for anti inflammatory drugs. Proceedings of Society of Biological Medicine III, 544–547.


Effects of Fagonia cretica L. constituents on various haematological parameters in rabbits M. Asif Saeed∗ , A. Wahid Sabir Department of Pharmacy, University of the Punjab, Allama Iqbal Campus, Lahore 54000, Pakistan Received 24 March 2002; received in revised form 11 April 2002; accepted 21 November 2002

Abstract Effects of powdered Fagonia cretica plant and its two major triterpenoid saponins (saponin-I and saponin-II), isolated from its ethanolic extract, on red blood cells (RBC) count, haemoglobin concentration (HC), mean corpuscular haemoglobin (MCH) and on total leucocyte (WBC) count of normal male rabbits were investigated. Saponins treated three dose groups of animals indicated significant decrease in RBC count during the experimental period of 16 days. This effect was more pronounced in animals treated with saponin-II than saponin-I. The 0.50 and 1.0 g crude drug treated animals and 10 and 20 mg of both the saponins treated animals, indicated a decreasing tendency of HC up to the 4th day, which increased afterwards up to the 16th day. The third dose of these materials (1.50 g of crude drug and 30 mg of both saponins) exhibited a highly significant decreasing pattern, up the 16 days. The decreasing effect of haemoglobin concentration was more distinct in the saponin-II treated animals than saponin-I and the crude drug. The MCH followed a reverse pattern than RBC and HC. A continued decreasing trend was found in total WBC during the 16 days treatment. An amount of 1.50 g of the crude drug and 30 mg of both the saponins had highly significant decreasing effects on the amount of total leukocyte count of rabbit’s blood. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Triterpenoid saponins; Haematological parameters; Fagonia cretica L.

1. Introduction Fagonia cretica L. (family Zygophyllaceae) is a small spiny under-shrub, mostly found in dry calcareous rocks throughout Pakistan (Chopra et al., 1956, 1982; Hooker, 1975). It is reputed to be a medicinal plant in scientific and folkloric literature and its medicinal values are well documented (Chopra et al., 1956, 1982; Saeed, 1969; Hooker, 1975). An aqueous decoction of the aerial parts of the plant is a popular remedy in the indigenous system of medicine for cancer in its early stages and for the treatment of various other diseases of digestive and blood vascular system (Saeed, 1969). The medicinal properties of the plant were attributed due to its variety of active phytochemical constituents. Although the plant had received a great interest for the phytochemical investigation since many years, yet the chemical structures of most of its constituents were established during the last 15 years. The entire plants of various Fagonia species were investigated mainly for the presence of two major types of

∗ Corresponding

author. E-mail address: [email protected] (M. Asif Saeed).

phytochemical compounds, i.e. flavonol glycosides and terpenoid glycosides. Several flavonol glycosides were isolated from various Egyptian Fagonia species and their phylogenetic affinities were investigated (El-Nagoumy et al., 1986; Al-Wakeel et al., 1987, 1988; El-Hadidi et al., 1988; Saleh et al., 1988, 1990; Al-Wakeel and Shahnaz, 1992). On the other hand, several saponins or triterpenoid glycosides were also separated and characterised (Ansari et al., 1987, 1988). Other constituents like docosyl docosanoate from hexane extract (Hamid et al., 1989) and water soluble proteins from aqueous extract of air-dried F. cretica plants were isolated (Shaukat et al., 1981). Nahagenin (Atta et al., 1982), hederagenin, ursolic acid and pinitol from other Fagonia species were also separated and characterised (Atta et al., 1984). Antimicrobial activity of its flavonoid compounds has been explored previously (Hash and Nag, 1988). The nutritive values of the wild species, growing in Rajasthan areas of India, have also been evaluated (Harash et al., 1981). Although the folkloric medicinal literature claimed F. cretica as anti-carcinogen, yet no scientific attempt was made to evaluate the effects of its individual isolated compounds on different parameters of living organisms. In the first instance we have isolated two major triterpenoid saponins glycosides from its aerial parts and their effects on


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four of the blood parameters such as red blood cells (RBC) count, haemoglobin concentration (HC), mean corpuscular haemoglobin (MCH) and on total leukocyte count (WBC) of normal male rabbits were investigated under normal laboratory conditions.

2. Materials and methods 2.1. General Unless otherwise stated, all the chemicals used were of analytical grade. Concentrations were performed under reduced pressure at bath temperatures not exceeding 55 ◦ C. Separations were performed on SE-54 fused-silica capillary columns using authentic hexitol hexa-acetate as reference. The TLC spots were visualised under UV lamp (TL 900 Camag Ltd.). IR spectra of the compounds were found on Pye-Unicam SP-8-400 and UV spectra were measured on Hitachi 270-30 spectrophotometer. 1 H NMR spectra were obtained at 400 MHz and 13 C NMR spectra at 25 MHz on JEOL GX-400 and JEOL FX-100 spectrophotometers using tetramethylsilane as an internal reference. Electron impact mass spectra (EIMS) were recorded on a Varian MAT-311A instrument using the direct inlet method. 2.2. Plant materials The F. cretica plants were collected from the waste and uncultivated areas of Lahore, Fasilabad and Rawalpindi (i.e. from the central Punjab plain areas of Pakistan), in January–February 1998. These were authenticated by the Herbarium staff, Department of Botany, University of the Punjab, Lahore. The voucher specimen no. P-cog 0139 was deposited in the herbarium of Pharmacognosy Section, Department of Pharmacy, University of the Punjab, Lahore.

vent system when evaporated, yielded a mixture of two saponins (3.12 g), one of which was present in major quantity. Other pooled fraction obtained with the second solvent system, afforded a mixture of compounds (7.23 g). Three grams of the dried product from the first fraction was re-chromatographed on second silica gel column (180 g), eluted with CHCl3 :MeOH:EtOAc (5:5:7) gave almost pure saponin-I (93 mg). This compound exhibited [α]24 D = +1 (c = 0.4; MeOH) and IR absorption maxima at 2940 (broad and strong OH absorption), 1700 (strong), 1460, 1380, 1280 (strong), 910 and 810 (medium) cm−1 . The structural evident indicated that the saponin-I was similar compound to saponin-A (previously reported by Ansari et al. (1987)). 2.5. Isolation of saponin-II Crude amorphous product (16 g), from the solvent extraction was chromatographed on a Sephadex LH-20 (5.6 cm × 85 cm) column. Elution was performed with EtOH:H2 O (1:1). First fraction contained a mixture of saponins. This mixture was passed through silica gel column (150 g) and eluted with CHCl3 :MeOH:EtOAc (5:6:7) to remove any yellow colouring matter. After evaporating the solvent, a white saponin mixture (6.3 g) was obtained. It was re-chromatographed on Biogel P-2 and eluted with H2 O. Pure saponin-II (81 mg) was obtained from a pooled fraction after evaporating the solvent. This compound exhibited [α]24 D = +1 (c = 0.3; MeOH); UV λmax = 230 nm and IR absorption maxima at 3480, 2940 (strong), 1700 (strong), 1650, 1460, 1380, 1260 (strong), 1050, 820 and 730 (medium) cm−1 . The structural evident of the saponin-II also showed that it was a similar compound to saponin-B (reported earlier by Ansari et al. (1987)). 2.6. Bioassay

2.4. Isolation of saponin-I

2.6.1. Procurement and maintenance of animals Sixty healthy adult male rabbits of albino strain of species Oryctolagus cuniculus and subspecies Caprolagus hispidus, weighing 1.0–1.5 kg were purchased from an animal supplier and acclimatised in the animal house of the Department of Pharmacy, University of the Punjab, Lahore, for 1 week. The animals were provided with fresh green fodder (clover) and tap water ad libitum. They were grouped into ten by random selection, each consisting of six animals. The groups were labelled as follows: (1) labelled as C (for untreated control group); (2–4) labelled as CT1 , CT2 , CT3 (for 1st, 2nd and 3rd crude drug treated groups); (5–7) labelled as S1 T1 , S1 T2 , S1 T3 (for 1st, 2nd and 3rd saponin-I treated groups); (8–10) labelled as S2 T1 , S2 T2 , S2 T3 (for 1st, 2nd and 3rd saponin-II treated groups).

Crude amorphous powder (25 g) was fractionated by flash chromatography on silica gel (400 g) and eluted with CHCl3 :MeOH:EtOAc (5:4:8) and CHCl3 :MeOH:EtAc:H2 O (5:6:8:2). The pooled fraction obtained with the first sol-

2.6.2. Dosage form Each rabbit of group C was orally administered 20 ml of distilled water through Ryle’s tube. The doses of crude F. cretica powder for CT1 , CT2 , CT3 were calculated on the

2.3. Extraction The aerial parts of the fresh plants (13 kg) were finely chopped and soaked in EtOH (3 × 15 l) for 20 days. Extracts were concentrated in a cyclone evaporator maintaining the temperature between 45 and 50 ◦ C. A dark green semi-solid residue (93 g) was obtained, which was dissolved in MeOH (3 × 500 ml). Me2 CO (2 × 500 ml) was added to it that resulted in precipitation. The precipitates were filtered, washed first with petroleum ether (40–60, 800 ml), then with CHCl3 (700 ml) and EtOAc (400 ml). Finally on drying, it yielded amorphous material (43.45 g).


197

basis of 0.5, 1.0 and 1.5 g/kg body weight of the animals respectively. For both saponin-I and saponin-II treated groups of the animals, i.e. for S1 T1 , S1 T2 , S1 T3 and for S2 T1 , S2 T2 , S2 T3 , the doses were calculated on the basis of 10, 20 and 30 mg/kg body weight of the animals, respectively. Each sample of the powdered crude drug dose and its isolated compound’s dose was suspended in 20 ml of distilled water and administered to the animals through Ryle’s tube. 2.6.3. Blood sampling Blood sampling was done from the ear veins of the rabbits at zero hours (before the treatment), then after 1st, 2nd, 4th, 8th, 12th and 16th day of the treatment. An amount of 1.5 ml of blood was collected each time in a vial containing 2.5 ␮g of ethylene diamine tetraacetic acid (EDTA) as an anticoagulant. 2.6.4. Haematological parameters The samples were investigated for red blood cell and white blood cell counts by dilution method using Neubaur’s scale (Green, 1978). Haemoglobin (Hb) was estimated photometrically with the help of haemoglobin kit. Mean corpuscular haemoglobin was calculated by the following equation (Green, 1978): Hb (in g)/100 ml of blood MCH = number of RBC/100 ml of blood 2.7. Statistical analysis In all the experiments, data was expressed as the mean plus standard error of the mean (mean ± S.E.M.) and tabulated in Tables 2–4. Significant differences between the controlled and treated groups were calculated by Student’s t-test (Snedecor and Cochran, 1967; Milton and Tsokos, 1983).

3. Results and discussion 3.1. Identification of extracted compounds The ethanolic extract of the aerial parts of F. cretica afforded a fraction containing saponins upon precipitation with acetone. One of the saponins was isolated and purified by repeated chromatography on silica gel. This saponin was designated as saponin-I (Fig. 1). Another saponin was isolated and purified after repeated chromatography on Sephadex LH-20 and Biogel P-2. This saponin was denoted as saponin-II (Fig. 1). Both the saponins were analysed by IR, UV, 1 H NMR and 13 C NMR spectroscopy. These compounds were identified after comparing their values of 1 H NMR and 13 C NMR chemical shifts (values of δ) and their coupling constants (values of J) with the previously known compounds (reported by Ansari et al. (1987)). Both saponin-I and saponin-II (Fig. 1) corresponded to the saponin-A and saponin-B of Ansari et al. (1987).

Fig. 1. Structures of the compounds isolated from F. cretica.

3.2. Effects on RBC The results indicated variations in RBC count in all the groups of saponin treated animals, which are outlined in Table 1. Generally it was found that RBC count has undergone a sharp decrease in the crude drug treated groups than the RBC counts of the controlled group of animals during the whole experimental period of 16 days. This trend was more pronounced in the animals of 1.0 g crude drug treated group than the other two dose groups of animals (Table 1). Both the saponins treated three dose groups showed a significant decrease in RBC count during the whole experimental period of 16 days (Table 1). This effect was more pontificated in animal groups treated with saponin-II than with the animals of saponin-I treated groups. The sharp and significant decrease in RBC count seemed to be more affirmed by the 30 mg dose of saponin-II as compared to the same dose of saponin-I (Table 1). 3.3. Effects on HC Haemoglobin concentration in the blood also followed a similar trend as was seen in case of RBC count. Minor variations however, was noted in this study. The 0.50 and 1.0 g crude drug treated groups of animals, indicated a decreasing tendency of haemoglobin concentration in their bloods up to 4th day, which gradually increased afterwards up to the 16th day. The third dose of the crude drug (1.50 g) on the other hand, showed a continued decreasing trend up to the 16th day (Table 2). Both the saponin-I and saponin-II treated groups also exhibited a decreasing pattern with the first two doses (i.e. 10 and 20 mg) up to the 4th day, which later on,

198


Table 1 Effects of crude powdered F. cretica, saponin-I and saponin-II on the red blood cells count (×106 cells/mm3 ) in rabbits Drugs

Dosage form

Days of treatment

Control

0

1

2

4

6.43 ± 0.06

6.40 ± 0.07

6.41 ± 0.04

6.42 ± 0.03

± ± ± ±

0.03 0.04 0.07 0.01

6.48 7.42 7.49 6.25

± ± ± ±

0.04 0.03 0.4 0.02

6.37 7.40 7.40 5.14

± ± ± ±

0.07 0.05 0.03 0.04

6.30 7.29 7.32 4.39

8

0.08∗

± ± 0.02 ± 0.06 ± 0.05

6.39 ± 0.02 0.03∗

± ± 0.06∗ ± 0.03 ± 0.06

12

16

6.40 ± 0.06

6.42 ± 0.04

± ± ± ±

± ± ± ±

Powdered F. cretica

0.50 g 1.00 g 1.50 g 10 mg

6.49 7.48 7.52 6.27

Saponin-I

20 mg 30 mg 10 mg

6.25 ± 0.2 6.41 ± 0.3 6.28 ± 0.04

6.21 ± 0.01 6.10 ± 0.04 6.27 ± 0.07

5.39 ± 0.02 5.29 ± 0.03 6.22 ± 0.06

5.31 ± 0.03 5.20 ± 0.4 5.21 ± 0.02

5.24 ± 0.05 5.03 ± 0.03 3.81 ± 0.03

5.00 ± 0.03 4.20 ± 0.05 2.19 ± 0.03∗∗

5.01 ± 0.08 3.69 ± 0.06∗∗∗ 2.13 ± 0.02∗∗∗

Saponin-II

20 mg 30 mg

6.17 ± 0.02 6.23 ± 0.03

6.07 ± 0.02 6.13 ± 0.03

6.01 ± 0.07 5.89 ± 0.05

3.29 ± 0.03 4.00 ± 0.08

2.52 ± 0.06 1.44 ± 0.05

1.33 ± 0.05∗ 1.42 ± 0.06∗∗∗

1.30 ± 0.03∗ 1.16 ± 0.05∗∗∗

6.29 7.10 7.12 4.30

5.80 6.00 6.21 4.12

0.05 0.02 0.05∗∗∗ 0.02

5.60 5.21 6.00 4.01

0.03 0.06 0.3∗∗∗ 0.01

Data represent the mean ± S.E.M. (n = 6). ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

slightly increased up to the 16th day (Table 2). The third dose of these compounds (i.e. 30 mg) displayed a highly significant decreasing pattern, through out the experimental period (Table 2). The effect of the third dose appeared to be more distinct in the saponin-II treated animal group (i.e. decrease from 5.89 ± 0.02 to 2.31 ± 0.02 g/100 ml) when compared with the saponin-I and crude drug treated animal groups (Table 2).

after the 1st day and was continued up to the 4th day, when compared with the controlled animal group. Later on, it continued decreasing, up to the end of the experimental period. This tendency seemed to be more effectual in 1.50 mg crude drug animal group (Table 3). A similar, but more consistent, tendency in MCH was also exhibited by both the saponin-I and saponin-II treated animal groups. The 30 mg treated animal groups of these saponins exhibited well-marked and significant increasing trends in MCH up to 4 days, while a decreasing pattern was seen in the later week; when compared with the animals of controlled group, crude drug treated animal groups and other two doses treated groups of both the saponins (Table 3). This increase in MCH concentration, after the 4th day reached at its maximum (28.9 ± 0.2) in 30 mg group of saponin-I as compared to the other saponin group (Table 3).

3.4. Effects on MCH First increasing then decreasing trends were seen in the mean corpuscular haemoglobin concentration which followed a reverse pattern than RBC counts in all the animal groups (Table 3). In the animals of all three crude drug dose groups, a significant increasing trend in MCH was started

Table 2 Effects of crude powdered F. cretica, saponin-I and saponin-II on haemoglobin concentration (HC in g/dl) in rabbits Drugs

Dosage form

Days of treatment 0

1

2

9.32 ± 0.042 9.28 ± 0.035

Control

± ± ± ±

0.02 0.04 0.03 0.045

± ± 0.025∗∗ ± 0.04∗∗∗ ± 0.26

9.31 ± 0.062 0.045∗

± ± 0.04∗∗∗ ± 0.06 ± 0.36

8

9.30 ± 0.31

9.31 ± 0.087

± ± ± ±

0.031∗∗

± ± 0.051∗∗ ± 0.025∗∗ ± 0.037

12

16

9.32 ± 0.063

9.31 ± 0.042

± ± ± ±

± ± ± ±

0.03∗∗∗ 0.012 0.036∗∗ 0.02

Powdered F. cretica

0.50 g 1.00 g 1.50 g 10 mg

9.89 9.82 9.98 8.54

Saponin-I

20 mg 30 mg 10 mg

8.69 ± 0.25 8.41 ± 0.36 8.58 ± 0.36 7.89 ± 0.58∗∗ 7.55 ± 0.041 6.25 ± 0.023

8.21 ± 0.56 6.28 ± 0.37∗∗ 6.21 ± 0.011

7.45 ± 0.56∗ 6.25 ± 0.02∗∗∗ 6.20 ± 0.024

7.89 ± 0.056 5.01 ± 0.036∗ 6.25 ± 0.021

7.33 ± 0.023 4.24 ± 0.012∗∗ 6.50 ± 0.021

7.21 ± 0.02 4.03 ± 0.24 6.61 ± 0.041

Saponin-II

20 mg 30 mg

6.58 ± 0.046 6.22 ± 0.012 5.89 ± 0.023 4.26 ± 0.013∗∗

6.02 ± 0.023 3.26 ± 0.02∗∗∗

6.00 ± 0.021 3.11 ± 0.031

6.21 ± 0.020 3.00 ± 0.031

6.52 ± 0.032 2.28 ± 0.025∗∗

6.22 ± 0.020 2.31 ± 0.02∗∗∗


7.52 8.56 9.10 8.24

0.47∗∗

4

7.89 9.00 8.87 7.58

7.06 8.55 7.89 7.44

0.086 0.06∗∗ 0.03 0.31

8.25 8.33 7.84 7.59

8.15 8.20 7.52 8.23

0.049 0.051 0.025 0.036

8.02 8.00 7.16 7.65


199

Table 3 Effects of crude powdered F. cretica, saponin-I and saponin-II on mean corpuscular haemoglobin (MCH in pg) in rabbits Drugs

Dosage form

Days of treatment

Powdered F. cretica

0.50 g 1.00 g 1.50 g 10 mg

18.07 18.05 17.91 17.8

Saponin-I

20 mg 30 mg 10 mg

17.9 ± 0.5 17.6 ± 0.8 17.1 ± 0.5

19.8 ± 0.9 18.5 ± 0.5 17.9 ± 0.42

22.5 ± 0.29 23.6 ± 0.27 18.6 ± 0.24

27.9 ± 0.5∗ 28.9 ± 0.2∗∗ 20.5 ± 0.21

22.3 ± 0.36 28.4 ± 0.42 20.2 ± 0.36

21.8 ± 0.37 22.4 ± 0.45 19.5 ± 0.35

21.0 ± 0.41 21.8 ± 0.31 18.7 ± 0.21∗

Saponin-II

20 mg 30 mg

17.5 ± 0.3 18.6 ± 0.7

19.5 ± 0.85 21.5 ± 0.36∗∗

22.5 ± 0.63∗∗ 24.5 ± 0.85∗∗

22.8 ± 0.52∗∗ 24.8 ± 0.27

24.5 ± 0.71∗∗ 23.8 ± 0.42∗

23.8 ± 0.81 18.5 ± 0.71

21.9 ± 0.24 17.2 ± 0.85∗∗

0

1

18.06 ± 0.12 18.07 ± 0.26

Control

± ± ± ±

0.5 0.7 0.5 0.8

21.06 22.16 19.8 19.6

± ± ± ±

2

4

19.01 ± 0.18

20.01 ± 0.29

0.16∗∗ 22.00 ± 0.35 0.13∗∗ 23.16 ± 0.20∗∗∗ 0.9∗∗∗ 24.0 ± 0.13∗∗∗ 0.5 21.58 ± 0.25

22.3 24.0 25.6 27.2

± ± ± ±

8

12

20.02 ± 0.43

20.02 ± 0.15

0.61∗ 21.61 ± 0.71∗∗ 0.5∗∗ 23.0 ± 0.48∗∗ 0.0.1∗∗∗ 23.1 ± 0.2∗∗ 0.8∗ 26.4 ± 0.25∗

20.5 21.4 23.0 24.6

16

± ± ± ±

20.02 ± 0.21

0.13∗∗∗ 20.11 ± 0.11∗∗ 0.12∗∗∗ 21.00 ± 0.17∗∗ 0.17 22.8 ± 0.13∗ 0.63∗∗ 23.2 ± 0.32


tard erythrophagocytosis may be a factor in the production of polycythemia. First decreasing, then increasing action of the saponins from F. cretica on RBC count and haemoglobin concentration of blood were similar to that of glucocorticoids. The glucocorticoids were also known to affect circulating white blood cells (Green, 1978). Administration of glucocorticoids leads to an increase in the number of polymorphonuclear leucocyte in the blood because of increased rate of entrance into the blood from the bone marrow and a diminished rate of removal from circulation (Hunter and Bomford, 1968). Moreover, the number of eosinophil, monocytes and basophils of the blood decreases, after the administration of glucocorticoids (Hunter and Bomford, 1968). The thymus derived lymphocytes (T-cells) are also known to decrease proportionally more than those derived from the bone marrow (B-cells), indicating that subpopulations of

3.5. Effects on total WBC The results further showed a continued decreasing trend in the amount of total WBC during the 16 days of experimental period (Table 4). The effects of the crude drug and its isolated compounds on total WBC count were also appeared to be dependent on their doses used. This effect was highly significant in case of higher dose levels. Further, it appeared that both the purified saponins had much more distinctive action in decreasing the amount of total WBC than the crude drug itself, even at lower dose levels (Table 4). It is known that glucocorticoids increase the haemoglobin and red blood count, as evidence from frequent occurrence of polycythemia in Cushing’s syndrome and in Addison disease (Green, 1978). The capacity of these steroids to re-

Table 4 Effects of crude powdered F. cretica, saponin-I and saponin-II on total white blood cell count (cells/mm3 ) in rabbits Drugs

Dosage form

Control

Days of treatment 0

1

2

13250 ± 436

13249 ± 334

13250 ± 330

± ± ± ±

262 112 134 163

± ± ± ±

164 274 421∗∗ 154

13062 12800 10861 11578

± ± 220∗ ± 245∗∗ ± 123

8

13251 ± 351

13251 ± 432

± ± ± ±

154∗

± ± 124∗ ± 294∗ ± 251

12

16

13250 ± 168

13252 ± 251

± ± ± ±

± ± ± ±

Powdered F. cretica

0.50 g 1.00 g 1.50 g 10 mg

13208 13303 13902 13221

Saponin-I

20 mg 30 mg 10 mg

13251 ± 201 13760 ± 152 13220 ± 113

12300 ± 231 10356 ± 142∗∗ 12500 ± 571

11752 ± 321 9868 ± 423∗∗ 12501 ± 201

12504 ± 302 9750 ± 103 12200 ± 103

12474 ± 321 9807 ± 102∗ 12250 ± 210

11200 ± 108 8609 ± 104∗ 10861 ± 52∗∗

11321 ± 105 7906 ± 321 9780 ± 89

Saponin-II

20 mg 30 mg

13254 ± 152 13800 ± 231

11581 ± 124 7861 ± 235∗∗

11400 ± 125 7558 ± 102

11368 ± 142 7042 ± 230∗

11368 ± 321∗ 7438 ± 105∗∗

10524 ± 172 6521 ± 320∗∗

10235 ± 241 6447 ± 321∗∗

Data represent the mean ± S.E.M. (n = 6). ∗ P < 0.05. ∗∗ P < 0.01.

13128 13150 11309 12316

234∗

4

12860 13000 8800 11352

162 194 142 432

12306 12600 8679 10952

11600 11550 8656 10825

184 501 297∗∗ 312

9700 11900 8650 8900

521 214 148∗∗ 109

200


lymphocytes are differentially affected by the steroids (Hunter and Bomford, 1968). It is also known that glucocorticoids cause a rapid lysis of lymphatic tissues in rats and mice, while there is no comparable effect in man (Hunter and Bomford, 1968). Although glucocorticoids do not produce a sudden massive lysis of lymphoid tissue in man, yet the cells of acute lymphoblastic leukaemia and in some cases, cells of other lymphatic malignancies are destroyed by glucocorticoids (Hunter and Bomford, 1968). A similar mechanism was probably operative for the action of saponins from natural sources, such as triterpenoid saponins from F. cretica plants. On the basis of results, we concluded that the triterpenoid saponin-I and saponin-II, isolated from F. cretica had first decreasing, then increasing effect on RBC count and haemoglobin concentration of rabbit’s blood. On the other hand, a decreasing trend in MCH and a continued lowering pattern for the total WBC was observed during the 16 days of experimental period. The mechanism of action of these saponins on various haematological parameters including not only the total WBC but also the individual leukocytes count of blood, i.e. WBC differential count, however, needs an amplification and further investigations, particularly in relation to the structure–activity relationship of these compounds. Moreover, there is a need of magnifying the sphere of activities of these saponins from F. cretica in some diseased model and also on both the healthy and diseased bone marrow tissues of animals or from human beings.

References Al-Wakeel, S.A.M., Shahnaz, A.M., 1992. Significance of flavonoid chemistry in the Egyptian Fagonia glutinosa and F. isothricha complex. Biochemical Systematic Ecology 20, 259–264. Al-Wakeel, S.A.M., El-Nagoumy, S.I., El-Hadidi, M.N., Saleh, N.A.M., 1987. Flavonoid pattern in Fagonia mollis complex. Biochemical Systematic Ecology 15, 459–460. Al-Wakeel, S.A.M., El-Garf, I.A., Saleh, N.A.M., 1988. Distribution of flavonoids in Fagonia thebica complex. Biochemical Systematic Ecology 16, 57–58.

Ansari, A.A., Kenne, L., Atta, U.R., Wehler, T., 1987. Isolation and characterization of two saponins from Fagonia indica. Phytochemistry 26, 1487–1490. Ansari, A.A., Kenne, L., Atta, U.R., Wehler, T., 1988. Isolation and characterization of a saponin from Fagonia indica. Phytochemistry 27, 3979–3982. Atta, U.R., Ansari, A.A., Drexlex, S.A., Clardy, J., 1982. The isolation and structure of nahagenin. Heterocycles 19, 217–220. Atta, U.R., Ansari, A.A., Kenne, L., 1984. Hederagenin, ursolic acid and pinitol from Fagonia indica. Journal of Natural Products 47, 186–187. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. CSIR, New Delhi, India, p. 116. Chopra, R.M., Handa, K.L., Kapur, L.D., Chopra, I.C., 1982. Indigenous Drugs of India, second ed. Academic Press, New Delhi, India, p. 507. El-Hadidi, M.N., Al-Wakeel, S.A.M., El-Garf, I.A., 1988. Systematic significance of the flavonoid constituents in Fagonia indica complex. Biochemical Systematic Ecology 16, 293–297. El-Negoumy, S.I., Al-Wakeel, S.A.M., El-Hadidi, M.N., Saleh, N.A.M., 1986. The flavonoids of the Fagonia arabica complex. Phytochemistry 25, 2423–2434. Green, J.H., 1978. The blood. In: An Introduction to Human Physiology. Oxford University Press, New York, USA, pp. 5–22. Hamid, A., Majid, A.C.M., Atta, U.R., 1989. Isolation of docosyl docosanoate from Fagonia cretica L. Arabian Gulf. Journal of Scientific Research A 7, 29–34. Harash, M.L., Purohit, G.R., Mathur, C.S., Nag, T.N., 1981. Nutritive value of dried terrestrial plants growing in Rajasthan—chemical composition. Physiological Ecology 6, 30–32. Hash, M.L., Nag, T.N., 1988. Flavonoids with antimicrobial activities of arid zone plants. Geobios 15, 32–35. Hooker, J.D., 1975. Flora of British India. Reeva, London, p. 425. Hunter, D., Bomford, R.R., 1968. Hutchison’s Clinical Methods, 15th ed. Bailliere Tindall & Cassel, London, pp. 120–124. Milton, J.S., Tsokos, J.D., 1983. Statistical Methods in Biology and Health Sciences. McGraw-Hill, Tokyo. Saeed, M.A., 1969. Hamdard Pharmacopoeia of Eastern Medicine. Hamdard Academy, Karachi, Pakistan, pp. 41–43. Saleh, N.A.M., El-Hadidi, M.N., Al-Wakeel, S.A.M., 1988. Phytochemistry and the evolution of Fagonia species. Bulletin of Liaison-Groupe Polyphenols 14, 46–49. Saleh, N.A.M., El-Hadidi, M.N., Al-Wakeel, S.A.M., Shahnaz, A.M., 1990. Phytochemistry and phylogenetic affinities among Egyptian species of Fagonia. Biochemical Systematic Ecology 18, 49–52. Shaukat, G.A., Malik, M.A., Ahmad, M.S., 1981. Water soluble protein from Fagonia cretica L. Pakistan Journal of Botany 13, 99–101. Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods, 6th ed. Iowa State University Press, Ames, IA, USA.


Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits S.B. Sharma a,∗ , A. Nasir a , K.M. Prabhu a , P.S. Murthy b , G. Dev c a

Department of Biochemistry, University College of Medical Sciences and GTB Hospital, Delhi 110095, India b B-164, Sector 14, Noida, India c Department of Pathology, University College of Medical Sciences and GTB Hospital, Delhi 110095, India Received 28 February 2002; received in revised form 20 September 2002; accepted 28 November 2002

Abstract The hypoglycaemic and hypolipidemic effect of ethanolic extract obtained from seeds of E. jambolana was investigated in alloxaninduced diabetic rabbits. Hypoglycaemic activity was assessed by reduction in fasting blood glucose (FBG) at 90 min and also fall in peak blood glucose during glucose tolerance test (GTT) in sub-diabetic and mild diabetic (MD) rabbits, but in severe diabetic (SD) rabbits by reduction in FBG at 90 min. Ethanolic extract (100 mg/kg body weight) when given orally to sub-diabetic (AR) for 1 day, MD for 7 days and SD for 15 days showed significant fall in FBG at 90 min (12% AR, 18.9% MD and 29% SD) and also produced 16.9% fall in peak blood glucose in AR and 21% in MD rabbits during GTT. When administered daily for 15 days to MD and SD rabbits, significant fall in FBG (41.3% MD, 31.6% SD) and glycosylated haemoglobin (GHb) levels (23.3% MD, 26.6% SD) were observed, while serum insulin level showed significant increase (32.8% MD, 26.9% SD). Liver and muscle glycogen content also increased. The ethanolic extract of seeds also exhibited significant hypolipidemic effect as evident from fall in total serum cholesterol (TC)/high density lipoprotein cholesterol (HDL-c) ratio, serum low density lipoprotein cholesterol (LDL-c) levels and decreased activity of HMG-CoA reductase. The histopathological studies of liver, pancreas and aorta in alcoholic extract treated diabetic groups revealed almost normal appearance. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hypoglycaemic; Diabetes; Eugenia jambolana; Hypolipidemic; Medicinal plants

1. Introduction Plants have been the major source of drug for the treatment of diabetes mellitus in Indian system of medicine and other ancient systems in the world. Out of many, only a few have been evaluated as per modern system of medicine. Eugenia jambolana known as Jamun (Hindi), Black plum (English) and Neredu (Telugu) is also reported to possess antidiabetic property (Bansal et al., 1981; Achrekar et al., 1991). Aqueous and ethanolic extract of seeds administered orally to experimental animals and to human adults at variable dose levels were found to be active (Anon, 1968; Kohli and Singh, 1985; Achrekar et al., 1991). Hypoglycaemic effect of E. jambolana was also reported in

∗ Corresponding author. Tel.: +91-228-2971–74x211 (O)/212-1087-229-5663 (R); fax: +91-11-229-0495. E-mail address: [email protected] (S.B. Sharma).

fructose-fed rats (Vikrant et al., 2001). However, tea prepared from leaves did not produce any antihyperglycaemic effect in experimental and clinical models (Claudio et al., 2000). Since data are available on hypoglycaemic effect of E. jambolana in different animals, but not in different types of diabetes, we compared the glycaemic control of ethanolic extract of seeds from the fruits in different diabetic models of rabbits. We studied its effect in severe diabetic (SD) rabbits (Type 1 or IDDM) in which pancreas is almost totally destroyed or with very few ␤-cells and in mild diabetic (MD) rabbits (type 2 or NIDDM) having functional ␤-cells. While working on the hypoglycaemic activity, we found that ethanolic extract of seeds possess not only hypoglycaemic activity but also improved lipid profile and also decreased the activity of key enzymes of cholesterol biosynthesis (HMG-CoA reductase). However, no reports are available in the literature about its hypolipidemic effect, which is also reported here.


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2. Material and methods

2.4. Measurement of hypoglycaemic activity of ethanolic extract of seeds

2.1. Plant material Fruits of E. jambolana (Myrtaceae) were procured from the Azadpur Mandi (herbal market) at Delhi. The identity was made with the help of a botanist using taxonomic rules (Voucher specimen no. P-96/7) and specimen is kept for future references in Indian Botanic Garden, Kolkata, India. 2.2. Preparation of the extract from seeds of E. jambolana fruits The fruits of E. jambolana (1 kg) were first washed well and pulp was separated from the seeds. Seeds were washed several times to remove the traces of pulp from seeds. The seeds of E. jambolana were then dried at room temperature and ground in an electric grinder to have coarse powder. Then 100 g seed powder of E. jambolana was suspended in distilled water (250 ml) and allowed it to stand overnight in refrigerator and then strained through several layers of muslin cloth. The filtrate (water extract) was discarded. Residue was extracted with petroleum ether to remove lipids. It was then filtered and filtrate was discarded. The residue was then mixed with ethanol (80%) and allowed to stand for 48 h in refrigerator and then extracted with 95% ethanol. The extract was filtered through muslin cloth and allowed to stand for 72 h in refrigerator and then concentrated under reduced pressure. The yield of alcoholic extract of E. jambolana seeds was 1.5 g/100 g of the dried powdered seeds. 2.3. Experimental animals Male albino rabbits weighing between 1 and 1.5 kg were used and were maintained on commercial diet (Hindustan Lever Ltd., Bombay) and water ad libitum. They were acclimatised to the laboratory conditions at least for 1 week before carrying out any experimental work. Diabetes mellitus was induced by alloxan. Alloxan monohydrate (80 mg/kg, Sigma Chemicals, USA) (Shukla et al., 1995) dissolved in citrate buffer (pH 4.0) was injected intravenously to the overnight fasted rabbits through their marginal ear vein. Food was provided to them 2 h after the injection. After 1 month, the rabbits showing stabilised diabetes having fasting blood glucose (FBG) values 250 mg/dl or above were considered as SD group and those with FBG values between 120 and 250 mg/dl and having abnormal glucose tolerance in glucose tolerance test (GTT) were as MD. In some of rabbits the FBG values increased initially but returned over a period of 1 month to normal or slightly elevated FBG values (120 mg/dl or below) but showed abnormal glucose tolerance pattern. They were designated as alloxan recovered (AR) sub-diabetic rabbits. Rabbits that did not show any increase in FBG levels even initially after alloxan injection were considered as totally resistant and excluded from studies.

For oral administration, dried ethanolic extract was suspended in water using Tween 80 and given orally at a dose ranging from 50 to 200 mg/kg. Control group of five rabbits received the same volume of vehicle (Tween 80 in distilled water). The extract was given to a group of five rabbits, each of sub-diabetic (AR) for 1 day, MD for 7 days and SD for 15 days and hypoglycaemic activity was assessed by improvement of GTT in sub-diabetic rabbits (because the increase in FBG is only slight), by fall in FBG and improvement in GTT in MD rabbits and by lowering FBG in SD rabbits. Blood samples were collected in fasting, 90 min after giving drug and 1–3 h after glucose load. Tolbutamide 250 mg/kg (Hoechst Pharmaceutical Ltd., Bombay) (Rahman et al., 1985) was used as a standard drug in another group of five rabbits for comparison. After assessment of hypoglycaemic activity in AR, MD and SD rabbits with different doses of ethanolic extract of E. jambolana seeds, the dose of 100 mg/kg of ethanolic extract was found to have maximum hypoglycaemic activity. Therefore, subsequent studies were carried out after giving 100 mg/kg of ethanolic extract for 15 days in MD and SD rabbits. Blood samples were collected from the lateral marginal ear vein of overnight fasted rabbits in glass vial containing an anticogulant and blood glucose was estimated by glucose oxidase method (Braham and Trinder, 1972). Plasma insulin levels were estimated by ELISA method (Burgi et al., 1988). Blood collected in EDTA vial was used for estimation of glycosylated haemoglobin (GHb) by using ion-exchange resin (Willey et al., 1984). Total serum cholesterol (TC) estimated by Allain et al. (1974) method. High density lipoprotein cholesterol (HDL-c) estimated by the method of Burstein et al. (1970). Low density lipoprotein cholesterol (LDL-c) and very low density lipoprotein cholesterol (VLDL-c) levels were calculated by using the formula of Friendwald et al. (1972). Serum triglycerides (sTG) were determined colorimetrically with enzymatic method (Fossati and Lorenzo, 1982). Indirect assessment of HMG-CoA reductase activity in liver tissue was by the method of Rao and Ramakrishan (1975) and total lipids from animal tissue was estimated by Folch et al. (1957). Liver and muscle glycogen was estimated with anthrone reagent by the method of Carroll et al. (1956). 2.5. Histopathological studies Histopathological studies of the liver, heart, aorta and pancreas were conducted in control and diabetic rabbits treated with control vehicle or ethanolic extract of E. jambolana seeds at a dose of 100 mg/kg for 1 month. The tissues were cut and fixed in 10% formalin and the sections stained with haematoxylin and eosin were studied under light microscope.


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effect, i.e. 18.9% fall at 90 min and 21% fall in peak blood glucose during GTT. In SD rabbits, 50 mg/kg of ethanolic extract of seeds showed 19.0% fall in blood glucose after 90 min of oral administration (P < 0.001). Dose of 100 mg/kg produced maximum effect, i.e. 29% fall in blood glucose levels which was statistically significant (P < 0.001). With 200 mg/kg, ethanolic extract of seeds did not show any further improvement and it produced 28.8% fall in 90 min after the oral administration of extract. The effect with ethanolic extract was better than that with tolbutamide.

2.6. Statistical analysis Results were expressed as mean±S.D. The significance of the difference between the means of test and control studies was established by Student’s t-test. P values less than 0.05 were considered significant.

3. Results 3.1. Blood glucose and glucose tolerance test

3.2. Fasting blood glucose, glycosylated haemoglobin and plasma insulin

Table 1 depicts hypoglycaemic effect of oral administration of ethanolic extract of E. jambolana seeds in sub-diabetic, MD and SD rabbits. In sub-diabetic rabbits, ethanolic extract of seeds at dose of 50 mg/kg showed significant change (P < 0.01) and it produced 8.0% fall in blood glucose after 90 min and 13% fall in peak blood glucose during GTT. A higher dose (100 mg/kg) produce 12% fall in 90 min after giving extract and 16.9% in peak blood glucose levels. The change was highly significant (P < 0.001). However, increase in the dose to 200 mg/kg produced only slightly higher fall, i.e. 13.2% fall after 90 min and 18% fall in peak blood glucose. Oral administration of 250 mg/kg tolbutamide in MD rabbits produced 26.3% fall in blood glucose after 90 min and 33% fall in peak blood glucose during GTT. In MD rabbits, significant hypoglycaemic activity was observed with 50, 100 and 200 mg/kg of ethanolic extract of E. jambolana. However, 100 mg/kg dose showed maximum

Ethanolic extract also exhibits remarkable glycaemic control in MD and SD rabbits when administered for 15 days as evident by decrease in FBG (MD 41.3%, SD 31.6%) and GHb (MD 23.3%, SD 26.6%) and significant increase in serum insulin levels (MD 32.8%, SD 26.9%) (Table 2). 3.3. Serum lipid profile Table 3 shows the effect of treatment with ethanolic extract of seeds of E. jambolana on serum lipid profile in MD and SD rabbits. In diabetic animals, before treatment there was an overall increase in serum lipids, except HDL-c which was decreased. After the administration of ethanolic extract of seeds at the dose of 100 mg/kg for 15 days, there was fall in TC, TC/HDL-c ratio, LDL-c, VLDL-c and sTG

Table 1 Hypoglycaemic effect of ethanolic extract of E. jambolana seeds in various diabetic models of rabbits Experimental groups

Dose (mg/kg)

Blood glucose (mg/dl) (mean ± S.D.) Fasting

Sub-diabetic Control (vehicle treated) Tolbutamide Ethanolic extract

Mild diabetic Control (vehicle treated) Tolbutamide Ethanolic extract

Severe diabetic Control (vehicle treated) Tolbutamide Ethanolic extract

After 90 min treatment

Percent fall in peak blood glucose during GTT Percent fall

250 50 100 200

108 107 102 107 110.8

± ± ± ± ±

5.3 5.0 4.0 4.9 5.2

109 87 94 94 96.2

± ± ± ± ±

7.3 3.0∗∗∗ 4.3∗∗ 5.1∗∗∗ 5.1∗∗∗

Nil 18.6 8.0 12.0 13.2

Nil 28.0 13.0 16.9 18.0

250 50 100 200

158.0 152.2 152 153 163.0

± ± ± ± ±

5.5 3.2 6.9 5.9 5.9

155.0 112 133 124 135

± ± ± ± ±

4.3 4.4∗∗∗ 4.6∗∗ 5.1∗∗∗ 3.2

Nil 26.3 12.5 18.9 16.8

Nil 33.0 18.3 21.0 20.8

261 277.6 260.4 255.8 269.4

± ± ± ± ±

13.5 13.3 15.4 17.0 14.3

259 219 210 181.6 191.8

± ± ± ± ±

12.8 14.3∗∗∗ 13∗∗∗ 13.2∗∗∗ 12.9∗∗∗

Nil 21.0 19.0 29.0 28.8

Nil

250 50 100 200

The administration period in each group is 1–2 min. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

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Table 2 Glycaemic control by ethanolic extract of E. jambolana seeds (100 mg/kg) in mild and severe diabetic rabbits after 15 days treatment Experimental groups FBG (mg%)

GHb (%)

Insulin (␮u/ml)

Mild diabetic Control Before treatment After treatment % change

89.1 ± 2.0 154 ± 6.8 90 ± 4.7∗∗∗ 41.3

34.7 ± 0.7 6.0 ± 0.4 4.6 ± 0.3∗∗∗ 23.3

35.2 ± 1.6 19.5 ± 1.0 25.9 ± 1.1∗∗∗ 32.8

Severe diabetic Control Before treatment After treatment % change

90 ± 10.5 3.8 ± 0.3 266 ± 23.1 6.8 ± 0.20 182 ± 11.8∗∗∗ 5.0 ± 0.30∗∗∗ 31.6 26.6

34.5 ± 2.4 16.3 ± 0.60 20.7 ± 0.50∗∗∗ 26.9

Values are mean ± S.D. of five animals in each group. ∗∗∗ P < 0.001.

levels. However, remarkable decrease (>20%) was found in TC/HDL-c ratio (atherogenic index) and LDL-c levels in both MD and SD rabbits. HDL-c also increased in both MD (12%) and SD (17%) rabbits. 3.4. HMG-CoA reductase, total lipids and liver and muscle glycogen Untreated MD and SD rabbits showed hyperlipidemia as compared to normal rabbits (Table 4). Feeding of ethanolic extract to diabetic rabbits also caused reduction in the total lipid content of the liver tissues in MD (12%) and SD (14%) rabbits. Activity of HMG-CoA reductase, key enzyme of cholesterol biosynthesis was increased in the liver tissue of the diabetic rabbits. However, administration of alcoholic

Table 3 Effect of ethanolic extract of E. jambolana seeds (100 mg/kg) on serum lipid profile in alloxan-induced mild and severe diabetic rabbits after 15 days treatment Experimental group

Serum lipid profile (mean ± S.D.) TC (mg/dl)

Mild diabetic Healthy control

72.0 ± 10.7

Diabetic Before Tt After Tt % change

105.5 ± 11.4 92.3 ± 10.4∗∗ 12.5

Severe diabetic Healthy control Diabetic Before Tt After Tt % change

68.0 ± 9.5 120.0 ± 14.1 96.0 ± 11.6∗∗ 20

HDL-c (mg/dl) 35.2 ± 3.7 22.2 ± 2.3 24.9 ± 3.4∗∗∗ 12.2 29.7 ± 2.5 23.5 ± 2.1 27.5 ± 3.0∗∗∗ 17

TC/HDL-c ratio 2.04 ± 0.2 4.7 ± 0.2 3.7 ± 0.5∗∗∗ 21.0 2.30 ± 0.2 5.1 ± 0.40 3.5 ± 0.5∗∗∗ 31.3

LDL-c (mg/dl) 88.4 ± 8.3 116.3 ±12.2 88.6 ± 7.2∗∗∗ 25.5 78.1 ± 11.6 145.0 ± 14.1 108.5 ± 10.7∗∗∗ 25.1

VLDL-c (mg/dl)

STG (mg/dl)

23.8 ± 0.5

117 ± 13.5

33.0 ± 0.9 28.6 ± 7.2∗∗∗ 13.3

165 ± 14.5 143 ± 2.3∗∗∗ 13.3

22.4 ± 2.2

112.0 ± 14.6

48.6 ± 2.9 40.0 ± 3.8∗∗ 17.9

243.0 ± 16.4 200.0 ± 9.4∗∗ 17.9

Tt: treatment. ∗∗ P < 0.001. ∗∗∗ P < 0.001. Table 4 Effect of ethanolic extract of E. jambolana seeds (100 mg/kg) on HMG-CoA reductase, total lipids and liver and muscle glycogen after 15 days treatment in mild and severe diabetic rabbits Experimental groups

HMG-CoA reductasea

Total lipids (mg/g tissue)

Liver glycogen (mg/g tissue)

Muscle glycogen (mg/g tissue)

Mild diabetic Healthy control

5.7 ± 0.40

26.3 ± 3.5

33.1 ± 6.2

12.3 ± 1.6


8.6 ± 0.60 7.8 ± 0.9b 9.3

32.5 ± 2.4 28.6 ± 3.1∗∗∗ 12

15.8 ± 3.1 21.6 ± 4.5∗∗∗ 27

6.8 ± 0.8 9.1 ± 1.1∗∗∗ 25

Severe diabetic Healthy control

5.9 ± 0.2

25.7 ± 8.9

31.8 ± 5.8

11.1 ± 1.5


10.1 ± 0.2 9.0 ± 0.2b 10.9

39.7 ± 1.6 34.1 ± 4.3∗∗∗ 14.0

10.9 ± 4.4 16.0 ± 5.2∗∗∗ 31.9

4.6 ± 2.6 6.5 ± 1.6∗∗∗ 29.0

a

HMG-CoA reductase activity is expressed as a ratio between HMG-CoA and mevalonate levels. Significantly different from its before treatment value in fasting. ∗∗∗ P < 0.001. b


extract of seeds orally for 15 days leads to reduction in the activity of HMG-CoA reductase (MD 9.3%, SD 10.9%) but values were still higher than normal. Glycogen content of the liver and muscle tissues in these groups is shown in Table 4. In diabetic control, hepatic and skeletal muscles glycogen content decreased significantly. However, treatment with alcoholic extract of seeds of E. jambolana led to increase in liver (MD 27%, SD 31.9%) and muscle glycogen contents (MD 25%, SD 29%). 3.5. Histopathological studies Histological studies of heart, aorta, liver and pancreas were carried out in alloxan-induced diabetic rabbits which were sacrificed after 1 month of the experiment. Group I consisting of untreated diabetic rabbits (n = 5, control) received only water showed mild congestion of central veins with surrounding hepatocytes showing fatty change. Aorta showed mild thickening of intima with lipid insudation. Vacuolation of the myocytes was also seen. Pancreatic islets are partly hylinised with reduced number of islet cells with an occasional cell and showed vacuolation. After 1 month of administration of ethanolic extract of seeds of E. jambolana, to Group II rabbits, liver showed only mild fatty changes in hepatocytes. No vacuolation was seen in aorta and heart. In the pancreas of the extract treated animals vacuolation of the islets cells was also not prominent. The pancreas appeared normal.

4. Discussion Ethanolic extract showed a dose-dependent effect on FBG up to a dose of 100 mg/kg. However, higher dose of 200 mg/kg did not show any dose-dependent effect although it caused a significant decrease in the FBG level. It is likely that the bigger doses could not produce the expected higher hypoglycaemic effect by the presence of some other substances in the ethanolic extract, which interfere with the hypoglycaemic effect. So detailed studies were carried out with a dose of 100 mg/kg. Ethanolic extract of seeds of E. jambolana caused significant hypoglycaemic effect in MD and SD rabbits after 15 days treatment. Although, the percent fall in blood glucose was found to be more in MD rabbits which have functioning pancreatic ␤-cells, significant fall in SD rabbits suggests that the active hypoglycaemic compound present in the ethanolic extract of seeds does not necessarily require the presence of functioning ␤-cells for its favourable action seen in type I and type II. It means that it can act in a variety of diabetic conditions with or without functioning pancreatic ␤-cells. GHb has been found to be increased over a long period of time in diabetes (Bunn et al., 1978). Therefore, measurement of GHb is supposed to be very sensitive index for glycaemic control. Ethanolic extract of seeds also exhibited significant reduction in glycosylated haemoglobin in

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both MD and SD rabbits. The extract was not only effective in lowering blood glucose level but also caused significant increase in plasma insulin levels which could correct other essential metabolic alterations. It is also possible that the ethanolic extract increases sensitivity of insulin in diabetic rabbits because it showed favourable effect in MD rabbits having adequate though reduced levels of plasma insulin. The rise in blood sugar is accompanied with the increase in TC, LDL-c, VLDL-c, TG and fall of HDL-c. However, ethanolic extract of seeds at a dose of 100 mg/kg exhibited hypocholesterolemic and hypotriglyceridemic effects while at the same time increasing HDL-c. The activity of HMG-CoA reductase in liver, key enzyme for cholesterol biosynthesis was also found to be decreased after treatment (Kedar and Chakrabarti, 1982). This implies that E. jambolana seeds can prevent or be helpful in reducing the complications of lipid profile seen in some diabetics in whom hyperglycaemia and hypercholesterolemia coexist quite often. Welihinda and Karunanayake (1986) also reported that hepatic and skeletal muscle glycogen content is reduced significantly in diabetes as compared to nondiabetic control. Treatment with ethanolic extract of seeds prevented this alteration in glycogen content but could not normalise it in 15 days treatment. This prevention of depletion of glycogen in the liver and muscles is possibly due to stimulation of insulin release from ␤-cells (Lolitkar and Rao, 1966). Therefore, the mechanism of action appears to be both pancreatic (Bansal et al., 1981) and extra pancreatic as E. jambolana is reported to inhibit insulinase activity in both liver and kidney (Achrekar et al., 1991). This shows that ethanolic extract is effective in decreasing blood glucose not only in NIDDM but also in IDDM. Another interesting feature is that the histopathological abnormalities seen in the pancreas, liver, aorta and heart of diabetic animals are also reversed giving almost normal appearance.

Acknowledgements The authors thank Director-General, Indian Council of Medical Research, New Delhi (India) for providing financial assistance during study.

References Achrekar, B., Kakij, G.S., Pote, M.S., Kelkar, S.M., 1991. Hypoglycaemic activity of Eugenia jambolana and Ficus bengalensis. In vivo 5, 143– 147. Allain, C.C., Poon, L.C., Chan, C.S.G., Richmond, W., Fu, C., 1974. Enzymatic determination of total cholesterol. Clinical Chemistry 20, 470–473. Anon, 1968. Hypoglycaemic medicament based on Syzygium jambolanum (Java plum). Patent FrM-6114, 4 pp. Bansal, R., Ahmad, N., Kidwai, J.R., 1981. Effect of oral administration of Eugenia jambolana seeds and chlorpropamide on blood glucose level and pancreatic cathepsin B in rats. Indian Journal of Biophysics 18, 377–380.

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Braham, D., Trinder, P., 1972. Estimation of glucose by glucose oxidase method. Analyst 97, 142–145. Bunn, H.G., Gabbay, K.H., Gallop, P.M., 1978. The glycosylation of haemoglobin: relevance to diabetes mellitus. Science 200, 21– 27. Burgi, W., Briner, M., Franken, N., Kessler, A.C.H., 1988. One step sandwich enzyme immunoassay for insulin using monoclonal antibodies. Clinical Biochemistry 21, 311–314. Burstein, M., Scholnick, H.R., Morgin, R., 1970. Rapid method for the isolation of lipoprotein from human serum by precipitation with polyanion. Journal of Lipid Research 11, 1583–1586. Carroll, N.V., Lonely, R.W., Roa, J.H., 1956. The determination of glycogen in liver and muscle by the anthrone reagent. Journal of Biological Chemistry 220, 561–583. Claudio, C.T., Carlos, A.R., Parila, M.D.S., Raquel, M., Rodrigo, A., Fabio, A., Claudia, R.C.A., Flavio, D.F., 2000. Absence of antihyperglycaemic effect of jambolan in experimental and clinical models. Journal of Ethnopharmacology 71, 343–347. Folch, J., Lee, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509. Fossati, P., Lorenzo, P., 1982. Serum triglycerides determined colorimetrically with an enzyme that produce hydrogen peroxide. Clinical Chemistry 28, 2077–2080. Friendwald, W.T., Levy, K.J., Frederickson, D.S., 1972. Estimation of concentration of LDL in plasma without use of preparative ultracentrifuge. Clinical Chemistry 18, 499–502.

Kedar, P., Chakrabarti, C.H., 1982. Effect of Bitter gourd (Momordica charantia) seed and glibenclamide in streptozotocin-induced diabetes mellitus. Indian Journal of Experimental Biology 20, 232–235. Kohli, K.R., Singh, R.H., 1985. Eugenia jambolana: a plant drug with potential antidiabetic properties. Journal of Science Research and Plant Medicine 6, 1–4. Lolitkar, M.M., Rao, M.R.R., 1966. Pharmacology of hypoglycaemic principle isolated from the fruits of Charantia Linn. Indian Journal of Pharmacy 28 (5), 129–133. Rahman, H., Kashjudduja Tyeb, M., Saleemuddin, M., 1985. Hypoglycaemic activity of Tephrosea purpurea Linn. Indian Journal of Medical Research 81, 418–421. Rao, A.V., Ramakrishan, S., 1975. Indirect assessment of HMG-CoA reductase activity in liver tissue. Clinical Chemistry 21, 1523–1525. Shukla, R., Anand, K., Prabhu, K.M., Murthy, P.S., 1995. Hypolipidemic effect of water extract of Ficus bengalensis in alloxan-induced diabetes mellitus in rabbits. Indian Journal of Clinical Biochemistry 10, 119– 121. Vikrant, V., Grover, J.K., Tandon, N., Rathi, S.S., Gupta, N., 2001. Treatment with extracts of Momordica charantia and Eugenia jambolana prevents hyperglycaemia and hyperinsulinemia in fructose-fed rats. Journal of Ethanopharmacology 76, 139–143. Welihinda, J., Karunanayake, E.H., 1986. Extra pancreatic effect of M. charantia in rats. Journal of Ethanopharmacology 17 (3), 247–255. Willey, D.G., Rosenthal, M.A., Caldwell, S., 1984. Glycosylated haemoglobin and plasma glycoprotein assay by affinity chromatography. Diabetologia 27, 56–61.


An evaluation of the activity related to inflammation of four plants used in Thailand to treat arthritis P. Laupattarakasem a,1 , P.J. Houghton a,∗ , J.R.S. Hoult b,2 , A. Itharat a,c a

b

Pharmacognosy Research Laboratories, Department of Pharmacy, King’s College London, 150 Stamford Street, Franklin Wilkins Building, London SE1 9NN, UK Messengers and Signalling Research Group, GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London SE1 9RT, UK c Pharmacognosy Department, Faculty of Pharmacy, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Received 1 July 2002; received in revised form 13 November 2002; accepted 28 November 2002

Abstract The leaves of Acanthus ebracteatus, stembark of Oroxylum indicum and the stems of Cryptolepis buchanani and Derris scandens are used as traditional remedies in Thailand for arthritis. Aqueous and alcoholic extracts were tested using three different in vitro systems for effects relevant to anti-inflammatory activity. The aqueous extracts of O. indicum and D. scandens significantly reduced myeloperoxide release. Eicosanoid production was reduced only by the aqueous extracts of A. ebracteatus and D. scandens. D. scandens extract showed potent inhibitory activity against generation of leukotriene B4 and also displayed antioxidant activity. In the rat hind paw edema test, D. scandens extract showed significant activity when given intraperitoneally but did not produce a significant reduction when given orally. The results therefore supported to some extent the traditional use of D. scandens for arthritic conditions and provided slight indication of activity which could explain the use of O. indicum and A. ebracteatus. No relevant activity was demonstrated in any of the tests for C. buchanani extracts. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acanthus ebracteatus; Oroxylum indicum; Cryptolepis buchanani; Derris scandens; Anti-inflammatory; Eicosanoid inhibition; Antioxidant

1. Introduction Inflammation is a natural response of the mammalian body to a variety of hostile agents including parasites, pathogenic micro-organisms, toxic chemical substances and physical damage to tissue. The processes associated with the inflammatory response are complex but important aspects which have been exploited for screening for anti-inflammatory compounds are the various functions of neutrophils, the metabolic products of arachidonic acid and the role played by reactive oxygen species (ROS). Neutrophils are the first cells which are recruited to sites of tissue injury to deal with the causes of inflammation. They destroy invading pathogens and compounds by phagocytosis or opsonisation and these processes involve ∗ Corresponding author. Tel.: +44-20-7848-4775; fax: +44-20-7848-4775. E-mail address: [email protected] (P.J. Houghton). 1 Present address: Pharmacology Department, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 2 Deceased 24th April 2001.

the production of ROS and release of tissue-damaging enzymes such as proteases and myeloperoxidase (MPO). Arachidonic acid (AA) is released from cell membranes by phospholipase A2 (PLA2 ) under the stimulus of several factors associated with inflammation (Scott et al., 1999). The products of metabolism of AA are collectively known as eicosanoids and the two most important groups are the prostaglandins and leukotrienes, formed by the actions of cyclo-oxygenases (COX) and lipoxygenases (LOX), respectively. The prostaglandins amplify the pain mechanism and enhance vascular permeability whilst the leukotrienes contract the smooth muscles of blood vessels, enhance vascular permeability and mediate pro-inflammatory and allergic responses (Bisgaard, 2000; Bley et al., 1998). In recent years, two COX enzymes, COX-1 and COX-2, have been discovered and it is agreed, subject to some debate, that inhibition of COX-2 is more selective for anti-inflammatory effects (Smith et al., 1998; Hawkey, 1999; Bauer, 1999). ROS in excess cause tissue damage by peroxidation and subsequent degradation of several cell components such as membrane lipids, proteins and DNA (Hogg, 1998; Halliwell and Gutteridge, 1998). Tissue damage and adverse effects due to excess inflammation may therefore be reduced by the


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use of suitable anti-oxidants which prevent the formation of oxygen free radicals, or scavenge them once they are formed and before they react with sites such as unsaturated lipids in the cell membranes (Cuzzocrea et al., 2001). A common disease state where excess inflammation presents a problem is arthritis, and other joint pains, so examination of plants used to treat such conditions may reveal anti-inflammatory compounds. Traditional practitioners are still active in many parts of Thailand and represent a large reservoir of relatively untapped information of interest to ethnopharmacologists. In this study, four species were selected from the answers given by Thai traditional healers to a questionnaire about plants used to treat pain in the bones and joints. Extracts of these species were investigated using a battery of tests since this approach is more likely to check the veracity of any reputed activity, determine its mechanism of action and, by bioassay-guided fractionation, determine its chemical basis, than one single bioassay (Houghton, 2000). A variety of methods have been employed to test for anti-inflammatory activity. In vivo tests using animal models give the most meaningful results when testing plant extracts for anti-inflammatory activity but they are unsuitable for bioassay-guided fractionation to determine the active compounds present and, in many countries, any use of animals is minimised for economic and ethical reasons. Several models have been developed and used but probably the one most often reported is the rat hind paw oedema model developed by Winter et al. (1962) and is the one employed here. In vitro tests are becoming increasingly popular in research aimed at elucidating the compounds in an extract responsible for an observed biological activity. Many disease states, and particularly inflammation, are related to deficiencies or disruption of more than one biochemical process, as discussed above, and so it is preferable to use a battery of in vitro methods which relate to different aspects of the disease. A test which measures the ability of test substances to alter neutrophil secretory activity utilises rat peritoneal leukocytes challenged with the calcium ionophore A23187 (Paya et al., 1994; De las Heras and Hoult, 1994). The amount of elastase myeloperoxidase can be measured and additional measurement of lactase dehydrogenase serves to indicate cellular integrity.

Tests for inhibition of the enzymes involved in the eicosanoid pathway determine inhibition of LOX and COX by radioimmunoassay of their products. A method, using mammalian enzymes contained within peritoneal leukocytes, has been used extensively to evaluate the potential anti-inflammatory activity of a wide range of plant extracts and derived natural products. In this technique, the leukocytes are stimulated to produce eicosanoids by the use of A23187 and the levels of the prostaglandins thromboxane B2 (TXB2 ), prostaglandin E2 (PGE2 ), and the leukotriene B4 (LTB4 ) measured by radioimmunoassay as an indication of COX and 5-LOX activity, respectively (Moroney et al., 1988; Hoult et al., 1995). A number of tests for activity having a net anti-oxidant effect have been introduced in recent years but work reported here has used two systems. In the hypoxanthine/xanthine oxidase system, the effect of superoxide radicals on ferric cytochrome c is measured spectrophotometrically (Paya et al., 1992). The scavenging ability of compounds or extracts for peroxide is measured in another spectrophotometric system using guaiacol, which gives a coloured complex with peroxide (Paya et al., 1992).

2. Methodology 2.1. Survey of traditional healers The Personnel Development Sections of all Provincial Public Health Offices in Thailand were contacted to each select five experienced and knowledgeable folk doctors from each province and 216 healers from 65 different provinces of Thailand. These healers were interviewed for their use of plants for treating bone and joint pain using a standard form (Fig. 1). Each healer had to conform to the criterion of at least 10 years’ experience in using medicinal plants. The purpose of the study was explained to them and they were than allowed to complete the questionnaires on their own. Data analysis of the reports obtained from all 216 healers selected each species separately from each recipe and these were then arranged in order of frequency of use. From the 10 species used most frequently (Table 1), the 4 itemised

Table 1 The 10 most commonly used plants in recipes obtained from Thai traditional healers for treating conditions associated with inflammation Scientific name of plant

Family

Part used

Preparation

C. buchanani Roem. D. scandens Benth. Piper nigrum L. Cleome viscosa L. A. ebracteatus L. Zingiber officinale Rose. Tamarindus indica L. O. indicum Vent. Citrus hystris DC. Morinda citrifolia L.

Periplocaceae Papilionaceae Piperaceae Cleomaceae Acanthaceae Zingiberaceae Caesalpiniaceae Bignoniaceae Rutaceae Rubiaceae

Stem Stem Stem Whole plant Whole plant Root Whole plant Stem bark Leaves Leaves

Macerate in alcohol Boil in water Boil in water Boil in water Boil in water Boil in water Boil in water Boil in water Boil in water Boil in water


below were selected which had a reputation for similar activity in other areas or as a result of other surveys. 2.2. Plant material and preparation of extracts All four species were collected in Songkla province in southern Thailand and identified by Dr. Arunporn Itharat.

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Voucher specimens of Acanthus ebracteatus (SKP A 0010101), Oroxylum indicum (SKP A 0250101), Cryptolepis buchanani (SKP A 0180101) and Derris scandens (SKP A 1410101) are deposited in the Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Science, Prince of Songkla University, Hat Yai, Thailand.

Fig. 1. Form used in survey of traditional healers.

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Fig. 1. (Continued )


Fig. 1. (Continued )

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The materials were dried in the shade. Each species was extracted by two methods (except for C. buchanani which was extracted only with alcohol since this is used traditionally for preparing the medicine). For the first method, the dried material (100 g) was macerated in boiling water for 30 min and the mixture was then filtered. After cooling, the filtrate was freeze-dried. For the second method the dried material (100 g) was percolated with ethanol 96% for 5 days. The filtrate was evaporated to dryness under reduced pressure. The weights obtained and percentage yield are given in the Section 2.7. For testing, the extracts were dissolved in water or 10% methanol with the aid of sonication. 2.3. In vitro test for release of myeloperoxidase (MPO) from rat peritoneal leukocytes A suspension of leukocytes containing approximately 85% polymorphonuclear leukocytes (PMNs) and 15% mononuclear cells was elicited from male Wistar rats (200–300 g) by an i.p. injection of 10 ml of a solution of 6% oyster glycogen in saline (Hoult et al., 1994), followed 16–20 h later by 60 ml iced-cold modified Hank’s balanced salt solution free of Ca2+ and Mg2+ . After about 90 s of vigorous massage, the peritoneal washing was removed, centrifuged at 400 × g for 10 min at 4 ◦ C and the contaminating erythrocytes in the pellet lysed after resuspension in a small volume of 0.2% saline for 30 s. The isotonicity of the pellet suspension was then re-established by adding excess HBSS. After further centrifugation and washing, the mixed peritoneal leukocytes were resuspended in complete HBSS at 2.5 × 106 cells/ml containing 1.26 mM Ca2+ and 0.9 mM Mg2+ . Cell viability based on Trypan blue exclusion was greater than 95%. 0.5 ml of the cell suspension was then treated with the extract or solvent alone for 10 min at 37 ◦ C then stimulated for another 10 min by addition of A23187 calcium ionophore (1 ␮M). The cells were then pelleted by centrifugation at 4 ◦ C for 10 min at 400 × g and the supernatants then taken and stored at −20 ◦ C until ready for use. The determination was carried out in 96-well microplates, each determination being carried out in triplicate. To each well was added 50 ␮l of supernatant from the peritoneal leukocytes, 50 ␮l aq. O-dianisidine (0.67 mg/ml) and 50 ␮l 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer pH 6.0. 50 ␮l 0.003% hydrogen peroxide solution was added to commence the reaction and the absorbance at 450 nm was read using a Dynatech Plate Reader at 1 min and at timed intervals thereafter at room temperature with intermittent shaking. The value for total MPO release was obtained by using cells disrupted by two cycles of freeze-thawing.

dimethylsulphoxide (DMSO) or chloroform to the 3 ml polypropylene incubation tubes. In the case of solvents added in chloroform, the solvent was allowed to evaporate before adding the cells. After this drug preincubation, 1 ␮l of calcium ionophore A23187 was added in DMSO to give a final concentration of 1 ␮M for further 10 min of incubation. The cells were pelleted by centrifugation at 2500 × g for 10 min at 4 ◦ C, and the supernatants were decanted and frozen. Aliquots (5–15 ␮l) of the thawed samples were subjected to radioimmunoassay for TXB2 and LTB4 by making up to 100 ␮l with 50 ␮M phosphate buffer (pH 7.5) containing 0.1% human ␥-globulin and 0.9% saline, adding 200 ␮l polyclonal rabbit anti-eicosanoid serum (diluted 1:1500), 100 ␮l tracer containing 10 nCi 3 H8 -TXB2 or 4 nCi 3 H8 -LTB4 (NEN or Amersham), respectively, mixing and incubating at 4 ◦ C for 18 h. After this, bound label was separated from free using 200 ␮l dextran-coated charcoal and the bound dpm counted in a Packard model 1900TR liquid scintillation analyser. Indomethacin 5 ␮M was used as positive control for COX inhibition and the methoxyalkylthiazole ZM-211965 5 ␮M for 5-lipoxygenase inhibition (Bird et al., 1991). 2.5. In vitro anti-oxidant effects of extracts The effect of extracts on the hypoxanthine/xanthine oxidase system was carried out in micro wells using ferricytochrome c as a reducing agent. To each well was added 50 ␮l of extract or solvent, 50 ␮l ferricytochrome c 100 ␮M in Hank’s buffered saline solution (HBSS) and 50 ␮l xanthine oxidase 25 mU/ml in HBSS. 50 ␮l hypoxanthine 100 ␮M was added and the absorbance at 550 nm was read using a Dynatech Plate Reader at 1 min and at timed intervals thereafter at room temperature with intermittent shaking. 2.6. In vivo test for effect on induced edema in rats Male albino Sprague–Dawley rats (200 ± 50 g), captivereared in the Faculty of Medicine, Khon Kaen University and fed on standard laboratory food and water ad libitum, were used. Groups of five rats were given either intraperitoneally or orally a dose of 100 mg/kg or 500 mg/kg of the aqueous extract of D. scandens in a volume equivalent to 0.1 ml/100 g body weight. Control group rats received either normal saline or aspirin 300 mg/kg orally as a positive control. 30 min after dosing, the rats were injected subplantar with 0.1 ml 1% carrageenin in saline. The volume of edema was measured by water displacement using a plethysmometer (Model 7150, Ugo Basle). Measurements were taken at 1, 2 and 3 h after administration of the carrageenin.

2.4. In vitro test for eicosanoid synthesis inhibition

2.7. Results and discussion

Triplicate aliquots of 0.5 ml leukocytes obtained as described above were preincubated at 37 ◦ C for 10 min with the test extracts. They were added prior to the cells in 5 ␮l

2.7.1. Yield of extracts from plant materials A. ebracteatus water 6.88% (w/w), ethanol 5.25% (w/w), O. indicum water 7.03% (w/w), ethanol 6.14% (w/w), C.


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2.9. In vitro test for eicosanoid synthesis inhibition

Fig. 2. Release of myeloperoxidase (MPO) from rat peritoneal leukocytes by extracts.

buchanani water 5.37% (w/w), ethanol 4.95% (w/w) and D. scandens water 6.93% (w/w), ethanol 5.99% (w/w). 2.8. In vitro test for release of myeloperoxidase (MPO) from rat peritoneal leukocytes It was observed that the absorbances measured reached a plateau after 20 min and so the values at that time were used to indicate the amount of MPO released. Results are shown in Fig. 2. It can be seen that the addition of the calcium ionophore considerably increased the release of MPO from the cells. None of the ethanolic extracts gave a significant reduction in MPO release but the aqueous extracts of both O. indicum and D. scandens had a significant reducing effect (64 and 88%, respectively) and this may indicate that a reduction in inflammation in vivo may occur if an aqueous extract is applied topically or taken internally.

Results are shown in Figs. 3 and 4 for TXB2 and LTB4 generation, respectively. The positive controls used gave the expected results. None of the ethanolic extracts affected COX activity, i.e. TXB2 production, and only the highest dose of D. scandens aqueous extract gave a statistically significant reduction (95%). However, a more obvious inhibitory effect on 5-lipoxygenase activity, indicated by a significant reduction in LTB4 production, was shown by both the A. ebracteatus (64% for 500 ␮g/ml ethanol extract, 44% for 500 ␮g/ml water extract) and D. scandens extracts (29% for 500 ␮g/ml ethanol extract, 89% for 500 ␮g/ml water extract) with a weaker activity shown by C. buchanani (31% for 500 ␮g/ml ethanol extract). The D. scandens extract exhibited the greater activity and at 500 ␮g/ml gave an effect (89% reduction) equivalent to 5 ␮M ZM-211965, the positive control. Leukotrienes play a more important part in the pain and inflammation associated with arthritis so the inhibition of lipoxygenase observed with these extracts may be translated in vivo to a reduction in the symptoms of this condition. 2.10. In vitro anti-oxidant effects of extracts Results are shown in Fig. 5. Only the highest dose of the D. scandens extract showed a significant antioxidant effect (23% reduction) which implies that any anti-inflammatory activity observed is not likely to be due to antioxidant effects. 2.11. In vivo test for effect on induced edema in rats Results are shown in Fig. 6. A significant dose-related reduction in edema (82% for 100 mg/kg; 91% for 500 mg/kg) was shown when the aqueous extract of D. scandens was

Fig. 3. Thromboxane release from peritoneal lymphocytes by extracts (Ac: Acanthus bracteatus 10, 100, 500 ␮g/ml; Or: Oroxylum indicum 10, 100, 500 ␮g/ml; Cr: Cryptolepis buchanani 10, 100, 500 ␮g/ml; De: Derris scandens 10, 100, 500 ␮g/ml).

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Fig. 4. Leukotriene release from peritoneal lymphocytes by extracts (Ac: Acanthus bracteatus 10, 100, 500 ␮g/ml; Or: Oroxylum indicum 10, 100, 500 ␮g/ml; Cr: Cryptolepis buchanani 10, 100, 500 ␮g/ml; De: Derris scandens 10, 100, 500 ␮g/ml).

given intraperitoneally but not when it was given by mouth. Indeed, the higher dose given by mouth yielded a lower reduction in swelling compared with the smaller dose but the difference was not statistically significant. This implies that the active compounds were not absorbed from the gastro-intestinal tract or that they were metabolised rapidly to inactive compounds before they could reach the area of inflammation. These results suggest that the traditional method of taking D. scandens extract orally to treat arthritic conditions is not likely to be effective although differences in absorption and metabolism between rats and humans may mean that the extract could be effective in humans. Clinical studies would be needed to ascertain if any such oral efficacy exists. It is also important to realise that the

Fig. 6. Effect of D. scandens extracts 100 and 500 ␮g/ml on rat paw edema.

extracts of the other three species were not tested in vivo because they showed insufficient activity in vitro. However, the conclusion should not be drawn that they are unlikely to display in vivo activity because they may do so by other anti-inflammatory mechanisms or compounds present may be metabolised in vivo to substances affecting eicosanoid synthesis.

3. Conclusion

Fig. 5. Antioxidant activity of extracts 500 ␮g/ml.

Of the four species examined, D. scandens shows the greatest evidence of activity in vitro, which, if it is exerted in vivo, could account for its use in treating arthritis. The in vivo experiments carried out showed that, in fact, the extract had an antiinflammatory effect, but not when it was given orally, so the traditional means of taking this extract appears


not to be supported. To our knowledge, no previous work has been carried out on this aspect of the pharmacology of this species and work is in progress to determine the compounds responsible, especially for the lipoxygenase inhibition. A. ebracteatus aqueous extract displayed a weaker inhibition of lipoxygenase so any derived clinical effect is less likely. No previous pharmacological studies have been reported from this plant. O. indicum displayed no relevant activity except for inhibition of release of MPO by the aqueous extract. A previous study has shown that a lipophilic extract of O. indicum stembark gave 100% inhibition of leukocyte lipoxygenase at a concentration of 50 ␮g/ml which was mainly due to lapachol (Ali et al., 1998). It is unlikely that significant amounts of this compound are present in the aqueous extract showing activity in this study. The aqueous extract of C. buchanani gave a weak inhibition of lipoxgenase but this was not dose-dependent and so any relevance of this activity to the traditional use in arthritis is questionable.

Acknowledgements The Faculty of Medicine, Khon Kaen University, Thailand, is thanked for a Ph.D. scholarship for Dr. Laupattarakasem and the Department of Pharmacognosy and Pharmaceutical Botany, Prince of Songkla University, Hat Yai, Thailand for supply of plant materials.

References Ali, R.M., Houghton, P.J., Hoult, J.R.S., 1998. Antimicrobial and antiinflammatory activities of extracts and constituents of Oroxylum indicum (L.). Ventional Phytomedicine 5, 375–381. Bauer, R., 1999. Cycloxygenase and 5-lipoxygenase as targets for medicinal plant research. In: L. Bohlin, J.G. Bruhn (Eds.), Bioassay Methods in Natural Products Research and Drug Development. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 119− 142. Bird, T., Bruneau, P., Crawley, G., Edwards, M., Foster, S., Girodeau, J.-M., Kingston, J., McMillan, R., 1991. Methoxyalkylthiazoles: a new series of potent, selective and orally active 5-lipoxygenase inhibitors

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displaying high enantioselectivity. Journal of Medicinal Chemistry 34, 2176–2186. Bisgaard, H., 2000. Role of leukotrienes in asthma pathophysiology. Pediatric Pulmonology 30, 166–176. Bley, K.R., Hunter, J.C., Eglen, R.M., Smith, J.A.M., 1998. The role of IP prostanoid receptors in inflammatory pain. Trends in Pharmacological Sciences 19, 141–147. Cuzzocrea, S., Riley, D.P., Caputi, A.P., Salvemini, D., 2001. Antioxidant therapy: a new pharmacological approach in shock, inflammation and ischemia/reperfusion injury. Pharmacology Reviews 53, 135–159. De las Heras, B., Hoult, J.R.S., 1994. Non-cytotoxic inhibition of macrophage eicosanoid biosynthesis and effects on leukocyte functions and reactive oxygen species of two novel anti-inflammatory plant diterpenoids. Planta Medica 60, 501–506. Halliwell, B., Gutteridge, J.M.C., 1998. Free Radicals in Biology and Medicine, 3rd ed. Oxford University Press, Oxford. Hawkey, C.J., 1999. COX-2 inhibitors. Lancet 353, 307–331. Hogg, N., 1998. Free radicals in disease. Seminars in reproductive. Endocrinology 16, 241–248. Houghton, P.J., 2000. Use of small scale bioassays in the discovery of novel drugs from natural sources. Phytotherapy Research 14, 419–423. Hoult, J.R.S., Moroney, M.A., Paya, M., 1994. Actions of flavonoids and coumarins on lipoxygenase and cycloxygenase. Methods in Enzymology 234, 443–454. Hoult, J.R.S., Pang, L.-H., Bland-Ward, P., Forder, R., Williams, C., Harborne, J.B., 1995. Inhibition of leukocyte 5-lipoxygenase and cyclooxygenase but not constitutive nitric oxide synthase by tanetin, a novel flavonoid derived from feverfew Tanacetum parthenium. Pharmaceutical Sciences 1, 71–74. Moroney, M.A., Alcaraz, M.J., Forder, R.A., Carey, F., Hoult, J.R.S., 1988. Selectivity of neutrophil 5-lipoxygenase and cyclooxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids. Journal of Pharmacy and Pharmacology 40, 787– 792. Paya, M., Halliwell, B., Hoult, J.R.S., 1992. Interactions of a series of coumarins with reactive oxygen species. Biochemical Pharmacology 44, 205–214. Paya, M., Goodwin, P.A., de las Heras, B., Hoult, J.R.S., 1994. Superoxide scavenging activity in leukocytes and absence of cellular toxicity of a series of coumarins. Biochemical Pharmacology 48, 445–451. Scott, K.F., Bryant, K.J., Bidgood, M.J., 1999. Functional coupling and differential regulation of the phospholipase A2-cycloxygenase pathways in inflammation. Journal of Leukocyte Biology 66, 535–541. Smith, C.J., Zhang, Y., Koboldt, C.M., Muhammad, J., Zweifel, B.S., Shaffer, A., Talley, J.J., Masferrer, J.L., Seibert, K., Isakson, P.C., 1998. Pharmacological analysis of cyclooxygenase-1 in inflammation. Proceedings of the National Academy of Sciences of the USA 95, 13313–13318. Winter, C., Risley, E., Nuss, G., 1962. Carrageenin-induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs. Proceedings of the Society for Experimental Biology and Medicine 111, 544–547.


Evaluation of the anti-inflammatory properties of Sclerocarya birrea (A. Rich.) Hochst. (family: Anacardiaceae) stem-bark extracts in rats John A.O. Ojewole∗ Department of Pharmacology, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa Received 21 March 2002; received in revised form 29 July 2002; accepted 16 December 2002

Abstract This study was designed to evaluate the anti-inflammatory effect of Sclerocarya birrea (family: Anacardiaceae) stem-bark aqueous and methanolic extracts in rats. Young adult, male Wistar rats (Rattus norvegicus) weighing 250–300 g were used. The anti-inflammatory effects of aqueous and methanolic stem-bark extracts of the plant (SB, 500 mg/kg p.o.) were examined on rat paw oedema induced by subplantar injections of fresh egg albumin (0.5 ml/kg). Acetylsalicylic acid (ASA, 100 mg/kg p.o.) was used as the reference anti-inflammatory agent for comparison. Both the aqueous and methanolic stem-bark extracts of S. birrea (SB, 500 mg/kg p.o.) progressively and time-dependently reduced rat paw oedema induced by subplantar injections of fresh egg albumin. However, the methanolic extract of the plant produced relatively greater and more pronounced anti-inflammatory effect than its aqueous extract counterpart in the experimental animal model used. The two extracts of S. birrea stem-bark were found to be markedly less potent than ASA as anti-inflammatory agent. Although both the aqueous and methanolic extracts of S. birrea stem-bark are less potent than ASA as anti-inflammatory agent, the results of this experimental animal study indicate that the extracts possess anti-inflammatory activity, and thus lend credence to the suggested folkloric use of the plant in the management and/or control of arthritis and other inflammatory conditions in certain communities of South Africa. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sclerocarya birrea stem-bark; Aqueous and methanolic extracts; Acetylsalicylic acid; Anti-inflammatory activity

1. Introduction South Africa is endowed with a rich floral biodiversity which is reflected in the existence of over 30,000 species of higher plants in the country. Many of the plant species are used for ornamental, nutritional, symbolic, ritual, spiritual, magical, and medicinal purposes. Approximately 3000 higher plant species are commonly used as medicines, and these plants constitute the floristic wealth of South Africa. Of these 3000 plants, however, only about 400 species constitute the most frequently-used and traded medicinal plants of the country. The focus of this study is on one of such medicinal plants, namely; Sclerocarya birrea (A. Rich.) Hochst., subspecies caffra (Sond.) Kokwaro (family: Anacardiaceae). S. birrea (popularly known as: ‘cider tree’ or ‘marula’ (in English), ‘maroela’ (in Afrikaans), or ‘umganu’ (in Zulu)) is a medium-sized, single-stemmed tree of up to 15 m in height. The rough stem-bark is flaky, with a mottled appearance due to con-

∗ Tel.:

+27-31-204-4356; fax: +27-31-204-4907. E-mail address: [email protected] (J.A.O. Ojewole).

trasting grey and pale-brown patches. The leaves are divided into 10 or more pairs of leaflets, each about 60 mm long, dark-green above, much paler below, with the tip abruptly narrowing to a sharp point. The flowers are borne in small, oblong clusters. Male and female flowers occur separately, usually but not always on separate trees. The flowers are small, with red sepals and yellow petals. Large, rounded, slightly flattened fruits of about 30 mm in diameter are borne in profusion in late summer to mid-winter. The fruits are much sought after for their delicious pulp, high vitamin C content and edible nuts. It has become a commercial fruit crop in recent years, the fruit pulp being used to produce a jelly and to flavour liqueur (Van Wyk et al., 1997). The stem-bark, roots and leaves are the usual medicinal products of the plant. These morphological parts of the plant have been reported to possess medicinal and other uses in addition to the nutritional values of the fruits and seeds of the plant. In South Africa and in some other African countries, the stem-bark, roots and leaves of S. birrea are used for an array of human ailments, including: malaria and fevers, diarrhoea and dysentery, stomach ailments, headaches, toothache, backache and body pains, infertility, schistosomiasis, epilepsy, diabetes mellitus, etc. (Watt and


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Breyer-Brandwijk, 1962; Van Wyk et al., 1997; Hutchings et al., 1996; Pujol, 1993). This study was prompted by the claim of some Zulu traditional healers (in South Africa) that aqueous and methanolic extracts of S. birrea stem-bark are useful in the management and/or control of arthritis and other inflammatory conditions in man. As far as we are aware, however, there is no published data on the anti-inflammatory effect of S. birrea (family: Anacardiaceae) in the medical literature. This study was, therefore, undertaken to examine the anti-inflammatory properties of S. birrea stem-bark extracts in rats, a typical mammalian experimental model, with a view to providing a pharmacological justification (or otherwise) for the folkloric use of the plant in arthritis and other inflammatory conditions in some communities of South Africa.

2. Materials and methods The experimental protocol used in this study was approved by the Ethics Committee of the University of DurbanWestville, Durban 4000, South Africa; and conforms with the “Guide to the Care and Use of Animals in Research and Teaching” (published by the University of Durban-Westville). 2.1. Plant material Pieces of fresh stem-barks of S. birrea were collected from the University of Durban-Westville’s campus, between January 2000, and June, 2001. They were identified by Prof. H. Baijnath, the Chief Taxonomist/Curator of the University of Durban-Westville’s Department of Botany, as those of S. birrea (A. Rich.) Hochst. subspecies caffra (Sond.) Kokwaro (family: Anacardiaceae). 2.2. Preparation of extracts

of both sexes weighing 250–300 g, were used. The animals were kept and maintained under laboratory conditions of temperature, humidity, and light; and were allowed free access to food (standard pellet diet) and water ad libitum. The animals were divided into drug-treated ‘test’ and distilled water-treated ‘control’ groups of eight animals per group. All the animals were fasted for 12 h, but still allowed free access to water, before the commencement of our experiments. The mice were used for the ‘acute toxicity’ testing of the plant extracts, while the rats were used for the anti-inflammatory evaluation of the S. birrea stem-bark extracts. 2.4. Acute toxicity testing The median lethal doses (LD50 ) of the plant extracts were determined as described in detail by Lorke (1983). Mice fasted for 12 h were randomly divided into groups of eight per group. Aqueous or methanolic extracts of S. birrea (SB) 125, 250, 500, 1000 and 1500 mg/kg were separately administered orally to the mice in each of the ‘test’ groups. Each of the mice in the ‘control’ group was treated with distilled water (2 ml/kg p.o.). The mice in both the ‘test’ and ‘control’ groups were then allowed free access to food and water, and observed over a period of 24 h for signs of acute toxicity. The number of deaths (caused by the extracts) within this period of time was recorded. Log-dose response plots were constructed for each extract, from which the median lethal doses (LD50 ) of the extracts were obtained. 2.5. Anti-inflammatory examination This was done by using fresh egg albumin-induced rat hind paw oedema (Muko and Ohiri, 2000) as a model of acute inflammation. Young, adult male Wistar rats were used. Acute inflammation of the hind paw was induced in each of the rats by injecting 0.5 ml/kg of fresh egg albumin into the subplantar surface of the right hind paw. Linear

One kilogram (1 kg) pieces of air-dried stem-bark of S. birrea were cut into small pieces, homogenised and Soxhlet extracted twice, on each occasion with 2.5 l of either 99.5% uniAr methanol or de-ionised water, at room temperature for 24 h with shaking. The combined methanol or de-ionised water extracts were filtered and concentrated to dryness under reduced pressure at 30 ± 1 ◦ C. Freeze–drying and solvent elimination under reduced pressure finally yielded crude, dark-brown, powdery methanolic (5.67%) and aqueous (4.85%) S. birrea stem-bark extracts, respectively. Aliquot portions of these extract residues were weighed and dissolved in distilled water for use on each day of our experiment. 2.3. Animal material Balb C male and female mice (Mus domesticus) weighing 20–25 g, and young adult, Wistar rats (Rattus norvegicus)

Fig. 1. Effects of ( ) 2 ml/kg p.o. distilled water (control); ( ) 500 mg/kg p.o. methanolic extract of S. birrea; (䊐) 500 mg/kg p.o. aqueous extract of S. birrea; and ( ) 100 mg/kg p.o. of ASA on 0.5 ml/kg fresh egg albumin-induced rat paw oedema. Each histogram represents the mean of eight observations, while the vertical bars denote standard errors of the means (S.E.M.).


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Table 1 Known chemical constituents and ethnomedical uses of S. birrea (A. Rich.) Hochst. (family: Anacardiaceae) Chemical constituents

Ethnomedical uses

Several chemical compounds have been isolated from the various morphological parts of S. birrea (family: Anacardiaceae) (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; Van Wyk et al., 1997). The compounds include: gallotannins; tannic, mallic, gallic and citric acids; proanthocyanins and procyanidins; catechins; flavonoids, terpenoids, sesquiterpenoids, coumarins and phytosterols. The oil-rich seeds contain oleic acid, myristic, stearic and amino acids, with a predominance of glutamic acid and arginine. Ross (2001) has reported the occurrence of the following chemical compounds in Anacardium occidentale (Linn.), another plant in the family ‘Anacardiaceae’: acetophenone, ␣-amyrin, anacardic acid, anacardol, apigenin, arachidic acid, ascorbic acid, benzaldehyde, benzoic acid, campesterol, capric acid, cardanol, cardol, catechin, cholesterol, cycloartenol, cyclohexane, digallic acid, ethyl acetate, fatty acids, furfural, gallic acid, kaempferol, lauric acid, leucocyanidin, limonene, linoleic acid, mallic acid, myricetin, myristic acid, naringenin, occidentoside, oleic acid, palmitic acid, phenolic compounds, protocatechuic acid, quercetin, resorcinol derivatives, salicylic acid and its derivatives, salipurposide, ␣-selinene, ␤-sitosterol, squalene, stearic acid, stigmasterol, tannin, ␣-terpinene, tocopherol, toluene and xylene

Various morphological parts of S. birrea, especially stem-barks, roots and leaves of the plant, have been reported (Watt and Breyer-Brandwijk, 1962; Pujol, 1993; Hutchings et al., 1996; Van Wyk et al., 1997) to be useful as effective remedies for diarrhoea and dysentery, constipation, abdominal cramps and some other unspecified gastro-intestinal problems; sores, boils, carbuncles, abscesses and certain other bacterial infections; malaria, fevers and pyrexia; sore eyes; heart pain; snakebites; diabetes mellitus; schistosomiasis; headaches and body pains; toothaches and swollen or infected gums; convulsions; impotence and sterility; and blood tonic

circumference of the injected paw was assessed for 3 h at 30 min intervals after the administration of the phlogistic agent. Increase in the linear circumference was taken as a measure of paw oedema. The increases in right hind paw circumferences induced by injections of fresh egg albumin were compared with those of the non-injected left hind paw circumferences (Muko and Ohiri, 2000; Oriowo, 1982; Hess and Milong, 1972). S. birrea stem-bark aqueous and methanolic extracts were separately administered orally, at a dose of 500 mg/kg (a dose that was found to produce 50–75% of the maximal anti-inflammatory effects in our pilot experiments with the extracts) to each of the rats in two of the ‘test’ groups, 1 h before inducing inflammation with the injection of fresh egg albumin. Rats in the reference, comparative ‘test’ group received acetylsalicylic acid (ASA, 100 mg/kg p.o.); while rats in the ‘control’ group received distilled water (2 ml/kg p.o.) only.

increase in the rat paw circumference. Maximal swelling and/or oedema was observed approximately 90 min post egg albumin administration. Both the aqueous and methanolic extracts of S. birrea stem-bark produced relatively moderate anti-inflammatory effects when compared with ASA. However, the methanolic extract of the plant’s stem-bark produced a relatively stronger and/or greater anti-inflammatory activity than the aqueous extract of the stem-bark (see Fig. 1). S. birrea has been reported to contain many chemical compounds, including: tannins and flavonoids, sterols, triterpenoids, sesquiterpenes, ascorbic acid, oleic acid, myristic, stearic, glutamic and amino acids, etc. (see Table 1). However, the exact chemical constituent of the plant that is responsible for the observed anti-inflammatory activity of the extracts remains speculative. Further studies are certainly warranted to purify, isolate, characterise and identify the anti-inflammatory compound(s) of the plant extracts.

2.6. Data analysis Results are expressed as means (±S.E.M.). In each case, Student’s t-test (Snedecor and Cochrane, 1967) was used to determine the level of significance of the difference between the ‘test’ and ‘control’ group data. Values of P ≤ 0.05 were taken to imply statistical significance.

3. Results Intraperitoneal administrations of graded doses of S. birrea stem-bark aqueous and methanolic extracts in mice gave LD50 values of 1215 ± 38 mg/kg and 1087 ± 41 mg/kg, respectively. These acute toxicity test results probably suggest that the plant is relative ‘safe’ in mammals. Subplantar injection of fresh egg albumin (0.5 ml/kg) produced marked, sustained, time-related and progressive

4. Discussion and conclusion The high LD50 values (of 1215 ± 38 mg/kg and 1087±41 mg/kg, respectively) obtained for the aqueous and methanolic extracts of S. birrea stem-bark probably suggest that the plant extracts are relatively safe and/or non-toxic to mice. Several investigators have isolated various secondary metabolites and bio-active chemical compounds from different morphological parts of S. birrea (see Table 1). It is not unlikely, however, that some of the chemical constituents of the plant extracts will be toxic to mammals. Nevertheless, the results of the present experimental animal study indicate that both the aqueous and methanolic extracts of S. birrea stem-bark possess anti-inflammatory activity in the mammalian animal model used. Treatment of the animals in the ‘control’ group with distilled water

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(2 ml/kg p.o.) alone did not produce any significant change in the fresh egg albumin-induced increases in the rat paw (oedema) circumferences (data not shown). S. birrea has been widely reported to contain many chemical compounds, including: coumarins, terpenoids, flavonoids, tannins, ␤-sitosterol, oils, organic acids and inorganic substances (Table 1). At present, the exact chemical constituent(s) of the extracts that is/are specifically responsible for the anti-inflammatory activity of S. birrea stem-bark aqueous and methanolic extracts remains speculative. Furthermore, it is difficult, at this stage, to draw any logical conclusion on the mechanism of anti-inflammatory action of such a diverse mixture of chemical compounds contained in the S. birrea stem-bark extracts examined in this study. The next step in our experimental studies will be to probe each of the chemical constituents of S. birrea stem-bark aqueous and methanolic extracts for anti-inflammatory activity, and thereafter establish the mechanisms of their anti-inflammatory action. All told, the experimental evidence obtained in this laboratory animal study indicates that S. birrea stem-bark aqueous and methanol extracts possess anti-inflammatory activity, and thus lends credence to the folkloric use of the plant in the management and/or treatment of arthritis and other inflammatory conditions in certain communities of South Africa.

Acknowledgements I am grateful to Prof. H. Baijnath for the identification of Sclerocarya birrea stem-bark used in this study. I also

like to thank Prof. C.O. Adewunmi for his valuable and constructive comments; Dr. E.K. Mutenda for her assistance in the extraction processes; Mr. S.O. Adewale for his able technical assistance; Mrs. P. Koloko for her interest in the study; and the Council of the University of Durban-Westville for the provision of a Research Grant [R642] to carry out a part of this study.

References Hess, S.M., Milong, R.C., 1972. In: Pow, L.E., Ward, P.A. (Eds.), Inflammation Mechanism and Control. Academic Press, New York, pp. 1–21. Hutchings, A., Scott, A., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants—An Inventory. Natal University Press, Pietermaritzburg, South Africa, p. 177. Lorke, D., 1983. A new approach to practical acute toxicity testing. Archives of Toxicology 54, 275–287. Muko, K.N., Ohiri, F.C., 2000. A preliminary study on the anti-inflammatory properties of Emilia sonchifolia leaf extracts. Fitoterapia 71, 65–68. Oriowo, M.A., 1982. Anti-inflammatory activity of piperonyl-4-acrylic isobutyl amide, an extractive from Zanthoxylum zanthoxyloides. Planta Medica 44, 54–56. Pujol, J., 1993. Naturafrica–The Herbalist Handbook. Jean Pujol Natural Healers’ Foundation, Durban, South Africa. Ross, I.A., 2001. Medicinal Plants of the World—Chemical Constituents, Traditional and Modern Uses. Humana Press, Totowa, NJ, pp. 43–53. Snedecor, G.W., Cochrane, W.G., 1967. Statistical Methods, 6th ed. The Iowa State University Press, Ames, Iowa, USA. Van Wyk, B.E., Van Oudtshoorn, B., Gericke, N., 1997. Medicinal Plants of South Africa, 1st ed. Briza Publications, Pretoria, South Africa, p. 234. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd ed. Livingstone, London.


The analgesic, antipyretic and anti-inflammatory activity of Diospyros variegata Kruz. S. Trongsakul, A. Panthong∗ , D. Kanjanapothi, T. Taesotikul Department of Pharmacology, Faculty of Medicine, Chiang Mai University, 50200 Chiang Mai, Thailand Received 30 July 2002; received in revised form 11 December 2002; accepted 16 December 2002

Abstract Pharmacological studies were conducted with the hexane extract of the dry stem of Diospyros variegata Kruz. (Ebenaceae) on experimental animals for evaluating the analgesic, antipyretic and anti-inflammatory activities. In the analgesic test, the hexane extract elicited inhibitory intensity on acetic acid-induced writhing response and on the late phase of formalin test but possessed only a weak effect on the tail-flick response and on the early phase of formalin test. The hexane extract also elicited antipyretic action when tested in yeast-induced hyperthermia in rats. In addition, the hexane extract showed an anti-inflammatory effect when tested in ethyl phenylpropiolate (EPP)- and arachidonic acid (AA)-induced rat ear edema. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diospyros variegata Kruz.; Analgesic; Antipyretic; Anti-inflammatory activity

1. Introduction

2. Material and methods

Diospyros variegata Kruz. (Ebenaceae) is known in Thailand as “Phayaa-raak-dam”. In Thai folk medicine, the extract obtained by boiling stems of D. variegata in water is concentrated to a thick residue. On cooling this is cut into pieces which are used to prepare a tea for the management of pain and inflammatory condition (Panthong et al., 1986). Pharmacological study of the extract from the root of D. variegata revealed that the extract did not possess antihistamine or antispasmodic activity when tested using isolated ileum of guinea-pig and had no hypotensive action in anesthetized dogs (Mokkasamit et al., 1971a). Phytochemical study of the root of D. variegata showed that the plant did not contain any alkaloid substance (Congdon et al., 1981). The study of a crude-methanol extract of D. variegata in mice found no toxicity after an oral and intraperitoneal administration of different doses, i.e. 1, 3 and 10 g/kg (Mokkasamit et al., 1971b). The present study was, therefore, undertaken to further work out the analgesic, antipyretic and anti-inflammatory actions of D. variegata, using the hexane-soluble portion of the methanol extract of the stem part of the plant.

2.1. Animals

∗ Corresponding

author. Tel.: +66-53-945431; fax: +66-53-217144. E-mail address: [email protected] (A. Panthong).

Male Sprague–Dawley rats and Swiss albino mice purchased from National Laboratory Animal Center, Salaya, Mahidol University, Nakorn Pathom, Thailand, were used. Animals were kept in appropriate room with temperature of 24 ± 1 ◦ C on a 12 h light/dark cycle and had free access to food and water. All animals were acclimatized for at least 1 week before the beginning of experiments. 2.2. Plant material and preparation of extract Stems of D. variegata Kruz. were collected in Chiang Mai, Thailand. A voucher specimen of the plant (QBG 6742) was deposited at the Herbarium of the Queen Sirikit Botanical Garden, Chiang Mai, Thailand. One kilogram of dried stem was ground and macerated with methanol at room temperature overnight; the process was repeated three times. The crude-methanol extract was evaporated under reduced pressure using rotary evaporator at a temperature of 55 ◦ C to give a syrupy residue, which was then partitioned three times with hexane in equal volume using separatory funnel. The combined hexane fractions were evaporated to dryness under vacuum (rotary evaporator) to give a solid residue (15.74 g), designated


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as hexane extract. Just prior to use of the extract for the pharmacological experiments, extract was suspended in 5% Tween 80 and administered intraperitoneally to animals, except in the ear edema model in which test drugs were dissolved in acetone and applied topically. 2.3. Analgesic tests 2.3.1. Acetic acid-induced writhing Male albino mice, weighing 30–40 g, were used. The method was essentially as described by Nakamura et al. (1986). The writhing response was elicited by an intraperitoneal injection of 0.75% acetic acid at the dose of 0.1 ml/10 g body weight. Test substances and control vehicle were intraperitoneally injected into the mice 30 min before acetic acid and the number of writhes was noted for 15 min beginning 5 min after acetic acid injection. 2.3.2. Tail-flick test Male rats weighing 180–200 g, six in each group, were used. The method was assessed using a tail-flick apparatus following the method of D’Amour and Smith (1941) as modified by Gray et al. (1970). The rat was placed on the tail-flick unit (Ugo Basile), so that the tail occluded a slit over a photocell. Heat was applied by a 100-W lamp mounted in a reflector. The apparatus was arranged so that when the operator turned on the lamp a timer was activated. When the rat felt pain and flicked its tail, light fell on the photocell then the timer was automatically stopped. The light intensity was adjusted to give a normal reaction time of 2–4 s. A 10-s cut-off time was used in order to prevent tissue damage. Two control readings, taken 30 min apart, were averaged and constituted the control reaction time. The extract or drug was administered (i.p.) immediately after this step, and 30 min later, the post-drug reaction time was measured. The analgesic response was calculated as a percentage of the maximum possible response time (Harris and Pieron, 1964). 2.3.3. Formalin test The formalin test possesses two distinctive phases, possibly reflecting different types of pain. The method of Hunskaar and Hole (1987) was used. Males Swiss albino mice, 30–40 g, were injected intraperitoneally with extract or drugs. Thirty minutes afterwards, 20 ␮l of 1% formalin were injected into the dorsal surface of the right hindpaw. Mice were observed in the chambers. The time spent licking the injected paw (licking time) was recorded. Mice were observed for the first 5 min post-formalin (early phase) or for 10 min starting at the 20th minute post-formalin (late phase). 2.4. Anti-inflammatory test The method of Brattsand et al. (1982) and Young et al. (1984) was used with slight modification. Male rats weighing 40–60 g were used. Ethyl phenylpropiolate (EPP) and arachidonic acid (AA) at the dose of 1 mg per ear were

dissolved in acetone and topically applied to the inner and outer surfaces of both ears. Extract and drugs were dissolved in acetone and applied topically to the ear just before the irritants. The thickness of each ear was measured with the vernier calipers before and at 15, 30, 60 and 120 min after edema induction. The increase in ear thickness was compared with the control group and percent inhibition was calculated. 2.5. Antipyretic test Antipyretic property of the extract was tested in rats weighing 200–220 g in which hyperthermia had been induced following the method of Teotino et al. (1963). Initial rectal temperatures of the rats were recorded using a 10-channel electric thermometer connected with probes. Rats were made hyperthermic by subcutaneous injection of 18% yeast suspension at a dose of 1 ml/100 g body weight. When the temperature was at peak (18 h after yeast injection) the rectal temperatures were again recorded. The rats that showed a rise in rectal temperature of more than 1 ◦ C were used. Test substances and vehicles were given intraperitoneally and the rectal temperatures of animals were recorded at 30 min intervals for 2 h following the administration of drug or extract. 2.6. Chemicals The following chemicals were used: Brewer’s yeast, ethyl phenylpropiolate, arachidonic acid, phenylbutazone, phenidone, aspirin were from Sigma. Morphine sulfate was purchased from The Pharmaceutical Organization, Bangkok, Thailand. All other chemicals were of analytical grade. 2.7. Statistical analysis Student’s t-test was applied to determine a significant between the control group and experimental group.

3. Results 3.1. Analgesic activity 3.1.1. Acetic acid-induced writhing The effect of the hexane extract of D. variegata on writhing response in mice is shown in Table 1. It was found that the hexane extract caused an inhibition on the writhing response induced by acetic acid. An increase in the doses of the plant extract resulted in a greater inhibition. Dose of 150 mg/kg of the hexane extract and aspirin could block the writhing response by 93 and 91%, respectively. 3.1.2. Tail-flick test The inhibitory effects of hexane extract and reference drugs on the tail-flick reflex in rats are shown in Table 2.

S. Trongsakul et al. / Journal of Ethnopharmacology 85 (2003) 221–225 Table 1 Effect of aspirin, morphine as well as hexane extract of D. variegata on acetic acid-induced writhing response in mice Group

Number of writhes

Control

58.33 ± 1.82

Aspirin 37.5 mg/kg 75.0 mg/kg 150.0 mg/kg Morphine 10.0 mg/kg Hexane extract 37.5 mg/kg 75.0 mg/kg 150.0 mg/kg

Inhibition (%)

3.1.3. Formalin test The effects of hexane extract in early and late phases of the formalin test are shown in Table 3. Aspirin and hexane extract were active in both early and late phases of the formalin test. The results obtained from the early phase showed that aspirin and the hexane extract possessed only mild inhibitory effect on licking response at the dose of 75 and 150 mg/kg. On the late phase, aspirin and hexane extract exhibited moderate analgesic effect at all doses used.

17 64 91

0∗

100

42.83 ± 1.07∗ 17.83 ± 2.70∗ 3.66 ± 0.88∗

26 69 93

Test drugs were administered intraperitoneally 30 min before acetic acid injection. Values were expressed as mean ± S.E.M. (n = 6). ∗ Significantly different from control group (P < 0.05).

Table 2 Effect of aspirin, morphine as well as hexane extract of D. variegata on the tail-flick test in rats Tc (s)

Control Aspirin, 150 mg/kg Morphine, 10 mg/kg Hexane extract, 150 mg/kg

2.77 2.63 2.93 2.63

± ± ± ±

Tr (s) 0.16 0.19 0.26 0.18

Morphine at a dose of 10 mg/kg completely inhibited the tail-flick response whereas aspirin and hexane extract at a dose of 150 mg/kg possessed slight analgesic effect of 16 and 21%, respectively.

–

47.66 ± 2.99∗ 21.33 ± 2.83∗ 5.33 ± 0.76∗

Group

223

2.97 3.83 10.00 4.20

Inhibition (%) ± ± ± ±

0.20 0.22∗ 0.00∗ 0.32∗

– 16 100 21

Test drugs were administered intraperitoneally 30 min before experiment. Values were expressed as mean ± S.E.M. (n = 6). Tc = control reaction time; Tr = reaction time after injection of test drugs. ∗ Significantly different from T (P < 0.05). c

3.2. Anti-inflammatory activity Results obtained from EPP-induced rat ear edema are demonstrated in Table 4. Phenylbutazone at the dose of 1 mg per ear inhibited the edema formation significantly. The percentage inhibition were found to be 55, 52, 52 and 51 when measurement was made at 15, 30, 60 and 120 min after application of EPP. The hexane extract at the dose of 4 mg per ear significantly inhibited the ear edema formation with percentage inhibition of 58, 60, 58 and 55 at 15, 30, 60 and 120 min, respectively. Results from AA-induced rat ear-edema, presented in Table 5 shows that phenidone at the dose of 2 mg per ear inhibited edema formation with the percentage of 39, 47, 56 and 55. The hexane extract at the dose of 4 mg per ear was also elicited significantly inhibitory effect on the ear edema with the percentage of 26, 41, 52 and 50 at 15, 30, 60 and 120 min after AA. Phenylbutazone (1 mg per ear) could not prevent the edema formation except 15 min after AA application.

Table 3 Effect of aspirin, morphine as well as hexane extract of D. variegata on the early phase and late phase of formalin test in mice Group

Early phase Licking

Control Aspirin 37.5 mg/kg 75.0 mg/kg 150.0 mg/kg Morphine SO4 10.0 mg/kg Hexane extract 7.5 mg/kg 75.0 mg/kg 150.0 mg/kg

timea

Late phase (s)

104.33 ± 7.69 98.33 ± 3.54 79.17 ± 4.47∗ 65.17 ± 2.00∗ 0∗ 97.33 ± 3.77 86.83 ± 3.41∗ 65.17 ± 2.94∗

Inhibition (%)

Licking timeb (s)

Inhibition (%)

–

115.33 ± 10.20

–

6 24 38

73.17 ± 1.19∗ 50.17 ± 7.02∗ 31.67 ± 3.79∗

37 57 72

100 7 16 38

0∗ 73.67 ± 2.80∗ 46.00 ± 2.48∗ 31.33 ± 3.58∗

Test drugs were administered intraperitoneally 30 min before 1% formalin injection. Values were expressed as mean ± S.E.M. (n = 6). a Seconds between 0 and 5 min after formalin injection. b Seconds between 20 and 30 min after formalin injection. ∗ Significantly different from control group (P < 0.05).

100 36 60 73

224


Table 4 Effect of topical application of phenylbutazone, hexane extract of D. variegata on ethyl phenylpropiolate-induced ear edema in rats Group

Edema thickness (␮m)

Inhibition (%)

15 min

30 min

1h

2h

15 min

30 min

1h

2h

Control Phenylbutazone, 1 mg per ear Hexane extract, 4 mg per ear

97 ± 8 44 ± 4∗ 40 ± 7∗

210 ± 7 100 ± 4∗ 85 ± 13∗

213 ± 13 103 ± 6∗ 90 ± 9∗

190 ± 11 94 ± 3∗ 85 ± 6∗

– 55 58

– 52 60

– 52 58

– 51 55

Values were expressed as mean ± S.E.M. (n = 6). ∗ Significantly different from control (P < 0.05). Table 5 Effect of topical application of phenylbutazone, phenidone as well as hexane extract of D. variegata on arachidonic acid-induced ear edema in rats Group

Edema thickness (␮m) 15 min

Control Phenylbutazone, 1 mg per ear Phenidone, 2 mg per ear Hexane extract, 4 mg per ear

77 58 47 57

± ± ± ±

Inhibition (%)

30 min

11 15∗ 11∗ 6∗

207 206 110 123

± ± ± ±

1h 8 18 7∗ 12∗

273 250 120 130

2h ± ± ± ±

10 10 7∗ 9∗

247 243 110 123

± ± ± ±

4 10 4∗ 6∗

15 min

30 min

1h

2h

– 25 39 26

– 0 47 41

– 8 56 52

– 2 55 50

Values were expressed as mean ± S.E.M. (n = 6). ∗ Significantly different from control (P < 0.05). Table 6 Effect of aspirin, hexane extract of D. variegata on yeast-induced hyperthermia in rats Group

Rectal temperature (◦ C) Initial

Control Aspirin, 150 mg/kg Hexane extract, 150 mg/kg

38.30 ± 0.07 38.30 ± 0.10 38.28 ± 0.08

18 h after yeast injection

39.35 ± 0.06 39.35 ± 0.06 39.37 ± 0.12

Time after medication (min) 30

60

90

120

39.30 ± 0.09 38.37 ± 0.21∗ 38.43 ± 0.18∗

39.23 ± 0.07 38.09 ± 0.26∗ 37.87 ± 0.16∗

39.20 ± 0.08 37.67 ± 0.24∗ 37.43 ± 0.17∗

39.15 ± 0.08 37.58 ± 0.24∗ 37.30 ± 0.19∗

Drugs were given (i.p.) 18 h after yeast injection. Values were expressed as mean ± S.E.M. (n = 6). ∗ Significantly different from the rectal temperature 18 h after yeast injection (P < 0.05).

3.3. Antipyretic activity As shown in Table 6, aspirin and the hexane extract at the dose of 150 mg/kg caused a significant lowering in rectal temperature of hyperthermia rats. The decrease in rectal temperature still existed when assessment was made 2 h after test drug administration.

4. Discussion and conclusion The writhing response of the mouse to an intraperitoneal injection of noxious chemical is used to screen for both peripherally and centrally acting analgesic activity. Acetic acid causes algesia by liberating endogenous substances and many others that excite pain nerve endings (Raj, 1996). According to the percentage inhibition on the number of writhes obtained with the various doses of hexane extract, it was found that the intensity of the analgesic effect was similar to that of aspirin at the same doses. Aspirin and other NSAIDs can inhibit cyclooxygenase in peripheral tissues,

thus, interfering with the mechanism of transduction in primary afferent nociceptors (Fields, 1987). The mechanism of analgesic action of the hexane extract could probably be due to the blockade of the effect or the release of endogenous substances that excite pain nerve endings similarly to aspirin and NSAIDs. The tail-flick test is widely used to investigate the centrally acting analgesic activity. The tail-flick response appears to be a spinal reflex, which is modulated by a supraspinal inhibitory mechanism. Aspirin and hexane extract possessed slight analgesic effect on this tail-flick response. The results obtained from the writhing response and tail-flick tests suggest that the hexane extract seems to possess an intensity of analgesic effect that is mostly mediated via a peripheral mechanism by inhibition of the PGs-mediated potential of algesic action of bradykinin (Nakamura et al., 1986). The hexane extract also could partly act via the central nervous system in producing an analgesic effect like that of aspirin and other NSAIDs, which is suggested to be at the hypothalamic region (Vane, 1987).


The formalin test possesses two distinctive phases, possibly reflecting different types of pain. The earlier phase reflects direct effect of formalin on nociceptors (non-inflammatory pain, whereas the late phase reflects inflammation (Hunskaar and Hole, 1987). From this study, the hexane extract showed only slight analgesic activity on the early phase but exhibited pronounced effect on the late phase. The slight inhibition in the early phase may be due to direct effects on nociceptor. The effect on the late phase of hexane extract indicates its inhibitory activity on pain arised from inflammation, which reflects the effect on the synthesis and/or release of PGs. Edema caused by topically applied EPP is due to vasodilation and increased vascular permeability. This event is caused by the release of various inflammatory mediators such as histamine, 5-HT and PGs (Brattsand et al., 1982). The hexane extract markedly inhibited ear edema formation induced by EPP. It is suggested that the hexane extract probably has anti-inflammatory activity like phenylbutazone (cyclooxygenase inhibitor), through inhibition of the inflammatory mediators of the acute phase of inflammation. AA produces an intense inflammatory reaction in the mouse ear and subsequent experiments demonstrated that this response could be ameliorated by putative lipoxygenase inhibitors (Chang et al., 1986). Phenidone (a non-selective inhibitor of arachidonic-acid metabolism) and the hexane extract exerted significant inhibitory effect on ear edema formation induced by AA. It is, therefore, possible that hexane extract exhibits anti-inflammatory activity in part by inhibition of lipoxygenase pathway. Since antipyretic activity is commonly mentioned as a characteristic of drugs or compounds which have an inhibitory effect on prostaglandin-biosynthesis (Vane, 1987), the yeast-induced hyperthermia in rat model was employed to investigate the antipyretic activity of the hexane extract of D. variegata. It was found that the hexane extract caused a significant decrease in rectal temperature similar to aspirin. This result seems to support the view that the hexane extract has some influence on prostaglandin-biosynthesis because prostaglandin is believed to be a regulator of body temperature (Milton, 1982). In conclusion, It is possible that the non-polar substances present in the hexane extract from the stem of D. variegata are responsible for the analgesic, antipyretic and anti-inflammatory activities observed in this study. An extract from the stem of this plant has been used in indigenous medicine as a remedy for algesia and inflammation

225

(Panthong et al., 1986), thus, this present study supports the claimed uses of this plant in folk medicine.

References Brattsand, R., Thalen, A., Roempke, K., Kallstrom, L., Gruvstad, E., 1982. Influence of 16␣, 17␣-acetal substitution and steroid nucleus fluorination on the topical to systemic activity ratio of glucocorticoids. Journal of Steroid Biochemistry 16, 779–786. Chang, J., Carlson, R.P., O’Neill-Davis, L., Lamb, B., Sharma, R.N., Lewis, A.J., 1986. Correlation between mouse skin inflammation induced by arachidonic acid and eicosanoid synthesis. Inflammation 10, 205–214. Congdon, G., Sirirugsa, P., Lojanapiwatna, V., Wiriyachitra, P., 1981. A contribution to the Thai phytochemical survey II. Journal of the Science Society of Thailand 7, 87–90. D’Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74–79. Fields, H.L., 1987. Analgesic drugs. In: Day, W. (Ed.), Pain, 1st ed. MacGraw-Hill, USA, 272 p. Gray, W.D., Osterberg, A.C., Scute, J.T., 1970. Measurement of the analgesic efficacy and potency of pentazocine by the D’Amour and Smith method. Journal of Pharmacological and Experimental Therapeutics 172, 154–162. Harris, L.S., Pieron, A.K., 1964. Some narcotic antagonists in the benzomorphan series. Journal of Pharmacological and Experimental Therapeutics 143, 141–148. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Milton, A.S., 1982. Prostaglandins and fever. Trends in Pharmacological Sciences 40, 490–492. Mokkasamit, M., Ngarmwathana, W., Sawasdimonkol, K., Permphiphat, U., 1971a. Pharmacological evaluation of Thai medicinal plants. Journal of the Medical Association of Thailand 54, 490–540. Mokkasamit, M., Swatdimonkol, K., Satrawaha, P., 1971b. Study on toxicity of Thai medicinal plants. Bulletin of the Department of Medical Science 12, 36–65. Nakamura, H., Shimoda, A., Ishi, K., Kadokawa, T., 1986. Central and peripheral analgesic action of nonsteroidal anti-inflammatory drugs in mice and rats. Archives International of Pharmacodynamic 282, 16– 25. Panthong, A., Kanjanapothi, D., Taylor, W.C., 1986. Ethanobotanical review of medicinal plants from Thai traditional books. Part I. Plant with anti-inflammatory, anti-asthmatic and antihypertensive properties. Journal of Ethnopharmacology 18, 213–228. Raj, P.P., 1996. Pain mechanisms. In: Raj, P.P. (Ed.), Pain Medicine: A Comprehensive Review, 1st ed. Mosby-Year Book, Missouri, pp. 12–23. Teotino, U.M., Friz, L.P., Gandini, A., Bella, D.D., 1963. Thio derivatives of 2,3-dihydro-4H-1,3-benzoazin-4-one synthesis and pharmacological properties. Journal of Medicinal Chemistry 6, 248–250. Vane, J.R., 1987. The evolution of non-steroidal anti-inflammatory drug and their mechanisms of action. Drugs (supply) 33, 18–27.


Antioxidant activity of the ethanolic extract of Striga orobanchioides Shrishailappa Badami∗ , Mahesh Kumar Gupta, B. Suresh Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Rocklands, Ootacamund 643 001, Tamilnadu, India Received 18 August 2002; received in revised form 16 December 2002; accepted 17 December 2002

Abstract Plants containing flavonoids have been reported to possess strong antioxidant properties. The ethanolic extract of Striga orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures. The ethanol extract exhibited IC50 values of 18.65 ± 1.46 and 11.20 ± 0.52 ␮g/ml, respectively in DPPH and nitric oxide radical inhibition assays. These values were less than those obtained for ascorbic acid and rutin, used as standards. In the in vivo experiments the extract treatment at 100 mg/kg body weight dose caused a significant increase in the level of the catalase in the liver and the kidneys. A significant increase in the level of SOD in the liver was observed. The treatment also caused a significant decrease in the TBA-RS and increase in the ascorbic acid levels. These results suggest strong antioxidant potentials of the ethanolic extract of S. orobanchioides. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Striga orobanchioides; Antioxidant; DPPH; Nitric oxide; Peroxidation; Free radical scavenging

1. Introduction Striga Orobanchioides Benth (Family—Scrophulariaceae), is a parasitic plant, lives on the roots of various plants mainly on Lepidagathis, Euphoria antiguorum and Dysophylla and is distributed up to 6000 ft in the hills, usually on red and gravelly soils (Gamble, 1935). In Ayurvedic medicine, the plant is described as antidiabetic (Chopra et al., 1956). Earlier studies have shown significant anti-implantation and estrogenic activity of ethanolic and distilled water extracts of the whole plant S. orobanchioides (Hiremath et al., 1994). The ethanolic extract has also shown significant antiandrogenic (Hiremath et al., 1997), antibacterial (Hiremath et al., 1996a), antihistaminic and mast cell stabilising activities (Harish et al., 2001). From the ethanolic extract, two known flavonoids, apigenin and luteolin which have also shown antifertility properties have been isolated (Hiremath et al., 1996b; Hiremath et al., 2001). Except for these studies, so far, no other chemical and biological investigations have been carried out on this plant. Lipid peroxidation has gained more importance now a days because of its involvement in pathogenesis of many diseases like atherosclerosis, cancer, diabetes mellitus, my∗ Corresponding

author. E-mail address: [email protected] (S. Badami).

ocardial infarction, and also in ageing. Free radicals or reactive oxygen species (ROS) are produced in vivo from various biochemical reactions and also from the respiratory chain as a result of occasional leakage. These free radicals are the main culprits in lipid peroxidation (Cheeseman and Scater, 1993). Plants containing flavonoids have been reported to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, in the present study, the ethanolic extract of S. orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures.

2. Material and methods 2.1. Plant material The whole plant was collected from the fields in and around Gulbarga (Karnataka, India) during September– November 2001 and authenticated in the Herbarium, Department of Botany, Gulbarga University, Gulbarga, where voucher specimens are deposited (Voucher No. GU 232). 2.2. Preparation of the extracts and standards The ethanolic extract of the shade-dried powdered whole plant of S. orobanchioides was obtained as described by (Hiremath et al., 1994). The suspension of this extract was


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prepared in sodium carboxy methyl cellulose (CMC, 0.3%) using distilled water in the case of in vivo experiments, whereas, for in vitro experiments, a weighed quantity of the extract was dissolved in distilled dimethyl sulphoxide (DMSO) and used. Solutions of ascorbic acid and rutin used as standards for in vitro studies were prepared in distilled DMSO. 2.3. Chemicals 1,1,3,3-tetramethoxypropane and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals Co., St. Louis, MO. Sodium nitroprusside, methanol and dimethyl sulphoxide were obtained from Ranbaxy Fine Chemicals Ltd., Mohali, India. Sulphanilic acid was obtained from Himedia Laboratories Ltd., Mumbai, India. Naphthylethylenediamine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), thiobarbituric acid, trichloroacetic acid, sodium CMC were obtained from Loba Chemie, Mumbai, India. Ascorbic acid and rutin were obtained from S.D. Fine Chem., Biosar, India. Hydrogen peroxide solution (30%) was obtained from Qualigen Fine Chemicals, Mumbai, India. 2.4. Test animals Male Wistar strain rats (180–200 g) were obtained from the animal house, J.S.S. College of Pharmacy, Ootacamund, India and were maintained under standard environmental conditions and fed with Amrut rat feed, NAV Maharasthra Chakan Oil Mills Ltd., Pune, India and water ad libitum. 2.5. In vitro assays 2.5.1. DPPH method The antioxidant activity of the plant extract and the standards were assessed on the basis of the radical scavenging effect of the stable DPPH free radical (Bang et al., 2001). A total of 10 ␮l of the ethanolic extract (from 21 mg/ml to 40 ␮g/ml in DMSO solution) or standard was added to 200 ␮l of DPPH in methanol solution (100 ␮M) in a 96-well microtitre plate. After incubation at 37 ◦ C for 30 min, the absorbance of each solution was determined at 490 nm using Elisa microtitre plate reader (Bio Rad Laboratories Inc., CA; model 550). The corresponding blank readings were also taken and the remaining DPPH was calculated. IC50 value is the concentration of sample required to scavenge 50% DPPH free radical.

instead of 1-naphthylamine (5%). Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). The reaction mixture (3 ml) containing sodium nitroprussude (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and extract or standard solution (0.5 ml) was incubated at 25 ◦ C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthylethylenediamine dihydrochloride (1%) was added, mixed and allowed standing for 30 min. A pink coloured chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions in microtitre plate using Elisa reader. The IC50 value is the concentration of sample required to inhibit 50% of nitric oxide radical. 2.6. In vivo antioxidant activity Animals were divided into three groups comprising of six rats in each group. Group 1 served as control and was given the vehicle alone (sodium CMC, 0.3%). The second and third groups received ethanolic extract of S. orobanchioides orally at 50 and 100 mg/kg body weight, respectively. The treatments were given for 7 days and on the 8th day of the experiment, all the animals were sacrificed by decapitation. The liver and kidneys were removed, weighed and homogenised immediately with Elvenjan homoginizer fitted with teflon plunger, in ice chilled 10% KCl solution (10 mg/g of tissue). The suspension was centrifuged at 2000 rpm at 4 ◦ C for 10 min and the clear supernatant was used for the following estimations. Catalase was estimated by following the breakdown of hydrogen peroxide according to the method of Beer and Seizer (1952). Superoxide dismutase (SOD) was assayed according to Misra and Fridevich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. Lipid peroxidation was measured in terms of MDA content following the thiobarbituric acid (TBA) method of Buege and Aust (1978). Ascorbic acid was measured by the method of Natelson (1963). Statistical analysis was carried out using the Student’s t-test and the results were judged significant, if P < 0.05.

3. Results 3.1. In vitro assay

2.5.2. Nitric oxide radical inhibition assay Sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction (Garrat, 1964). In the present investigation, Griess Illosvoy reagent is modified by using naphthylethylenediamine dihydrochloride (0.1% w/v)

The ethanolic extract of S. orobanchioides exhibited strong antioxidant activity in the DPPH and the nitric oxide radical inhibition assay as evidenced by the low IC50 values (Table 1). The IC50 values obtained are 18.65 ± 1.46 and 11.20 ± 0.52 ␮g/ml, respectively in the DPPH and nitric oxide radical inhibition assays. These values were found

S. Badami et al. / Journal of Ethnopharmacology 85 (2003) 227–230 Table 1 Effect of the ethanolic extract of S. orobanchioides on free radical generation in vitro S. No.

1 2 3

Tested material

Ethanolic extract Ascorbic acid Rutin a

IC50 (␮g/ml) ± S.E.a DPPH method

Nitric oxide radical inhibition assay

18.65 ± 1.46 54.63 ± 3.92 43.60 ± 1.79

11.20 ± 0.52 – 37.31 ± 4.17

Average of 10 determinations.

to be less than those obtained for the reference standards, ascorbic acid and rutin. 3.2. In vivo assays The administration of ethanolic extract of S. orobanchioides to normal rats for 7 days induced a dose dependent increase in the level of catalase in the liver and kidneys. The results are significant at 100 mg/kg body weight dose of the treatment (P < 0.001 and 0.05, respectively for liver and kidneys when compared with control; Tables 2 and 3). The treatment caused a significant increase in the level of SOD in the liver (P < 0.01, when compared with control). However, the level of SOD in the kidneys of the treated rats was not dose related and found to be significantly decreased (P < 0.001 and 0.05). The treatment with ethanol extract also caused a significant and dose related decrease in the level of malondialdehyde (MDA, P < 0.05–0.01, when compared with control) formed in peroxidising system and a significant increase in the level of ascorbic acid (P < 0.05–0.001, when compared with control) in the liver and kidneys.

229

4. Discussion Free radical oxidative stress has been implicated in the pathogenesis of a wide variety of clinical disorders, resulting usually from deficient natural antioxidant defences (Hellwell and Gutteridge, 1989). Potential antioxidant therapy should, therefore, include either natural free radical scavenging enzyme or agents, which are capable of augmenting the activity of these enzymes, which include SOD and catalase (Cheeseman and Scater, 1993). The administration of the ethanol extract of S. orobanchioides at 100 mg/kg body weight significantly increased the level of catalase in liver (P < 0.001) and kidneys (P < 0.05) and the level of SOD (P < 0.01) in the liver. This shows the antioxidant nature of the extract. However, a decrease in the level of SOD was observed in the treated groups in the kidneys. Generally, results for the kidneys have shown fewer changes in antioxidant activity compared to the liver (Jadwiga et al., 2000), which could explain the present observations. The present study also showed the depletion in the lipid peroxidation as observed by the significant decrease in the MDA content of the liver and kidneys in the treated groups. Vitamin C is regarded as the first line natural antioxidant defense in plasma and powerful inhibitor of lipid peroxidation. It also regenerates the major antioxidant tocopherol in lipoproteins and cell membranes. Intracellular mechanisms exist which can regenerate ascorbate from its dehydroascorbate by reduced glutathione (Bhattacharya et al., 1999). The treatment with the ethanolic extract also caused a significant increase in the ascorbic acid level of the treated rats in the liver and kidneys. Frie (1991) has shown the ability of Vitamin C to preserve the level of other antioxidants in human plasma. Depletion of the ascorbic acid level in a biological system has been found to correlate to a loss in antioxidant capacity (Aartensson and Neister, 1991). It can be concluded that

Table 2 Effect of the ethanolic extract of S. orobanchioides on rat liver Treatment

Dose (mg/kg body weight)

Catalase (IU/mg tissue/min)

SOD (IU/mg tissue/min)

TBA-RS (nM/mg tissue)

Ascorbic acid (␮g/mg tissue)

Control Ethanolic extract

– 50 100

0.644 ± 0.024 0.941 ± 0.098∗ 1.352 ± 0.027∗∗∗

0.086 ± 0.008 0.091 ± 0.002 0.117 ± 0.002∗∗

0.287 ± 0.018 0.264 ± 0.012∗ 0.204 ± 0.009∗∗

4.610 ± 0.38 5.830 ± 0.39 7.440 ± 0.56∗∗∗

Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).

Table 3 Effect of ethanolic extract of S. orobanchioides in rat kidney Treatment

Dose (mg/kg body weight)

Catalase (IU/mg tissue/min)

SOD (IU/mg tissue/min)

TBA-RS (nM/mg tissue)

Ascorbic acid (␮g/mg tissue)

Control Ethanolic extract

– 50 100

1.556 ± 0.069 1.650 ± 0.045 1.921 ± 0.107∗

0.096 ± 0.008 0.056 ± 0.005∗∗∗ 0.082 ± 0.007∗

0.304 ± 0.024 0.275 ± 0.018∗∗ 0.262 ± 0.025∗∗

4.060 ± 0.230 5.190 ± 0.144 7.016 ± 0.181∗∗∗

Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).

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the ethanolic extract of S. orobanchioides possess strong antioxidant properties as evidenced by the significant increase in the level of catalase, SOD and ascorbic acid and decrease in the levels of TBA-RS. The in vitro studies confirm the same. The ethanolic extract is found to contain two flavonoids, apigenin and luteolin (Hiremath et al., 1996b). A large number of flavonoids including these is known to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, the antioxidant activity of S. orobanchioides is probably due the presence of these flavonoids in the ethanolic extract. Acknowledgements The authors wish to place on record their heart felt thanks to Jagadguru Sri Sri Shivarathreeshwar Deshikendra Mahaswamigalavaru of Suttur Mutt for providing the facilities. References Aartensson, J., Neister, A., 1991. Glutathione deficiency disease tissue ascorbate levels in newborn rats, ascorbate spares glutathione and protects. Proceedings of the National Academy of Sciences 88, A-656– A-660. Bang, Y.H., Hang, S.K., Jeong, H.L., Young, S.H., Jai, S.R., Kyong, S.L., Jung, J.L., 2001. Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus. Journal of Natural Products 64, 82–84. Beer, R.F., Seizer, T.W., 1952. A spectrophotometric method for measuring breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 115, 130–140. Bhattacharya, A., Chatterjee, A., Ghosal, S., Bhattacharya, S.K., 1999. Antioxidant activity of tannoid principles of Emblica officinalis (Amla). Indian Journal of Experimental Biology 37, 676–680. Buege, J.A., Aust, S.D., 1978. The thiobarbituric acid assay. Methods in Enzymology 52, 306–307. Cheeseman, K.H., Scater, T.F., 1993. Free radical in medicine. British Medical Bulletin, vol. 49. Churchill Livingstone, London, pp. 479–724.

Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 235. Frie, B., 1991. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage. American Journal of Clinical Nutrition 54, 113S. Gamble, J.S., 1935. Flora of the Presidency of Madras, vol. II. Adlard and Son, London, p. 967. Garrat, D.C., 1964. The Quantitative Analysis of Drugs, vol. 3. Chapman and Hall, Japan, pp. 456–458. Harish, M.S., Nagur, M., Badami, S., 2001. Antihistaminic and mast cell stabilizing activity of Striga orobanchioides. Journal of Ethnopharmacology 76, 197–200. Hellwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine, 2nd ed. Clarendon Press, Oxford. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., 2001. Antifertility and hormonal properties of flavones of Striga orobanchioides. European Journal of Pharmacology 391, 193–197. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1994. Antifertility activity of Striga orobanchioides. Biological and Pharmaceutical Bulletin 17, 1029–1031. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1997. Antiandrogenic activity of Striga orobanchioides. Journal of Ethnopharmacology 56, 55–60. Hiremath, S.P., Swamy, H.K.S., Badami, S., Meena, S., 1996a. Antibacterial and antifungal activity of Striga orobanchioides. Indian Journal of Pharmaceutical Sciences 58, 174–178. Hiremath, S.P., Badami, S., Swamy, H.K.S., 1996b. Flavones from Striga orobanchioides. Indian Drugs 33, 232. Jadwiga, J.L., Marek, M., Elzbeita, B., 2000. Effect of Sesquiterpene lactones on antioxidant enzymes and some drug metabolizing enzymes in rat liver and kidney. Planta Medica 66, 199–205. Marcocci, P.L., Sckaki, A., Albert, G.M., 1994. Antioxidant action of Ginkgo biloba extracts EGP761. Methods in Enzymology 234, 462– 475. Misra, H.P., Fridevich, I., 1972. The role of superoxide dismutase anion in the autooxidation of epinephrine and sample assay in superoxide dismutase. Journal of Biological Chemistry 241, 3170–3175. Natelson, S., 1963. Routine determinations: ascorbic acid (with dinitrophenyl hydrazine). In: Natelson, S. (Ed.), Microtechnique of Clinical Chemistry. Springfield, Illinois, p. 121. Raj, K.J., Shalini, K., 1999. Flavonoids—a review of biological activities. Indian Drugs 36, 668–676.


Antimicrobial activity of aqueous and methanol extracts of Juniperus oxycedrus L. b , M. Güllüce b,c , H. Öˇ ˙ Karaman a,b,∗ , F. Sahin I. ¸ gütçü c , c , A. Adıgüzel b,c M. Sengül ¸ a

Department of Biology, Faculty of Science, Gebze Institute of Technology, Kocaeli 41400, Turkey b Biotechnology Application and Research Center, Ataturk University, Erzurum 25240, Turkey c Department of Biology, Faculty of Art and Science, Ataturk University, Erzurum 25240, Turkey

Received 6 June 2002; received in revised form 27 November 2002; accepted 18 December 2002

Abstract Aqueous and methanol extracts of the leaves of Juniperus oxycedrus were investigated for their in vitro antimicrobial properties. The plant was collected from Pelitli Village of Gebze, Kocaeli, in the Marmara region of Turkey. Juniperus oxycedrus is widely used as traditional folk medicine in Turkey for treatment of different infectious diseases. The antimicrobial activity of the extracts against 143 laboratory strains belonging to 56 bacterial species, and 31 isolates of 5 fungi species were evaluated based on the inhibition zone using the disc-diffusion assay, minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) values. The aqueous extract of J. oxycedrus had no antimicrobial effect against the test microorganisms whereas the methanol extract had inhibitory effects on the growth of 57 strains of 24 bacterial species in the genera of Acinetobacter, Bacillus, Brevundimonas, Brucella, Enterobacter, Escherichia, Micrococcus, Pseudomonas, Staphylococcus, and Xanthomonas. In addition 11 Candida albicans isolates at a concentration of 31.25–250 ␮g/ml were also inhibited. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antimicrobial activity; Juniperus oxycedrus L.; Medicinal plant; Methanol extract

1. Introduction Juniperus oxycedrus L. (Cupressaceae) is 1 of 10 species in the genus Juniperus throughout the world (Adams, 1998). This shrub or tree has a typical Mediterranean distribution (Amaral Franco, 1964) and three subspecies have been recognized in the Iberian Peninsula, including J. oxycedrus ssp. oxycedrus, J. oxycedrus ssp. macrocarpa (Sibth & Sm.) Ball. and J. oxycedrus ssp. badia (H. Gay) Debeaux. The former is the most abundant in the Marmara and Aegean regions of Turkey (Amaral Franco, 1986). Methanol and dichloromethane extracts of leaves and stems of J. oxycedrus ssp. oxycedrus (from Spain) reduce the blood pressure of normotensive rats (Bello et al., 1997), inhibit the response to histamine, serotonin, and acetylcholine (Moreno et al., 1998a,b), and exhibit significant anti-inflammatory activity (Moreno et al., 1998a,b). Juniperus oxycedrus is used to prepare an empyreumatic oil by destructive distillation of ∗ Corresponding author. Tel.: +90-442-231-2610; fax: +90-442-231-1469. ˙ Karaman). E-mail address: [email protected] (I.

the branches and wood of the plant, which is widely employed in human and veterinary dermatology to treat chronic eczema and other skin diseases (Bouhlal et al., 1988). Rectified cade oil, used as a fragrance component in soaps, detergents, creams, lotions, and perfumes (Leung and Foster, 1996), is also produced. Furthermore, this plant is also used as a folk remedy to treat various ailments, such as hyperglicemia, obesity, tuberculosis, bronchitis, and pneumonia (Sanchez de Medina et al., 1994; Swanston-Flatt et al., 1990; Duke, 1985; WHO, 1980). In recent years, multiple drug resistance in human pathogenic microorganisms have developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases. This situation forced scientists for searching new antimicrobial substances from various sources, like medicinal plants, which are the good sources of novel antimicrobial chemotherapeutic agents. The present study was conducted to investigate antimicrobial properties of methanol and aqueous extracts of J. oxycedrus against a wide range of bacteria, fungi, and yeast species which have not been evaluated in the previous studies.


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I˙. Karaman et al. / Journal of Ethnopharmacology 85 (2003) 231–235

2. Material and methods 2.1. Plant materials and extraction procedure The plants were collected from Pelitli Village of Gebze in Kocaeli located in the region of Marmara, Turkey. The taxonomic identification of plant materials was confirmed by a senior plant taxonomist, Meryem Sengül, ¸ at the Department of Biology, Ataturk University, Erzurum, Turkey. Collected plant material was dried in the shade, and the leaves of plant were separated from the stem, and ground in a grinder with a 2 mm diameter mesh. The dried and powdered leaves of plant (500 g) were extracted successively with 1 l of methanol by Soxhlet for 72 h at a temperature not exceeding the boiling point of the solvent (Lin et al., 1999). Crude aqueous infusion were also prepared by adding 1 l of boiling water to 500 g of powdered plant material in a glass 2.5-l flask and incubated at room temperature for 2 h on a rotating shaker (200 rpm). The aqueous extracts were filtered using Whatman No. 1 filter paper and then concentrated in vacuo at 40 ◦ C using a rotary evaporator. The residues obtained were stored in a freezer at −80 ◦ C until use. 2.2. Microorganisms A total of 178 microbial cultures belonging to 56 bacterial and 5 fungal species were used in this study (Table 1). Microorganisms were provided by Department of Clinical Microbiology, Faculty of Medicine, and Plant Diagnostic Laboratory, Faculty of Agriculture at Ataturk University, Erzurum, Turkey. Identity of the test organisms were confirmed by Microbial Identification System in Biotechnology Application and Research Center at Ataturk University. 2.3. Antimicrobial activity 2.3.1. Disc-diffusion assay The dried plant extracts were dissolved in the same solvent (methanol and distilled water) to a final concentration of 30 mg/ml and sterilized by filtration by 0.45 ␮m Millipore filters. Antimicrobial tests were then carried out by disc-diffusion method (Murray et al., 1995) using 100 ␮l of suspension containing 108 CFU/ml of bacteria, 106 CFU/ml of yeast, and 104 spore/ml of fungi spread on nutrient agar (NA), sabourand dextrose agar (SDA), and potato dextrose agar (PDA) medium, respectively. The discs (6 mm in diameter) were impregnated with 10 ␮l of the extracts (300 ␮g/disc) at the concentration of 30 mg/ml and placed on the inoculated agar. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. Ofloxacin (10 ␮g/disc), sulbactam (30 ␮g) + cefoperazona (75 ␮g) (105 ␮g/disc), and/or netilmicin (30 ␮g/disc) were used as positive reference standards to determine the sensitivity of one strain/isolate in each microbial species tested. The inoculated plates were incubated at 37 ◦ C for 24 h for clinical bacterial strains, 48 h for yeast, and 72 h for fungi

isolates. Plant-associated microorganisms were incubated at 27 ◦ C. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms. Each assay in this experiment was repeated twice. 2.3.2. Microdilution assay The minimal inhibition concentration (MIC) values were also studied for the microorganisms which were determined as sensitive to the methanol extract of J. oxycedrus in disc-diffusion assay. The inocula of microorganisms were prepared from 12-h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The J. oxycedrus extract dissolved in 10% dimethyl sulfoxide (DMSO) were first diluted to the highest concentration (500 ␮g/ml) to be tested, and then serial twofold dilutions were made in a concentration range from 7.8–500 ␮g/ml in 10-ml sterile test tubes containing nutrient broth. MIC and minimal bactericidal concentration (MBC) values of J. oxycedrus extract against bacterial strains and Candida albicans isolates were determined based on a micro-well dilution method (Zgoda and Porter, 2001) and described with some modifications as follows. The 96-well plates were prepared by dispensing into each well 95 ␮l of nutrient broth and 5 ␮l of the inoculum. A 100 ␮l from J. oxycedrus extract initially prepared at the concentration of 500 ␮g/ml was added into the first wells. Then, 100 ␮l from their serial dilutions was transferred into six consecutive wells. The last well containing 195 ␮l of nutrient broth without compound and 5 ␮l of the inoculum on each strip was used as negative control. The final volume in each well was 200 ␮l. Maxipime (Bristol-Myers Squibb) at the concentration range of 500–7.8 ␮g/ml was prepared in nutrient broth and used as standard drug for positive control. The plate was covered with a sterile plate sealer. Contents of each well were mixed on plate shaker at 300 rpm for 20 s and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by absorbance at 600 nm using the ELx 800 universal microplate reader (Biotek Instrument Inc., Highland Park, VT, USA) and confirmed by plating 5 ␮l samples from clear wells on NA medium. The extract tested in this study was screened two times against each organism. The MIC was defined as the lowest concentration of the compounds to inhibit the growth of microorganisms. MBCs were determined by plotting 5 ␮l samples from clear wells onto NA plates without plant extract. The MBC was the concentration at which there was no microbial growth.

3. Results and discussion In the present study, the antimicrobial compounds from the leaves of J. oxycedrus collected from Pelitli Village of Gebze, Kocaeli, in the Marmara region of Turkey, were extracted against wide range of microorganisms on the basis of disc-diffusion and microdilution assay.


233

Table 1 Antimicrobial activity of Juniperus oxycedrus extracts (300 ␮g/disc) against the bacterial strains tested based on disc-diffusion method Microorganisms

Bacteria Acinetobacter baumanii Acinetobacter calcoaceticus Acinetobacter lwoffii Acinetobacter johnsonii Alcaligenes pacificus Alcaligenes xylosoxydans Agrobacterium radiobacter Bacillus amyloliquefaciens Bacillus atrophaeus Bacillus cereus Bacillus lentimorbus Bacillus licheniformis Bacillus macerans Bacillus megaterium Bacillus pumilus Bacillus sphaericus Bacillus substilis Brevundimonas diminuta Brucella abortus Brucella melitensis Burkholdria cepacia Burkholdria gladioli Citrobacter freundii Clavibacter michiganense Curtobacterium flaccumfaciens Enterobacter agglomerans Enterobacter cloacae Enterobacter intermedius Enterobacter pyrinus Erwinia amylovora Erwinia carotovora Erwinia chrysanthemi Escherichia coli Flavobacterium blastinum Klebsiella pneumoniae Klebsiella trevisanii Kocuria varians Leclercia adecarboxylata Micrococcus luteus Neisseria spp. Pantoea agglomerans Plesiomonas shigelloides Proteus vulgaris Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomona huttiensis Pseudomonas putida Pseudomonas syringae pvs. Ralstonia pickettii Salmonella typhimurium Staphylococcus aureus Staphylococcus epidermis Stenotrophomonas maltophilia Streptococcus pneumoniae Streptococcus pyogenes Xanthomonas campestris pvs. Total 56 bacterial species a

Number of strains

3 2 1 2 1 2 1 1 2 2 1 1 2 4 2 1 4 1 15 11 1 1 1 1 2 1 2 2 1 1 1 1 1 1 2 1 1 1 1 1 3 1 1 20 5 2 1 5 1 1 5 5 2 2 2 4 143 strains

Inhibition zone diameter around test disc Methanol extract

Aqueous extract

NCa

Standard antibiotic discsb

8 mm/2 – – – – – 7 mm/1 9 mm/2 13 mm/2 9 mm/1 11 mm/1 7–8 mm/2 8–15mm/4 10 mm/2 10 mm/l 9–17 mm/4 8 mm/1 8–13 mm/15 – – – – – – 9 mm/1 – – 15 mm/1 – – – 9 mm/1 – – – 13 mm/1 – 13 mm/1 – – – – 10–15 mm/3 – – 9 mm/1 8–10 mm/2 – – 8–11 mm/5 9 mm/2 – – – 9 mm/1

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

NT 16 mm (OFX) 27 mm (OFX) 25 mm (OFX) 32 mm (OFX) 12 mm (SCF) 28 mm (SCF) 27 mm (SCF) 21 mm (SCF) 30 mm (OFX) 22 mm (SCF) (SCF) 26 mm (SCF) 9 mm (SCF) 23 mm (OFX) 18 mm (OFX) 28 mm (OFX) 34 mm (SCF) 12 mm (SCF) 18 mm (OFX) (SCF) 22 mm (NET) 23 mm (SCF) 25 mm (SCF) 37 mm (OFX) 12 mm (OFX) 10 mm (OFX) 27 mm (SCF) NT 12 mm (OFX) 30 mm (OFX) 25 mm(SCF) (OFX) 32 mm (OFX) 12 mm (OFX 23 mm (OFX) 18 mm (OFX) NT (OFX) (OFX) 24 mm (NET) (SCF) 12 mm (OFX) 22 mm (NET) 30 mm (OFX) 10 mm (OFX) 24 mm (SCF) 24 mm (OFX) 25 mm (OFX) 27 mm (SCF) 22 mm (SCF) NT NT (OFX) 10 mm (OFX) 20 mm (SCF)

7–17 mm/57 strains of 24 species

NC: negative control (MeOH). OFX: ofloxacin (10 ␮g/disc); SCF: sulbactam(30 ␮g) + cefoperazona (75 ␮g) (105 ␮g/disc); NET: netilmicin (30 ␮g/disc) were used as positive reference standard antibiotic discs (Oxoid); NT: not tested. b


234

Table 2 Antimicrobial activity of Juniperus oxycedrus extracts (300 ␮g/disc) against yeast and fungus isolates tested based on disc-diffusion method Microorganisms

Yeast Candida albicans Fungi Alternaria alternata Aspergillus flavus Fusarium oxysporum Penicillium spp.

Number of isolates

23 2 2 2 2

Total a b

Inhibition zone diameter around test disc Methanol extract

Aqueous extract

NCa

Standard antibiotic discsb

7–15 mm/11

–

–

SCF

– – – –

– – – –

– – – –

NT NT NT NT

31 isolates

7–5 mm/11 isolates

NC: negative control (MeOH). SCF: sulbactam (30 ␮g) + cefoperazona (75 ␮g) (105 ␮g/disc) was used as positive reference standard antibiotic discs (Oxoid); NT: not tested.

The antimicrobial activities of J. oxycedrus methanol and aqueous extracts against microorganisms examined in the present study and their potency were quantitatively assessed by the presence or absence of inhibition zones and zone diameters (Tables 1 and 2), MIC and MBC values (Table 3). The results showed that the methanol extract has inhibition effect on the growth of 11 of 23 yeast (C. albicans) isolates and 57 of 178 strains in 24 bacterial species which were Acinetobacter calcoaceticus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus cereus, Bacillus circulans, Bacillus lentimorbus, Bacillus licheniformis, Bacillus macerans, Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus substilis, Brevundimonas diminuta, Brucella abortus, Enterobacter agglomerans, Enterobacter pyrinus, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas

syringae, Staphylococcus aureus, Staphylococcus epidermidis, and Xanthomonas campestris (Tables 1 and 2). However, the aqueous extract of J. oxycedrus had no antimicrobial activity against any of the bacterial or fungal isolates tested in the present study. Maximal inhibition zones, MIC and MBC values for the microorganisms sensitive to the methanol extract of J. oxycedrus were in the range of 7–17 mm, 31.25–250 ␮g/ml and 62.5–500 ␮g/ml, respectively (Tables 1–3). This is the first study to demonstrate that methanol extract of J. oxycedrus contains antimicrobial substances with antibacterial and anticandidal effects. In addition, these results confirmed the evidence in previous studies reported that methanol is a better solvent for more consistent extraction of antimicrobial substances from medical plants compared to other solvents, such as water, ethanol, and hexane (Ahmad et al., 1998; Eloff, 1998; Lin et al., 1999).

Table 3 The MBC and MIC values of Juniperus oxycedrus methanol extract against the microorganisms tested in microdilution assay Microorganisms

Methanol extract MBC

Methanol extract MIC

Standard drug (maxipime)

Acinetobacter calcoaceticus Bacillus amyloliquefaciens Bacillus cereus Bacillus lentimorbus Bacillus licheniformis Bacillus megaterium Bacillus macerans Bacillus pumilus Bacillus substilis Bacillus sphaericus Brevundimonas diminuta Brucella abortus Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Pseudomonas syringae Pseudomonas putida Staphylococcus aureus Staphylococcus epidermidis Candida albicans

500 250 500 500 250 125 62.5 125 62.5 250 500 500 500 250 500 500 250 500 500 125

250 125 250 250 125 62.5 31.25 62.5 31.25 125 250 125 250 125 125 125 125 250 250 31.25

NT NT 250 NT NT NT NT 31.25 125 NT NT NT 15.62 NT NT 31.25 125 NT NT NT

MBC: minimal bactericidal concentration (␮g/ml); MIC: minimal inhibition concentration (␮g/ml); NT: not tested.


Our data showed that there was no uniform response within or between the bacterial strains of the same species and C. albicans isolates in terms of susceptibility to antimicrobial compounds in the methanol extract of J. oxycedrus (Tables 1 and 2). These kinds of differences in susceptibility among the microorganisms against antimicrobial substances in plant extracts may be explained by the differences in cell wall composition and/or inheritance genes on plasmids that can be easily be transferred among bacterial strains. The results may suggest that methanol extract of the J. oxycedrus possess compounds with antibacterial and anticandidal properties which can be used as antimicrobial agents in new drugs for therapy of infectious diseases References Adams, R.P., 1998. The leaf essential oils and chemotaxonomy of Juniperus sect Juniperus. Biochemical Systematics and Ecology 26, 637–645. Ahmad, I., Mehmood, Z., Mohammad, F., 1998. Screening of some Indian medicinal plants for their antimicrobial properties. Journal of Ethnopharmacology 62, 183–193. Amaral Franco, J., 1964. Juniperus L. In: Tutin et al. (Eds.), Flora Europaea, vol. 1. Cambridge University Press, Cambridge. Amaral Franco, J., 1986. Juniperus L. In: Castroviejo et al. (Eds.), Flora Iberica, vol. 1. Real Jardin Botanico, Madrid. Bello, R., Moreno, L., Beltran, B., Primo-Yufera, E., Esplugues, J., 1997. Effects on arteriol blood pressure of methanol and dichloromethanol extracts from J. oxycedrus L. Phytotherapy Research 11, 161–162.

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Bouhlal, K., Meynadier, J.M., Peyron, J.L., Peyron, L., Marion, J.P., Bonetti, G., Meynadier, J., 1988. Le cade en dermatology. Parfums, Cosmetiques et Aromes 83, 73–82. Duke, J.A., 1985. Handbook of Medicinal Herbs. CRC Press, Boca Raton, FL, p. 256. Eloff, J.N., 1998. Which extract should be used for the screening and isolation of antimicrobial components from plants? Journal of Ethnopharmacology 60, 1–8. Leung, A.Y., Foster, S., 1996. Encyclopedia of Common Natural Ingredients. Wiley, New York, p. 109. Lin, J., Opoku, A.R., Geheeb-Keller, M., Hutchings, A.D., Terblanche, S.E., Jager, A.K., van Staden, J., 1999. Preliminary screening of some traditional Zulu medicinal plants for anti-inflammatory and anti-microbial activities. Journal of Ethnopharmacology 68, 267–274. Moreno, L., Bello, R., Beltran, B., Calatayud, S., Primo-Yufera, E., Esplugues, J., 1998a. Pharmacological screening of different J. oxycedrus L. extracts. Pharmacology and Toxicology 82, 108–112. Moreno, L., Bello, R., Primo-Yufera, E., Esplugues, J., 1998b. In vitro studies of methanol and dichlorometanol extracts of J. oxycedrus L. Phytotherapy Research 11, 309–311. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H., 1995. Manual of Clinical Microbiology, vol. 6. ASM, Washington, DC. Sanchez de Medina, F., Gamez, M.J., Jimenez, I., Jimenez, J., Osuna, J.I., Zarzuelo, A., 1994. Hypoglycemic activity of juniper berries. Planta Medica 60, 197–200. Swanston-Flatt, S.K., Day, C., Bailey, C.J., Flatt, P.R., 1990. Traditional plant treatments for diabets. Studies in normal and streptozotocin diabetic mice. Diabetologia 33, 462–464. WHO Expert Committee on Diabetes Mellitus Second Report, 1980. Technical Report Series 646. WHO, Genova, p. 66. Zgoda, J.R., Porter, J.R., 2001. A convenient microdilution method for screening natural products against bacteria and fungi. Pharmaceutical Biology 39 (3), 221–225.


Effect of T. foenumgraecum on glycogen content of tissues and the key enzymes of carbohydrate metabolism V. Vats, S.P. Yadav, J.K. Grover∗ Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 31 May 2002; received in revised form 20 December 2002; accepted 20 December 2002

Abstract The Indian traditional system of medicine prescribed plant therapies for diseases including diabetes mellitus called madhumeh in Sanskrit. One such plant mentioned in Ayurveda is Trigonella foenumgraecum (FG). In the present study, FG (1 g/kg PO) was assessed for its effect on glycogen levels of insulin dependent (skeletal muscle and liver), insulin independent tissues (kidneys and brain) and enzymes such as glucokinase (GK), hexokinase (HK), and phosphofructokinase (PFK). Administration of FG led to decrease in blood glucose levels by 14.4 and 46.64% on 15th and 30th day of the experiment. Liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics (P < 0.0005) versus normal controls and this alteration in the renal weight (P < 0.0005) but not liver weight was normalized by feeding of FG. Renal glycogen content increased by over 10 folds while hepatic and skeletal muscle glycogen content decreased by 75 and 68% in diabetic controls versus controls and these alteration in glycogen content was partly prevented by FG. Activity of HK, GK and PFK in diabetic controls was 35, 50 and 60% of the controls and FG partially corrected this alteration in PFK, HK and GK. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T. foenumgraecum; Methi; Streptozotocin; Experimental diabetes; Carbohydrate metabolism

1. Introduction It is projected that incidence of diabetes is on rise. Present number of diabetics worldwide is 150 million and this is likely to increase to 300 million or more by the year 2025 (King et al., 1998). Reasons for this rise include increase in sedentary lifestyle, consumption of energy rich diet, obesity, higher life span, etc. (Yajnik, 2001). Though biguanides and sulfonylureas are valuable in treatment of diabetes mellitus, their use is restricted by their limited action, pharmacokinetic properties, secondary failure rates and accompanying side effects (Bailey et al., 1989). Moreover, these therapies only partially compensate for metabolic derangements seen in diabetics and do not necessarily correct the fundamental biochemical lesion (Taylor and Agius, 1988). Nature has been a source of medicinal treatments for thousands of years, and plants-based systems continue to play an essential role in the primary health care of 80% of the world’s underdeveloped and developing countries (King et al., 1998). ∗ Corresponding author. Tel.: +91-11-659-4897 (O)/461-5315 (R); fax: +91-11-686-2663. E-mail address: [email protected] (J.K. Grover).

Biguanides developed from a prototypic plant molecule is an excellent example of anti-diabetic drug development from plants. Thus, it is prudent in the current context to look for new and if possible more efficacious hits from the vast reserves of phytotherapy. Trigonella foenumgraecum Linn (FG) has been shown to possess hypoglycemic activity in experimental animals (Ghafghazi et al., 1977; Ribes et al., 1984, 1986; Swanston-Flatt et al., 1989; Ali et al., 1995; Khosla et al., 1995). However, very little is known about cellular and biochemical mechanism of the hypoglycemic or anti-hyperglycemic effect of FG. Although Raju et al. (2001) have shown that FG favorably effects glycolytic and gluconeogenic enzymes, they have worked with a whole seed powder diet. Since seeds have a very high fiber content, it is not possible to ascertain whether the anti-diabetic effect seen in their study was due to the effect of solid seed contents or some active pharmacological constituents. Therefore, this study was undertaken to assess the effect of FG on key enzymes of carbohydrate metabolism and glycogen content of various tissues. The dose was based on a previous pilot work that was undertaken to establish and assess the dose–response relationship (Vats et al., 2002).


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2. Materials and methods

2.5. Biochemical and enzymatic estimations

2.1. Preliminary phytochemical evaluation

2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak® , Bayer Diagnostics, Baroda).

It yielded moisture content of 10.3%, dry matter 89.7% (fiber 56.2%, protein 18.2%, mineral constituent 2.1%, alkaloidal content 0.011%, fixed oil in traces). The dose was 2 g/kg body weight. 2.2. Animals Male albino rats (150–200 g) were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before initiation and during the experiment, rats were fed standard chow diet. After randomization into various groups, the rats were acclimatized for 2–3 days in the new environment before initiation of experiment. Animals had free access to food and drinking water till before 30 min of sampling.

2.5.2. Hepatic phosphofructokinase activity (EC 2.7.1.11) The liver homogenate was prepared by grinding frozen tissue in ratio of 1:4 in 50 mM imidazole, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl and 30 mM beta-mercaptoethanol (pH = 7). The homogenate was centrifuged at 12,000 × g for 15 min and supernatant was removed and stored in ice until assay. The phosphofructokinase activity was assayed spectrophotometrically by the method of Racker (1947). 2.5.3. Hepatic glucokinase activity The above-mentioned sample was used and enzyme activity was based on spectrophotometric method assaying glucose phosphorylation described by Pilkis (1975).

2.3. Experimental design All the animals were randomly divided into 3 groups with 10 animals in each group. Group I (CNT) were normal and used as controls. Group II (DCNT) were used as diabetic control. Group III was daily treated with FG after 10 days of standing hyperglycemia for 30 days. Streptozotocin (STZ) (Sigma, USA) was administered in Groups II–IV by a single intravenous injection of STZ (65 mg/kg) given intraperitoneally. STZ was first weighed in individual Eppendorf’s according to the weight of the animal, then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Rats exhibiting plasma glucose levels >300 mg/dl, 48 h after administration of STZ were included in the study. They were allowed a window period of 10 days before start of treatment. 2.4. Sample collection 2.4.1. Blood sample It was collected retro-orbitally from the inner canthus of the eye under light ether anesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Blood was collected in fresh vials containing sodium fluoride and sodium oxalate as anti-coagulant/anti-glycolytic agents and plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min. 2.4.2. Collection of organs After 30 days of daily feeding of FG orally (40 days after STZ), the animals were euthanized by overdose of intraperitoneal anesthesia and blood and tissue samples were collected for the assessment of plasma glucose, hepatic glucokinase (EC 2.7.1.1), hexokinase (EC 2.7.1.1), and phosphofructokinase (EC 2.7.1.11) content along with glycogen content in different tissues, i.e. liver, brain, heart and skeletal muscle.

2.5.4. Skeletal hexokinase activity The enzyme activity was determined by the method of Chou and Wilson (1975) and was based on the reduction of NADPH coupled with hexokinase measured spectrophotometrically at 340 nm. 2.5.5. Glycogen content The tissue sample was digested in hot concentrated 30% KOH, precipitated with ethanol, hydrolyzed and finally determined as glucose in the hydrolyzate as reducing sugar (Hassid and Abraham, 1957).

3. Results The basal levels of plasma glucose of the rats in all groups prior to STZ administration were not significantly different. However, 48 h after STZ administration, plasma glucose levels were significantly higher in the rats selected for the study. In contrast, non-diabetic controls remained persistently euglycemic throughout the course of the study. 3.1. Glucose levels The anti-hyperglycemic effect of FG on the blood sugar levels of diabetic rats is shown in Table 1. Controls rats did not show any significant variation in the blood glucose throughout the experimental period. Administration of STZ (65 mg/kg) led to over four-fold elevation of blood glucose levels (P < 0.001) which was maintained over a period of 5 weeks. After 4 weeks of daily treatment with FG led to a fall in blood sugar levels by 14.4 and 46.64% on 15th and 30th day of the experiment, respectively (P < 0.01 and P < 0.0001, respectively).


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Table 1 Effect of FG administration (30 days) on glucose levels (mg/%) in STZ (65 mg/kg) diabetic rats

CNT DCNT FG

0 day

10th day

25th day

40th day

78.76 ± 5.38 (n = 10) 77.90 ± 4.21 (n = 10) 80.87 ± 8.01 (n = 10)

81.29 ± 5.44 (n = 10) 325.43 ± 18.95∗ (n = 8) 322.86 ± 14.45 (n = 10)

82.11 ± 4.81 (n = 10) 318.28 ± 18.76∗ (n = 7) 276.21 ± 34.67∗ (n = 9) (14.44%)

84.39 ± 6.77 (n = 10) 321.53 ± 16.52∗ (n = 6) 172.27 ± 21.63∗∗ (n = 8) (46.64%)

CNT: control; DCNT: diabetic control. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage lowering of plasma sugar in comparison to the previous reading. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with the corresponding values at 48 h. ∗ Statistically significant at P < 0.01. ∗∗ Statistically significant at P < 0.0001.

Table 2 Effect of PM on body, liver and kidney weight in rats

Body weight on 0 day Body weight on 15th day Body weight on 30th day Liver weight on 30th day (g) Liver weight/100 g body weight on 30th day Kidneys weight on 30th day (g) Kidneys weight/100 g body weight on 30th day

CNT

DCNT

FG

163.1 ± 7.88 191.4 ± 9.14 213.2 ± 6.98 7.62 ± 0.68 3.53 ± 0.25 1.54 ± 0.08 0.71 ± 0.02

163.9 ± 10.98 165.7 ± 3.94∗∗∗ 168.7 ± 5.06∗∗∗ 7.45 ± 0.38 4.36 ± 0.16∗∗∗ 1.56 ± 0.04 0.92 ± 0.02∗∗∗

167.7 ± 13.6 175.4 ± 11∗ 189 ± 11∗∗,∗∗∗ 7.95 ± 0.59 4.18 ± 0.15 1.58 ± 0.09 0.84 ± 0.04∗∗

Abbreviations as for Table 1. Values are given as mean ± S.D. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗ Statistically significant at P < 0.05. ∗∗ Statistically significant at P < 0.005. ∗∗∗ Statistically significant at P < 0.0005.

3.2. Body weight, renal and liver weight The effect of STZ and feeding FG on the body weight, renal and liver weight is shown in Table 2. There was no significant intra-group variation in the basal body weight on the 0 day of the experiment. While control rats gained significant weight in the 30-day period, there was no appreciable increase in the diabetic controls over the same period. On the other hand, treated rats gained significantly more weight than diabetic controls but the increase remained significantly lesser than the normal controls. There was no significant difference in the absolute weights of livers and kidneys among any of the experimental groups. However, liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics

(P < 0.0005) versus normal controls and this alteration in the renal weight but not liver weight was significantly reduced (P < 0.005) by feeding FG. 3.3. Glycogen content Glycogen content of various tissues (liver, skeletal muscle, heart, brain and kidneys) was estimated on the 30th day in normal controls, diabetic controls and FG-treated groups (see Table 3). As evident, the glycogen content decreased significantly by 75 and 68% in liver and skeletal muscle in diabetic controls as compared to non-diabetic controls. On the other hand renal glycogen content increased by over 10 folds in diabetic animals versus non-diabetic animals. Brain and heart glycogen content remained unaltered in diabetic

Table 3 Effect of FG administration (30 days) on glycogen content (mg/g tissue) of various tissues of STZ (65 mg/kg) diabetic rats Groups

Brain (mg/100 g)

Kidney (mg/g)

Heart (mg/g)

Muscle (mg/g)

Liver (mg/g)

CNT (n = 10) DCNT (n = 6) FG (n = 8)

40.34 ± 4.76 43.11 ± 6.41 40.33 ± 7.04

0.83 ± 0.22 11.5 ± 2.99∗∗ (↑1280%) 6.95 ± 1.48∗

1.81 ± 0.32 1.85 ± 0.58 1.8 ± 0.65

8.71 ± 1.22 2.81 ± 1.26∗∗ (↓68%) 4.6 ± 1.5∗

52.55 ± 5.22 13.25 ± 3.99∗∗ (↓75%) 30.15 ± 6.76∗∗

Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic control. ∗ Statistically significant at P < 0.005. ∗∗ Statistically significant at P < 0.0005.

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Table 4 Effect of administration (30 days) on levels of three important enzymes (%) involved in carbohydrate metabolism in STZ (65 mg/kg) diabetic rats Enzyme PFK GK HK

CNT (n = 10)

DCNT (n = 6)

FG (n = 8)

100 100 100

59.07∗∗∗

86.13∗∗ 73.20∗∗ 62.33∗∗

48.69∗∗ 35.56∗∗∗

Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗∗ Statistically significant at P < 0.001. ∗∗∗ Statistically significant at P < 0.0001.

animals while FG significantly attenuated the STZ-induced hepatic (P < 0.0005), and muscle (P < 0.005) renal (P < 0.005) glycogen content. 3.4. Hepatic enzymes The diabetic controls showed significant decrease in the values of PFK, GK and HK (see Table 4). The respective percentage decrease was approximately 40, 50 and 65% as compared to non-diabetic control values. As compared to diabetic controls, FG significantly altered these enzymes favorably but could not normalize them to the control values.

4. Discussion The present study was carried out to determine effect of feeding of FG on the key enzymes involved in carbohydrate metabolism in STZ (65 mg/kg) rats and glycogen content of insulin dependent (skeletal muscle and liver) and independent tissues (kidneys and brain). Diabetes mellitus is characterized by partial or total deficiency of insulin resulting in derangement of carbohydrate metabolism and a decrease in enzymatic activity of glucokinase, hexokinase and phosphofructokinase resulting in depletion of liver and muscle glycogen (Murphy and Anderson, 1974). GK and PFK are the most sensitive indicators of the glycolytic pathway in the diabetic state (Steiner and King, 1964). Since insulin administration normalizes these alterations in the enzymatic activities (Weber et al., 1966), these enzymes represent a method to assess peripheral utilization of glucose. In the present case, diabetic rats showed a significant reduction in all the three enzymes in comparison to the normal controls. Liver plays an important role in buffering the postprandial hyperglycemia and is involved in synthesis of glycogen. Diabetes mellitus is known to impair the normal capacity of the liver to synthesize glycogen (Hornbrook, 1970; Migliorini, 1971; Whitton and Hems, 1975). However, after food, diabetic animals fail to synthesize glycogen from glucose and gluconeogenic precursors. Synthase phosphatase activates glycogen synthase resulting in glycogenesis and

this activation appears to be defective in STZ-induced diabetic animals (Bishop, 1970; Tan and Nuttall, 1976; Golden et al., 1979). This inhibition of synthase phosphatase is almost complete after 3 days in alloxan-diabetic rats and in 1–2 weeks in STZ diabetes (Langdon and Curnow, 1983). Therefore, the drug treatment was started 10 days after STZ administration and continued for 4 weeks. STZ-induced diabetic animals tend to show renal hypertrophy. In the present case also, diabetic rats showed 29% increase in 2-kidney versus body weight ratio in comparison to controls. This degree of renal enlargement is lesser than 62 and 52% reported previously (Rasch, 1980; Nielsen et al., 1999). Probably degree of increase in renal weight is a time-dependent phenomenon as duration in the above-mentioned two studies was 6 and 2 months, respectively, as compared to 1 month in the present case. Forty days after STZ injection, GK and PFK levels of the diabetic rats were 48.69 and 59.07%, respectively of the controls and this is similar to previous finding (Steiner and King, 1964) in which both the dose of STZ and duration of diabetes is comparable with the present study. An increase in the activity of glucokinase and PFK in the FG-treated rats implies that cellular entry of glucose was facilitated by FG, which in turn stimulated the activity of these enzymes. This glucose influx could either be due to an insulin releasing or direct insulinomimetic effect of FG. Since STZ diabetes is an insulin deficient model, the probability of insulinomimetic effect seems more probable. Feeding of FG (2 g/kg) for 30 days caused a significant reduction in glucose levels by almost 50% though euglycemia was not achieved. In a recent study (Raju et al., 2001), complete normalization of blood glucose with feeding of 5% diet of powdered FG seeds was reported, a reduction that was not seen in the present study. This could be attributed to the presence of high fiber (>90%) and saponin content in the seeds of FG, which could have contributed partly to euglycemia in their study (Moorthy et al., 1989; Sauvaire et al., 1996; Buyken et al., 1999). Since we had used defatted extract (which is devoid of fiber content), it is possible that lack of fibers contributed to the lack of euglycemia. Moreover, in a previous study (Vats et al., 2002), we had shown that feeding single dose FG extract did not affect peak glucose levels in glucose-fed hyperglycemic rats implying that FG extract does not affect glucose absorption from the gut. STZ induced diabetes is characterized by severe loss in body weight (Chen and Ianuzzo, 1982; Raju et al., 2001) and this was also seen in the present study. While the normal rats registered approximately 30% growth in body weight diabetic rats showed no gain. FG prevented this loss in body weight significantly. However, it did not normalize the body weight completely as it remained significantly lesser than normal controls. Assessment of glycogen levels serves as a marker for studying insulinomimetic activity. Glycogen content of skeletal muscle and liver markedly decreases in diabetes and this alteration is normalized by insulin treatment (Prasannan


and Subrahmanyam, 1965; Welihinda and Karunanayake, 1986; Grover et al., 2000). In the diabetic controls after 5 weeks of STZ administration, glycogen content in liver and muscle was reduced by approximately 75 and 68%, respectively, in comparison to the non-diabetic controls. This is in line with previous studies utilizing similar dose of STZ and carried for approximately same period (Khandelwal et al., 1977; Ferrannini et al., 1990) except from the study of Chen and Ianuzzo (1982) who reported lesser reduction in hepatic glycogen content though the duration and dose of diabetogenic agent was comparable to the present study. Treatment with FG for 30 days significantly increased the glycogen content by approximately 50% in muscle and 120% in the liver but lesser than the normal controls indicating that the defective glycogen storage of the diabetic state was only partially corrected by the herb. The glycogen content of insulin-dependent tissues is known to decrease in STZ diabetes but the opposite happens in insulin-independent tissues like kidneys (Belfiore et al., 1986). In the present study, renal glycogen content of diabetic rats was elevated by almost 13-folds in comparison to normal controls and is likely due to increased glycogen deposition. This is in agreement with earlier findings (Ross and Goldman, 1971; Spiro and Spiro, 1971). Feeding of FG failed to normalize renal glycogen deposition though it significantly reduced its levels. This may be due to failure in achieving euglycemia by the FG extract as glucose levels on the 30th day were still higher in comparison to the normal controls. In the present study, brain glycogen levels were not affected either in diabetic controls or in the treated groups and this in agreement with our earlier findings (Grover et al., 2000) though the duration of the work in the present case was longer. Diabetic rats had significantly higher liver weight/100 g body weight and this alteration in the diabetics was not altered by treatment with FG. Literature shows conflicting reports on the effect of diabetes on liver weight. While some report an increase in liver weight in animals (Murphy and Anderson, 1974; Chen and Ianuzzo, 1982; Sadique et al., 1987) as well as humans (Van Lancker, 1976), no change in liver weight and decrease (alloxan 200 mg/kg induced diabetes, study duration 21 days) has also been reported (Gupta et al., 1999; Raju et al., 2001). Exact reasons of hepatic hypertrophy are not known. However, fat deposition has been proposed to be the cause (Van Lancker, 1976). Several probable mechanisms have been suggested to explain the mechanism of action of FG. These include insulin secretion (Riyad et al., 1988; Khosla et al., 1995), insulinomimetic activity or inhibition of intestinal glucosidase (Petit et al., 1993). The current study provides some useful insight into the molecular effect of feeding FG. However, we suggest that further work should be carried out with whole seed powder and not their extracts. FG is widely distributed throughout the country and people commonly use leaves and seeds as an edible item implies a relative lack of toxicity. Moreover, leaves have also been

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shown to have an anti-hyperglycemic effect (Abdel-Barry et al., 1997, 2000). These findings suggest that consumption of FG seeds and leaves should be promoted as a constituent of diet in higher proportion in diabetic patients as well as those prone to getting diabetes.

References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H., 1997. Hypoglycaemic and antihyperglycaemic effects of Trigonella foenumgraecum leaf in normal and alloxan induced diabetic rats. Journal of Ethnopharmacology 58, 149–155. Abdel-Barry, J.A., Abdel-Hassan, I.A., Jawad, A.M., Al-Hakiem, M.H., 2000. Hypoglycaemic effect of aqueous extract of the leaves of Trigonella foenumgraecum in healthy volunteers. Eastern Mediterranean Health Journal 6, 83–88. Ali, L., Khan, A., Hassan, Z., Mosihuzzaman, M., Nahar, N., Nasreen, T., Nur-e-Alam, M., Rokeya, B., 1995. Characterization of hypoglycemic effects of Trigonella foenumgraecum seed. Planta Medica 61, 358–360. Bailey, C.J., Flatt, P.R., Marks, V., 1989. Drugs inducing hypoglycemia. Pharmacology Therapeutics 42, 361–384. Belfiore, F., Rabuazzo, A.M., Iannello, S., Campione, R., Vasta, D., 1986. Anabolic response of some tissues to diabetes. Biochemical Medicine and Metabolic Biology 35, 149–155. Bishop, J.S., 1970. Inability of insulin to activate liver glycogen transferase D phosphatase in the diabetic pancreatectomized dog. Biochimica et Biophysica Acta 208, 208–218. Buyken, A.E., Toeller, M., Heitkamp, G., Vitelli, F., Stehle, P., Scherbaum, W.A., Fuller, J.H., 1999. Relation of fiber intake to HbA1c and the prevalence of severe ketoacidosis and severe hypoglycemia. EURODIAB IDDM complications study group. Diabetologia 41, 882–890. Chen, V., Ianuzzo, C.D., 1982. Dosage effect of streptozotocin on rat tissue enzyme activities and glycogen concentration. Canadian Journal of Physiology and Pharmacology 60, 1251–1256. Chou, A.C., Wilson, J.E., 1975. Carbohydrate metabolism. In: Wood, W.A. (Ed.), Methods in Enzymology, vol. XLII. Academic Press, New York, USA, pp. 20–21. Ferrannini, E., Lanfranchi, A., Rohner-Jeanrenaud, F., Van de Werve, G., 1990. Influence of long-term diabetes on liver glycogen metabolism in the rat. Metabolism 39, 1082–1088. Ghafghazi, T., Sheriat, H.S., Dastmalchi, T., Barnett, R.C., 1977. Antagonism of cadmium and alloxan-induced hyperglycemia in rats by Trigonella foenumgraecum. Pahlavi Medical Journal 8, 14–25. Golden, S., Wals, P.A., Okajima, F., Katz, J., 1979. Glycogen synthesis by hepatocytes from diabetic rats. Biochemistry Journal 182, 727–734. Grover, J.K., Vats, V., Rathi, S.S., 2000. Anti-hyperglycemic effects of Eugenia jambolana and Tinospora cordifolia in experimental diabetes and their effects on key metabolic enzymes involved in carbohydrate metabolism. Journal of Ethnopharmacology 73, 461–470. Gupta, D., Raju, J., Prakash, J., Baquer, N.Z., 1999. Change in the lipid profile, lipogenic and related enzymes in the livers of experimental diabetic rats: effects of insulin and vanadate. Diabetes Research and Clinical Practice 46, 1–7. Hassid, W.Z., Abraham, S., 1957. Chemical procedures for analysis of polysaccharides. Methods in Enzymology, vol. III. Academic Press, New York, USA, pp. 34–37. Hornbrook, K.R., 1970. Synthesis of liver glycogen in starved alloxan diabetic rats. Diabetes 19, 916–923. Khandelwal, R.L., Zinman, S.M., Zebrowski, E.J., 1977. The effect of streptozotocin induced diabetes and of insulin supplementation on glycogen metabolism in rat liver. Biochemistry Journal 168, 541–548. Khosla, P., Gupta, D.D., Nagpal, R.K., 1995. Effect of Trigonella foenumgraecum (fenugreek) on blood glucose in normal and diabetic rats. Indian Journal of Physiology and Pharmacology 39, 173–174.

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King, H., Aubert, R.E., Herman, W.H., 1998. Global burden of diabetes, 1995–2025: prevalence, numerical estimates, and projections. Diabetes Care 21, 1414–1431. Langdon, D.R., Curnow, R.I., 1983. Impaired glycogenic substrate activation of glycogen synthase is associated with depressed synthase phosphatase activity in diabetic rat liver. Diabetes 32, 1134–1140. Migliorini, R.H., 1971. Early changes in the levels of liver glycolytic enzymes after total pancreatectomy in the rat. Biochimica et Biophysica Acta 244, 125–128. Moorthy, R., Prabhu, K.M., Murthy, P.S., 1989. Studies on the isolation and effect of orally active hypoglycemic principle from the seeds of fenugreek (Trigonella foenumgraecum). Diabetes Bulletin 9, 69–72. Murphy, E.D., Anderson, J.W., 1974. Tissue glycolytic and gluconeogenic enzyme activities in mildly and moderately diabetic rats. Influence of tolbutamide administration. Endocrinology 94, 27–34. Nielsen, B., Gronbaek, H., Osterby, R., Orskov, H., Flyvbjerg, A., 1999. The calcium channel blocker nitrendipine attenuates renal and glomerular hypertrophy in diabetic rats. Experimental Nephrology 7, 242–250. Petit, P., Sauvaire, Y., Ponsin, G., Manteghetti, M., Fave, A., Ribes, G., 1993. Effect of fenugreek seed extract on feeding behaviour in the rat: metabolic endocrine correlates. Pharmacology, Biochemistry and Behavior 45, 369–374. Pilkis, S.J., 1975. Carbohydrate metabolism. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, vol. XLII. Academic Press, New York, USA, pp. 31–32. Prasannan, K.G., Subrahmanyam, K., 1965. Effects of insulin on the glycogen synthesis in vivo in the brain and liver of rats under different conditions. Indian Journal of Medical Research 53, 1003. Racker, E., 1947. Spectrophotometric measurement of hexokinase and phosphohexokinase activity. Journal of Biology and Chemistry 167, 843–854. Raju, J., Gupta, D., Rao, A.R., Yadava, P.K., Baquer, N.Z., 2001. Trigonella foenumgraecum (fenugreek) seed powder improves glucose homeostasis in alloxan diabetic rat tissues by reversing the altered glycolytic, gluconeogenic and lipogenic enzymes. Molecular and Cellular Biochemistry 224, 45–51. Rasch, K., 1980. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Albumin secretion. Diabetologia 18, 413–416. Ribes, G., Sauvaire, Y., Baccou, J.C., Valette, G., Chenon, D., Trimble, E.R., Loubatieres-Mariani, M.M., 1984. Effects of fenugreek seeds on endocrine pancreatic secretions in dogs. Annals of Nutrition Metabolism 28, 37–43. Ribes, G., Sauvaire, Y., Da Costa, C., Baccou, J.C., Loubatieres-Mariani, M.M., 1986. Antidiabetic effects of subfractions from fenugreek seeds in diabetic dogs. Proceedings of Society of Experimental Biology and Medicine 182, 159–166.

Riyad, M.A., Abdul-Salam, S.A., Mohammad, S.S., 1988. Effect of fenugreek and lupine seeds on the development of experimental diabetes in rats. Planta Medica 54, 286–290. Ross, J., Goldman, J.K., 1971. Effect of streptozotocin-induced diabetes on kidney weight and compensatory hypertrophy in the rat. Endocrinology 88, 1079–1082. Sadique, J., Begum, V.H., Thenmozhi, V., Elango, V., 1987. Hypoglycemic effect of cottonseed aqueous extract in alloxan induced diabetes mellitus in rats. Biochemical Medicine and Metabolic Biology 38, 104–110. Sauvaire, Y., Baissae, Y., Leconte, O., Petit, P., Ribes, G., 1996. Steroid saponins from fenugreek and some of their biological properties. Advances in Experimental Medicine and Biology 405, 37–46. Spiro, R.G., Spiro, M.J., 1971. Effect of diabetes on the biosynthesis of the renal glomerular basement membrane. Studies on the glucosyltransferase. Diabetes 20, 641–648. Steiner, D.F., King, J., 1964. Induced synthesis of hepatic uridine diphosphate glucose glycogen glycosyltransferase after administration of insulin to alloxan diabetic rats. Journal of Biology and Chemistry 239, 1292–1298. Swanston-Flatt, S.K., Day, C., Flatt, P.R., Gould, B.J., Bailey, C.J., 1989. Glycemic effect of traditional European plants treatments for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetes Research 10, 69–73. Tan, A.W., Nuttall, F.Q., 1976. Regulation of synthase phosphatase and phosphorylase in rat liver. Biochimica et Biophysica Acta 445, 118– 130. Taylor, R., Agius, L., 1988. The biochemistry of diabetes. Biochemistry Journal 250, 650–740. Trinder, P., 1969. Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology 22, 158–161. Van Lancker, J.L., 1976. Molecular and Cellular Mechanism in Diseases. Springer Verlag, New York, USA, p. 497. Vats, V., Grover, J.K., Rathi, S.S., 2002. Evaluation of antihyperglycemic and hypoglycemic effect of Trigonella foenumgraecum, Ocimum sanctum and Pterocarpus marsupium in normal and alloxanized diabetic rats. Journal of Ethnopharmacology 79, 95–100. Weber, G., Lea, M.A., Fisher, E.A., Stamm, N.B., 1966. Regulatory pattern of liver carbohydrate metabolizing enzymes insulin as an inducer of key glycolytic enzymes. Enzymology Biology Clinics (Basel) 7, 11–24. Welihinda, J., Karunanayake, E.H., 1986. Extra-pancreatic effects of Momordica charantia in rats. Journal of Ethnopharmacology 17, 247–255. Whitton, P.D., Hems, D.A., 1975. Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats. Biochemistry Journal 150, 153–165. Yajnik, C.S., 2001. The insulin resistance epidemic in India: fetal origins, later lifestyle, or both? Nutrition Reviews 59, 1–9.


Ethnomedical field study in northern Peruvian Andes with particular reference to divination practices Vincenzo De Feo∗ Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo, I-84084 Fisciano (Salerno), Italy Received 8 July 2002; received in revised form 20 December 2002; accepted 20 December 2002 In memory of Dr. Richard Evans Schultes.

Abstract The results of a field study carried out in August and September 1988, in the northern Peruvian Andes are described. The area of investigation extends from Ayabaca City (about 2900 m above sea level) to Haughtiness Lagunas (about 3800 m above sea level) in the Ayabaca District, Department of Piura. This is the first time that this location has been the subject of an ethnobotanical investigation. We have collected 46 plant species, belonging to 20 families, used in the treatment of various diseases. For each plant, we report the common/local names, the crude drug formulation, method of preparation, dosage and claimed toxicity. The disease concept of this Andean population concerning the “hot” and “cold” aspects of diseases and the plants to treat them, is also discussed. Very important appear to be the use and knowledge of psychoactive plants, in particular “cimoras,” Brugmansia and Trichocereus species. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Medicinal plants; Traditional medicine; Northern Peruvian Andes; Hallucinogens

1. Introduction The use of medicinal plants has always been a part of human heritage. Over the centuries, every population has developed its knowledge in recognizing, harvesting, and using plants to cure infirmities. One can still find this situation in communities that are culturally and geographically isolated, where it is difficult or impossible to find medical doctors who practice “official” medicine, and in those countries still economically emerging, where there are very few medical and social facilities due to the limitation of economic factors. In these areas, the treatment of diseases is based essentially, and sometimes exclusively, on medicines that have a natural origin; among these, vegetal drugs constitute the majority. The recognition and the use of medicinal plants is an untouchable heritage of most preliterate cultures. Therefore, in the past centuries, and presently in some cultures, the practice of using plants for medicine has assumed a “sacred” characteristic: it is secretly kept and conveyed by priests and other religious figures, who are very knowledgeable about ∗ Tel.:

+39-089-962824; fax: +39-089-962828. E-mail address: [email protected] (V. De Feo).

herbs and who combine their botanical, phytotherapeutical and toxicological knowledge with religious elements and rituals based on magic, superstition and ancestral beliefs (De Feo, 1992). In rural communities of the Andes, the herbalist or “curandero,” the individual who is knowledgeable about all healing and harmful plants, assumes a primary role. He is considered a priest, an intermediate figure between our world and the world of the spiritual forces. At the same time he is also a therapist and an expert on all healing plants, psychotropic plants (used to awaken religious spirits or to gain an altered state of mind) and harmful plants (Polia, 1988). There is a daily contact between the priest and the plant world, from which he receives most of his remedies; hence, his power over the rest of the community. The shamanic culture in the Andean area of Peru is very old. Its origins certainly predate the Columbian eras and, since then, have been enriched by continuous intercultural and interethnic relationships. In relatively recent times, it has also been enriched by the Spanish and the other European contacts and by academic medicine. Still today, this culture is alive and often represents the only medical practice that a population can refer to daily. This makes the “curandero”


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the only medical doctor available to the Andean population to treat illnesses. This research has been conducted in a high-altitude lake area, that stretches from the City of Ayabaca (about 2900 m above sea level) to the Prieta lake, in northern Peru. It is important to underline that this area is a sacred zone, claimed to be very effective for therapeutic-magic rituals, due to the presence of particularly strong spiritual forces (Polia, 1988; De Feo, 1992). The extension of the area studied is about 20 km2 and is situated between 79◦ 38◦ and 79◦ 28◦ west longitudes and between 4◦ 42◦ and 4◦ 46◦ south latitudes. The vegetation cover is commonly known as “Ceja de la montaña” and is characteristic of inland Cordilleras; it is made up primarily of bamboos and ferns and extends all the way up to 3700–4000 m. In the specific area of our study, however, the vegetation is somewhat different because most of the year the area is under fog banks and because there are many lagoons, near which small swamps are often found (Weberbauer, 1911). However, in the area of our study the vegetation is similar to that of humid forest in the lower tropical mountains with bushes, small evergreen trees, epiphtyes and herbaceous plants (Ferreyra, 1960; Tosi, 1960; Weber, 1969; Schnell, 1987). The principal and typical species found in this vegetation are Weinmannia ayavacensis (Cunoniaceae), Streptosolen jamesonii (Benth.) Miers (Solanaceae), Viburnum ayavacense HBK. (Caprifoliaceae), Monnina salicifolia R. et P. (Polygalaceae), Bomarea ayavacensis Kränzl (Amaryllidaceae), Oreopanax sp. (Araliaceae), Hesperomeles lanuginosa R. et P. (Rosaceae), Oreocallis grandiflora (Lam.) R. Br. (Proteaceae), and species belonging mainly to the families Melastomataceae, Ericaceae, Gentianaceae and Asteraceae.

2. Methodology Information was gathered through interviews with the population in the study area, chiefly with the “curanderos,” and through participation in the harvesting, preparation and administration of the vegetal drug in therapy. Much importance and care were given to the recording of data, in order to assure the separation of cultural-anthropological information from ethnobotanical data. A set of voucher herbarium specimens (identified by initials and listed in parentheses following common names in this paper) has been deposited in the herbarium of the Museo de Historia Natural “J. Prado” of the Universidad Nacional Mayor de San Marcos in Lima, while another set is deposited in the Pharmaceutical Botany Chair at the University of Salerno, Italy. Taxonomic determinations were performed at the herbarium of the Museo de Historia Natural, Lima, with the help of MacBride’s Flora of Peru (1938–1981) and Ferreyra’s Flora del Peru—Dicotiledoneas (1986) and by comparison with the rich collections of exiccata of this herbarium and that of the Facultad de Farmacia y Bioqu´ımica, Universidad Nacional Mayor de San Marcos, Lima.

3. Results Forty-six plants belonging to 20 families were collected during the field study. A list of these plants (alphabetical order by family) is presented below. For each of these plants, the following information is given: the botanical name, the local name, coded voucher specimen number, and observations on the prescription and the dosage form of the crude drug preparations.

Amaranthaceae I. herbstii Hook. “Cimora señorita” (DF/P/88/33) The leaves, whole or ground, are used externally against eczemas, sores and pimples. A cataplasm made from the leaf decoction (approximately 30 g in 1 l of water) is also used in treatment of these ailments. The plaster is applied externally for one night and its effects are interrupted, the next morning, with the “arranque” (see Tibouchina longifolia, Melastomataceae). In the Cujate District (about 3200 m above sea level) the plant is known as “Sangurache negro” and the leaf and flower decoction (about 50 g in 1 l of water) is used as an antipyretic, in a dosage of 2 cups a day. Anacardiaceae Schinus molle L. “Molle” (DF/P/88/49) A leaf and fruit decoction (about 40 g in 1 l of water) is prescribed for oral administration, 2–3 cups a day, to treat respiratory disorders and rheumatism. Externally, the same decoction is prescribed as a hot cataplasm or liniment. Asteraceae Aristiguietia pseudarborea (Hieron.) K. et R. “Pajaro bobo” (DF/P/88/42) A leaf decoction (about 40 g in 1 l of water) is used as a vaginal douche in case of infections, inflammations and white secretions. The same decoction is used as a remedy in treatment of hepatic inflammations, and as an antipyretic.


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Chuquiraga jussieui J.F. Gmelin “Chiquiragua” (DF/P/88/4) A preparation made by soaking the leaves and flowers in a woman’s or lamb’s milk is drawn-in through the nasal mucosa to cure hepatic disorders. Externally, the shredded plant is applied to skin eruptions caused by metabolic and hepatic disorders. Diplostephium foliosissimum Blake “Poleo del Inca” (DF/P/88/8) An infusion of the whole plant (about 40 g in 1 l of water, 2–3 cups a day or more) is drunk to calm intestinal and gastric pains. An alcoholic tincture of the whole plant (about 50 g in 1 l of alcohol for 1 h, 20–30 drops a day) is filtered and used to treat hypotension and systemic debilitation. Gynoxys sp. “Congona” (DF/P788/9) A leaf tincture (approximately 100 g in 1 l of alcohol) is used externally to massage the whole body in case of low blood pressure, psychological depression, and sight and respiratory problems. This tincture can be also applied through the nasal mucous membrane, a method called “shingada.” For the same complaints, 2–3 cups of leaf infusion (about 50 g in 1 l of hot water) can be administered. The plant tincture and infusion are also prescribed as sedatives for colic pains, along with “Cola de caballo” [Equisetum bogotense HBK. or E. giganteum L.] and “Grama dulce” [Cynodon dactylon (L.) Pers.]. G. oleifolia Muschler “Congona grande” (DF/P/88/12) The plant is used to regulate arterial pressure and to treat respiratory problems. A decoction is prepared by boiling approximately 50 g of leaves in 1 l of water for 15 min; the dosage is 2 cup a day by mouth. For the same disorders, others prescribe a more concentrated decoction (about 100 g of leaves in 1 l of water) externally, by massage and friction on the whole body. Loricaria sp. 1 “Cacho de venado” (DF/P/88/6) The alcoholic leaf and branch tincture is employed externally to treat low blood pressure, by massaging energetically on the whole body. The same tincture is used orally as an expectorant, mucolytic and antipyretic (2–3 small cups a day). Loricaria sp. 2 “Corona de Cristo” (DF/P/88/5) The upper parts of the plant are soaked in plenty of sugared water and employed to treat physical and psychological weakness and anemia (2–3 cups a day). The same preparation and dosage are prescribed in cases of menstrual irregularities. Salmea scandens (L.) DC. “Huayme-huayme” (DF/P/88/48) This plant is claimed to be effective in the treatment of female sterility: 2–3 cups a day of a fresh leaf infusion (about 50 g in 1 l of water) are administered by mouth. Senecio elatus HBK. “Hornamo amarillo” (DF/P/88/16) This plant is claimed to cause hallucinations and is used in preparations together with “San Pedro” (Trichocerues pachanoi Britt. & Rose or Thevetia peruvianus Britt. & Rose). An infusion of its aerial parts is used as a strong ritual purge. S. ericaefolius Benth. “Romerillo” (DF/P/88/1) The plant is used as a general tonic to ease weakness and as a cardiotonic. The upper parts of the plant are soaked in fresh water; the dosage is 3 cups a day, sweetened with white sugar or honey. Boraginaceae Myosotis sp. “Buena esperanza” (DF/P/88/46) A whole plant infusion (30–40 g in 1 l of water) is used as a general tonic in cases of physical debility (2–3 cups a day).

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Bromeliaceae Puya sp. “Achupalla del oso” A fresh leaf decoction (about 30 g in 1 l of water, 2–3 cups a day) is given orally to treat menstrual irregularities. The orange sap that exudes from the leaf base is used to treat stomach acidity and in cases of inflammation. Cactaceae T. pachanoi Britt. & Rose “San Pedro,” “Achuma” “Huachuma” This is the most important plant is Andean shamanism. It is the object of special ceremonies at crop time at the time of preparing the drug. It has hallucinating properties (Schultes and Hofmann, 1973) and is utilized in rituals. A decoction is made by placing rounded pieces of stem or trunk in water (about 200 g in 1 l of water) and is boiled for several hours, until the volume of water is reduced to 1/4 of the initial volume. The dosage by mouth is 3 cups of cooled decoction during the ritual (“mesada”) and may be given only by the “curandero.” Trichocereus peruvianus Britt. & Rose (“San Pedro” or “Aguacolla” or “Gigantón” or “Huando”) has a similar use and is claimed to produce the same effect. When a preparation of “San Pedro” is ingested, the next day the individual is prohibited from drinking alcoholic beverages. Caprifoliaceae Sambucus sp. “Tilo” (DF/P/88/35) The leaf and flower decoction (about 30 g in 1 l of water) is prescribed as an expectorant and mucolytic (3 cups a day) in cases of colds, bronchitis and all respiratory problems. Clusiaceae Hypericum strictum HBK. “Poleo verde” (DF/P/88/2) A decoction of the plant tops in half-a-liter of water is taken in cases of anemia. One cup of a well sweetened decoction is taken by mouth, twice a day, morning and night. Ericaceae Befaria cinnamomea Lindl. “Payama” (DF/P/88/19) A leaf and flower decoction is used as a vaginal douche, apparently to act as an antiseptic. The leaf and flower infusion, however, is taken orally (2–3 cups a day) for menstrual irregularities. Gentianaceae Halenia umbellata (R. et P.) Gilg “San Juan amarillo” (DF/P/88/13) A whole plant decoction (about 30 g in 1 l of water, boiled for 15 min, sweetened with honey) is valued in pediatrics as an anthelmintic. Gentianella sp. “Huancoya” (DF/P/88/18) The macerated obtained by soaking the whole plant in cold water for 1 h is used to treat diseases of domestic animals as a general tonic: externally, it is applied as washings and baths and internally given at a dose of 2–3 big cups a day. G. bicolor (Wedd.) Fabris “San Juan negro” (DF/P/88/17) The plant decoction (about 30 g in 1 l of water, boiled for 15 min) is used as an anthelmintic at a dose of 3 cups a day. Lamiaceae Coleus blumei Benth. “Cimorilla,” “Timorilla” (DF/P/88/24) The shredded leaves are employed topically on inflamed areas, either of traumatic or rheumatic origin. This plant is claimed to be very toxic, therefore its internal use is prohibited: its effect is usually interrupted by the “arranque” (see T. longifolia, Melastomataceae).


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S. sagittata R. et P. “Cahuina,” “Suburón” (DF/P/88/40) An alcoholic tincture of the whole plant is used as a massage in cases of rheumatic pains. The infusion (about 20 g of plant tops in 1 l of water, 2–3 cups a day) is taken to relieve headaches and gastric pains. Lycopodiaceae Huperzia sp. 1 “Cabello del bosque” (DF/P/88/37) The juice obtained from pounded fresh plant is applied as a rub on the scalp to strengthen the hair and help its growth. Huperzia sp. 2 “Huaminga” (DF/P/88/7) About 50 g of the whole plant is ground and placed in 1 l of boiling water, until the volume of water is reduced to one-fourth its initial volume; once cooled, a cup of this decoction is injested as a drastic purgative. A less concentrated decoction is employed as a vermifuge, the dosage being 2–3 cups a day for 2 days. Melastomataceae Miconia alypifolia Naud. “Hierba del susto” (DF/P/88/20) A leaf decoction (about 50 g in 1 l of water) is taken as a general tonic and for the treatment of respiratory disorders. The dosage is 2–3 cups a day orally. At the same time, the breast and shoulders may be rubbed with the same decoction. T. cymosa Cogn. “Huishco,” “Flor de gallinazo” (DF/P/88/45) A flower and leaf decoction is used as an emetic, especially in cases of poisoning. The dosage is 1 cup a day. T. longifolia (Vahl) Baill. “Palo del susto,” “Palo del espanto” (DF/P/88/10) This plant is prescribed for the treatment of a condition called “susto” or “espanto,” a complex pathology in which psychosomatic problems, originating from phobic factors, produce profound biological and psychological weakness. The leaves are soaked in water (approximately 50 g in 1 l) and then filtered. The dosage is 2–3 cups a day for a week along with a strictly vegetarian diet. After this period, one has to interrupt the plant’s effect (“cortar”) by drinking a mixture of water with corn, sugar, drops of “Limón agrio” [Citrus aurantifolia (Christm.) Swingle] and white rose petals. This preparation is called “corte” or “arranque.” In the treatment of respiratory disorders, 2–3 cups a day of a leaf decoction (approximately 30 g in 1 l of water) are taken. Externally, the leaves are used as a vulnerary. Onagraceae Ludwigia peruviana (L.) Hara “Arirumba amarilla” (DF/P/88/44) A whole plant decoction (about 30 g in 1 l of water) is prescribed (2–3 cups a day) to treat inflammations of the gastric mucosa and in treatment of headaches. Piperaceae Piper acutifolium R. et P. “Matico,” “Hierba del soldado” (DF/P/88/38) A decoction of the fresh leaves (about 50 g in 1 l of water) is employed as a wash (antiseptic) for sores and wounds and in douches for vaginal infections. Orally, the decoction, in a dosage of 2 cups a day, is indicated in the treatment of gastritis and menstrual irregularities. Polypodiaceae Pteridium aquilinum Kühn in Decken “Aragaya” (DF/P/88/52) This plant is valued as a vermifuge and as an antipyretic. Pityrogramma calomelanos Link “Gara gara” (DF/P/88/51) The tincture of the leafy branches (approximately 30 g in 1 l of alcohol, for 15 days) is used as a wash (antiseptic) and as a vulnerary for recalcitrant wounds and sores.

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Rosaceae H. lanuginosa R. et P. “Lancillo” (DF/P/88/15) A concentrated leaf decoction (about 200 g in 1 l of water) is used externally in the treatment of pulmonary disorders, by application to the shoulders. Orally, a hot leaf decoction (about 50 g in 1 l of water, then wine added and allowed to boil for 15 min) is prescribed orally to treat the same ailments. The dosage is 2–3 cups a day. Rubiaceae Arcytophyllum nitidum (HBK.) Schlecht “Hierba de la estrella,” “Trencilla de plata,” “Trencilla de la señorita” (DF/P/88/3) The whole plant is soaked in water and filtered; this preparation is prescribed for eye washes in cases of disturbed vision. Solanaceae Brugmansia arborea (L.) Lagerheim “Misha león” (DF/P/88/32) The fresh leaves or their alcoholic tincture (approximately 20 g in 1 l of alcohol) are used as a vulnerary and to cure pimples and other skin eruptions. It is claimed to be toxic if ingested and is considered the strongest “Misha” (Brugmansia spp.). The plant, like other species of Brugmansia, is often grown in the sacred gardens of the “curanderos.” It is also known as “Misha oso” (DF/P/88/25). The leaves, whole or shredded, are also valued externally, by applying them to aching areas, in cases of rheumatic inflammations or other traumas. B. arborea (L.) Lagerheim hybrid “Misha galga” (DF/P/88/23) This plant is used externally to relieve pains, in cases of traumatic and rheumatic inflammations. The effect of this and all other “mishas” can be interrupted by using the “arranque” or “corte,” which may be given orally or externally by rubbing on affected body parts. This plant is also claimed to have hallucinating properties. It is often grown around the city of Ayabaca and is known as “Misha oso” (DF/P/88/31). The vapors of the leaf decoction are a used as a vaginal cleanser (antiseptic). The plant is claimed to be toxic if ingested. B. aurea Lagerheim “Misha galga” (DF/P/88/28) The leaves, applied externally on aching body parts, are claimed to relieve pains. It is also used to treat headaches, by absorption of the tincture (made of 2 flowers and 1 leaf in 1 l of alcohol) through the nasal mucosa and at the same time by rubbing the head and limbs with the same preparation. The plant is considered as one of most potent “mishas.” Because of its claimed high toxicity, the plant is rarely used. B. candida (Pers.) Safford hybrid “Misha curandera” (DF/P/88/26) The fresh leaves and the tincture (about 100 g of leaves and branches in 1 l of alcohol, for 8 days) are used. The plant is valued as an analgesic against traumatic or rheumatic pains. The fresh leaves are placed on the aching part of the body for 8 days, changing them every so often to keep the heated part moist (when dry, the leaves lose most of their curative properties). The tincture is also used to relieve headaches by absorption through the nasal mucosa (“shingada”) and simultaneously by rubbing the head and limbs with the same preparation; these administrations are to repeated three times a day for 3 consecutive days. B. insignis (Barb.Rodr.) Lockwood hybrid “Misha rastrera” (DF/P/88/27) The leaves, whole or shredded, are used to relieve pains in cases of traumatic or rheumatic suffering, by applying them locally on the aching part. The leaf tincture is used magically only by the “curandero” to find lost things (“rastrear”) and, in some cases, to create an altered state of mind. It is claimed that one can get the same effect by drinking a “San Pedro” decoction and cross-tying 2 leaves of this “misha” on the back of the head. The plant’s effect may be interrupted by the “arranque,” but it may not be taken internally, because of its toxicity. B. sanguinea (R. et P.) D. Don “Misha toro” (DF/P/88/22)


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This plant is used externally (toxic internally) to relieve pains, especially in cases of arthritic inflammations and cramps. The ground leaves are wetted with alcohol or perfume and applied on the affected area overnight. Following this application, one must stay on a 5-day diet without fats or meat. In addition, during this period, the patient should stay out of water (keep dry). The pulverized leaves are used as a vulnerary on sores and wounds. The tincture (2 leaves soaked in 1 l of alcohol, for 15 days) is claimed to have hallucinogenic effects when absorbed through the nasal mucosa (“shingada”). Some “curanderos” call this plant “Misha toro curandera” (DF/P/88/30) and use it during the ritualistic ceremonies to help in divination. B. suaveolens (Willd.) Bercht. et Presl “Misha colambo” (DF/P/88/29) The leaves, whole or shredded, sometimes mixed with tobacco leaves (“Tabaco” = Nicotiana tabacum L.; “Tabaco cimarr o´ n” = N. paniculata L.), are used as a vulnerary for sores, ulcers and wounds that wouldn’t heal. The leaf decoction (approximately 100 g in 1 l of water, boiled for 30 min until the preparation becomes green) is used externally in cataplasms as an anti-inflammatory on traumatized body parts. The vapors of this decoction are used as a vaginal cleanser (antiseptic) in cases of dysmenhorrea and white secretions. The plant is claimed to be toxic if ingested. B. versicolor Lagerheim “Misha del Inca” (DF/P/88/34) It is the only “misha” taken internally. One cup of the tincture (2 flowers and 1 leaf in 1 l of alcohol) is prescribed as a sedative and general analgesic at bed time. It is claimed to have hallucinogenic properties; therefore, its effects have to be stopped in the morning by the “arranque.” The whole plant or the shredded leaves are applied locally, in cases of muscular pains of traumatic or rheumatic origin. Solanum oblongifolium HBK. var. soukupii Macbr. “Tululuche” (DF/P/88/39) Two or three cups a day of a leaf infusion (about 15 g in 1 l of water) are prescribed for colds and bronchitis; a more concentrated leaf decoction is used as an animal poison. S. jamesonii (Benth.) Miers “San Juan” (DF/P/88/50) In pediatrics the plant is used as an anthelmintic. A whole plant decoction (approximately 20 g in 1 l of water, boiled for half-an-hour) is administered on empty stomach, then, one must take a purgative, usually “Huaminga” (see Huperzia sp.). Valerianaceae V. adscendens Trel. “Hornamo morado” (DF/P/88/67) The aerial parts of the plant are added to “San Pedro” decoction to enhance its hallucinogenic power. A decoction of the whole plant is claimed to act as a drastic purge.

4. Observations on disease concept In the Andean folk medicine of northern Peru, medicinal plants (and all other plants) are divided into two groups: plants with “hot virtues” and plants with “cold virtues.” Following this division a distinction is also made as a consequence, between infirmities which are either “hot” or “cold.” “Hot infirmities” are claimed to be cured by “cold” plants and a “cold” vegetarian diet, while “cold infirmities” are claimed to be cured by “hot” plants and foods. For example, rheumatism, bronchitis and malaria are considered “cold” diseases which are to be cured with “hot” herbs. Stomach

acidity (indigestion) and inflammation are to be cured using “cold” remedies, being “hot” diseases. Some psychological problems, such as hysterical manifestations, are believed to be caused by an excess heat in the brain and in the blood stream; they are to be cured using “cold” remedies. The “susto,” a psychosomatic pathology, in which phobic events lead to a state of general organic debilitation, and other infirmities, which results in the loss of “vital forcem,” are to be cured by “hot” remedies. “San Pedro,” “mishas,” “cimorilla” and other plants that produce hallucinations are unanimously classified as “hot” plants. Based on this concept, we present below a classification of some vegetables, remedies and things used

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in the Andean popular medicine as either “cold” or “hot” species. Studies are under way to determine further the underlying reasons for this classification. In fact, this system is not limited to medicinal plants, but encompasses all other plants.

honey (“Miel de Mexico”); the goat cheese; the cow cheese; all bitter foods (“Todas cosas amargas”). The following ailments: tuberculosis; stomach acidity; all inflammations. 4.2. Plants and things considered “cold” (“frias”)

4.1. Plants and things considered “hot” (“calientes”) All hallucinogenic plants: San Pedro [T. pachanoi Britt. & Rose, T. peruvianus Britt. & Rose]; all mishas [Brugmansia spp.]: Misha colambo [B. suaveolens (Willd.) Bercht. et Presl.], Misha curandera [B. candida (Pers.) Safford hybrid], Misha del Inca [B. versicolor Lagerheim], Misha galga [B. arborea (L.) Lagerheim hybrid, B. aurea Lagerheim], Misha león [B. arnorea (L.) Lagerheim], Misha oso [B. arborea (L.) Lagerheim, B. arborea (L.) Lagerheim hybrid], Misha rastrera [B. insignis (Barb.Rodr.) Lockwood hybrid], Misha toro [B. sanguinea (R. et P.) D. Don], Misha toro curandera [B. sanguinea (R. et P.) D. Don]; Cimora [Sanchezia sp.]; Cimora lanza [Iresine sp.]; Cimora león [Acalypha macrostachya Jacq.]; Cimora oso [Coleus sp.]; Cimora señorita [I. herbstii Hook.], Cimorilla [C. blumei Benth.], Tabaco [N. tabacum L.]; Tabaco cimarrón [N. paniculata L.]. All purgative plants: Hornamo amarillo [S. elatus HBK.]; Hornamo morado [V. adscendens Trel.]; Huaminga [Huperzia spp.]. All magical “varas” (“canes”) and plants (De Feo, 1992): Ajo aspe [not identified]; Ajosquiro [not identified]; Chiquir huande [Aiphanes sp.]; Chonta [Bactris sp., Iriartea sp.]; Guayacan [Tababuya impetiginosa Standl.]; Hualtaco [Loxopteryngium huasango Spruce]; Membrillo [Cordia sp.]; Palo huaco [not identified]; “Montañesas:” Bejuco de la montaña [not identified]; Camalonga [T. peruviana (Pers.) Schum.]; Huarmi [not identified]; Huayme-huayme [S. scandens (L.) DC.]; Piri-piri [Cyperus spp.]. Medicinal plants: Cahuina [S. sagittata R. et P.], Congona [Gynoxys sp.], Hierba del susto [M. alypifolia Naud.], Molle [S. molle L.], Palo del espanto [T. longifolia (Vahl) Baill.], Poleo del Inga [D. foliosissimum Blake], San Juan amarillo [H. umbellata (R. et P.) Gilg], San Juan negro [G. bicolor (Wedd.) Fabris], Tululuche [S. oblongifolium HBK. var. soukupii Macbr.]. All things forbidden in ritual diet: garlic, Aj´ı [Capsicum annuum L.], onion, common salt, pork fat [“manteca de chancho”]. The following aromatic and food plants: basil; peas; almendra [Caryocar spp.]; anise; celery; coffee; clove; bean; fennel; orange; mango; chamomile; marjoram; nutmeg; paico [Chemopodium ambrosioides L.]; parsley; rosemary; balm-mint; cinnamon. The scented waters “Agua cananga” and “Agua florida;” the alum; puma and bear fats (“Cebo de león” and “Cebo de oso”); the sukstroke (“Locura de calor”); the calumny (“Difamación”); the man; non-Christian tombs (“Huacas gentileñas”); the hen; the goat milk; the puma; the Agave

The medicinal plants: Limón agrio [C. aurantifolia (Christm.) Swingle]; Limón dulce [C. limon (L.) Burm. f.]; Payama [B. cinnamomea Lindl.]; Chiquiragua [C. jussieui J. F. Gmelin]; Achupalla del oso [Puya sp.]; Sangurache negro [I. herbstii Hook.]; Maiz blanco [Zea mays L.]; Matico [P. acutifolium R. et P.]; Llantén [Plantago spp.]; Cola de caballo [E. bogotense HBK., E. giganteum L.]; Tabaco moro [N. rustica L.]; Grama dulce [Cynodon dactylon (L.) Pers.]. The following aromatic and food plants: rice; barley; chirimoya [Annona cherimolia Miller]; apple; melon; nut [Juglans neotropica Diels.]; avocado; potato; papaya; banana; sage; tomato; wheat; grapefruits; yuca [Manihot utilissima Pohl.]; zambumba [Cucurbita spp.]; lime; apple. The following ailments: “susto;” wind ailment (“aire de viento”); bronchitis; rheumatism; cold; cold ailments (“locuras del frio”); pneumonia; malaria. The river and lagoon waters (“agua de corriente” and “agua de laguna”); the ritual waters, “agua del Carmen,” “agua de San Pablo;” the cane alcohol; the perfumes; the snake fat (“cebo de macanche”); the women; the iron; the gold; the silver; the copper; the rabbit; the guinea-pig; the dog; the pig; the sheep; the cat; the duck; the turkey; all snakes; all fishes; all sweet foods (“todas cosas dulces”); bee’s honey; oil; sugar; the cow milk; the butter.

5. Discussion and conclusions The use of medicinal plants and phytotherapy is a phenomenon of primary importance in the medical practices of the high-Andean zones. This study is the first study ever conducted on the medical ethnobotany of the lake area of Ayabaca, where most diseases are treated with phytotherapy. In most cases the people uses native plants along with some naturalized and cultivated species. Some medicinal plants, particularly the “mishas” (Brugmansia spp.), grow wild around the towns and are cultivated by the healers (“curanderos”) in their gardens of magic and medicinal plants. In this study, the Asteraceae and the Solanaceae are the most represented families in medicinal flora. Ancestral beliefs and magical rituals accompany the prescription of the vegetal remedies. Medicinal plants enter into the Andean cosmovision, in which they possess a beneficent spirit, who exerts the cure. Some interesting notations are related to these observations. For example, before gathering medicinal plants, the “curandero” offers to the plant perfumes (“florecimientos”) and tobacco smoke (“fumes”) to propitiate the spirit of the plant.


The most important therapeutical and sacred time for Andean healers is constituted by the nights of Tuesday and Friday, during a magic/therapeutic rite, the “mesada.” Through the consumption of hallucinogenic plants, chiefly the “San Pedro” cactus (T. peruvianus Britt. et Rose, T. pachanoi Britt. et Rose), the “curandero” reaches altered states of consciousness and, in this shamanistic trance, diagnoses the illness and prescribes vegetal remedies. “San Pedro” cactus represents a central plant in Andean folk medicine: Saint Peter (San Pedro in Spanish) is responsible for opening the doors of the Paradise, the cactus permits to the “curandero” the enter in the world of the supernatural forces to see the causes of illnesses and the plants that may help in the cure. The Andean “curanderos” associate the action of this psychotropic plant with a supernatural depersonalization or dissociation of body and spirit; this conception has been reported for other cultures (McLaughlin, 1973). The “San Pedro” cactus contains the alkaloid mescaline and its derivatives and other phenethylamine derivatives (Poisson, 1960; Agurell, 1969; Crosby and McLaughlin, 1973; Jimenez, 1973; Pardanani et al., 1977; Smith, 1977; Shulgin, 1979; Pummangura et al., 1982; Davis, 1983; Gennaro et al., 1996) with well known hallucinogenic properties (Klüver, 1966; Shulgin, 1973). T. peruvianus, on a weight basis probably exceeds peyote (Lophophora williamsii) as a source of mescaline (Shulgin, 1979). Alkaloid level can be quite variable from cactus to cactus. Some of the San Pedro compounds are sympathomimetics. Other compounds have no apparent effects when ingested alone. It is possible, however, that in combination with the mescaline they exert a synergistic influence that alters the qualitative aspects of the hallucination. Mescaline is the key hallucinogenic alkaloid in San Pedro cacti. This compound exerts its main action in a stimulation of the visual and visuo-psychic areas of the cortex (Klüver, 1966) and is known for its visual hallucinations (Goodman et al., 1996), producing an altered state of consciousness. It is interesting to note that in traditional practices, for 2–3 h after “San Pedro” administration, it is severely forbidden to see lights: this finding is well correlated with literature that report a photophobia following the ingestion of mescaline (Shulgin, 1973). The ritual use of “San Pedro” cacti is very old: representations of the cacti have been found in Chavin engraved stones and textiles, Nazca, Moche and Chimú ceramics (Schultes and Hofmann, 1979). The plant was utilized as a hallucinogen by Nazcas (Dobkin de Rios and Cardenas, 1980). The curanderos of the northern Peruvian Andes use the term “San Pedro” indifferently both for T. peruvianus and T. pachanoi, although a folk taxonomy exists of the cacti ritually used. The wild “San Pedro” is preferred to cultivated cactus, due to its stronger properties. Sometimes, cacti with little spines are known as “San Pedro legitimo” (true “San Pedro”) and used only in curative rituals, whereas cacti with longer spines, known as “San Pedro cimarrón” (wild “San Pedro”) are use in sorcery. Branched “San Pedro” cactus is known as “San Pedro femina” (female) and non-branched

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specimens as “San Pedro macho” (male). “San Pedro” is a “hot species” and its hallucinogenic properties derive from its “hot” characteristics, for this reason “San Pedro misha,” variegated “San Pedro” with flowers both white and red in color, is the hottest, and therefore the most potent, “San Pedro.” The cacti are known also as “gigantón,” “aguacolla” and “huando,” three terms related to its size (“huando,” from the quechua “wantuq,” high); “huacuma,” related to the quechua term “kachum” (cactus) (Cabieses Molina, 1990) or to the Spanish term “achumarse” (to get inebriated); “remedio,” “cardo,” “cardo santo,” “yerba,” “palo,” “paja.” Another type of taxonomy of “San Pedro” cacti is related to the number of ribs: cacti with 4 or 7 ribs are claimed to be stronger in curative rituals; specimens with 5 ribs are claimed to be effective in protection rites; 12 ribs are characteristic of cacti useful in divination. The “San Pedro” folk uses were previously reported (Friedberg, 1960, 1963; Dobkin de Rios, 1968, 1969; Polia, 1988; Davis, 1983) and correlated with the use of other psychodysleptic plants used by natives of North America (Diaz, 1979). The decoction of the plant is used with some ritual purposes: (a) shamanistic diagnosis of the illnesses; (b) divination in past and future times; (c) location (“rastreo”) of peoples and things lost in time and/or in the space; (d) prescription of ritual therapies. Ritual rules control all the phases of “San Pedro” preparation and administration. For collecting the plant, the curandero respects the lunar cycle and observes the site in which the cactus grows (the wild “San Pedro” is more potent than the cultivated cactus; the plant growing on the rocks is more potent than the cactus growing in the soil; “San Pedro” cacti living in arid zones are stronger than cacti living in humid sites; cacti growing near fire have lost their potency). The cactus is analyzed in relation to the color of flowers, to the presence of spines, ribs, cut color; special offering and ritual formulas are made to the plant in order obtain the favour of the plant spirit. The knife and the people that pick the cactus need to be ritually pure. The ritual purity consists in the abstention from salt, aj´ı (C. annuum L.), garlic, onion, blood and pork fat and from sex. During the preparation of the decoction the “curandero” observes rituals about purity of the knife used to cut the cactus, the pot and the water used for the decoction. The time of the ritual administration appears to be very important: the best results will be obtained on Tuesday and Friday, from sunset to midnight. Sometimes, other magic plants can be added to the “San Pedro” decoction: Brugmansia species (to enhance the hallucinogenic power of the “San Pedro” decoction) or “purgas,” to ritually expel “bad spirits” form the patient’s body. If the decoction falls down on the fire the plant will lose its properties. The people that prepare the decoction need to be ritually pure. The “curandero” takes the decoction and a single cup of the San Pedro decoction is passed repeatedly in a clockwise direction, until each patient has drunk these cupfuls.

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The vomiting and diarrhoea caused by “San Pedro” administration are considered as a signal that the power of the plant has caused the expulsion of the illness. Generally, the “curandero” does not show this effect, maintaining control (Chiappe et al., 1985). At the end of the ceremony, after shamanistic diagnosis, the “curandero” blows over everyone, by mouth and by a brush of leaves of maize, a preparation, called “corte” or “arranque,” composed by only “cold” ingredients, able to counteract the hot power of the cactus. The “corte” is also drunk. In the day after the “San Pedro” administration it is severely forbidden to bathe, to see fire, to eat salt, onion, garlic, aj`ı, pork fat, animal blood. The “San Pedro” session represents a syncretic ritual, in which it is often difficult to distinguish one tradition from the other. Probably, the real effectiveness of “San Pedro” administration can be attributed to a synergism between pharmacological and cultural factors. I my first “mesada,” I drunk “San Pedro” decoction, but I had not any hallucinogenic effect. After 2 weeks of “immersion” in Andean culture and practices, after drunken decoction, I saw lights. Very few reports are available in ethnobotanical literature on the other hallucinogenic plants, normally used in association with “San Pedro.” For the first time in this study we identified some plants, important in magic/curative beliefs of northern Peruvian Andes: the “hornamo amarillo” as S. elatus HBK. (Asteraceae); the “hornamo morado” as V. adscendens Trel. (Valerianaceae); the “huaminga” as Huperzia sp. (Lycopodiaceae), previously reported by Friedberg (1963) as a Lycopodium species. Our research permitted us to identify different plants known by the name of “cimora,” belonging to different genera: “cimora” as Alternanthera sp. (Amaranthaceae); “cimora oso,” as Coleus sp. (Labiatae); “cimorilla,” as C. blumei Benth. (Labiatae); “cimora señorita,” as I. herbstii Hook. (Amaranthaceae), known also as “Singurache negro;” “cimora león,” as Acalypha sp. (Euphorbiaceae); “cimora macanche,” as Sanchezia sp. (Acanthaceae); “cimora lanza,” as Iresine sp. (Amaranthaceae), together with two unidentified species: “cimorilla dominadora” and “cimora colambu.” The term “cimora” appears to be a generic term, referring to “algo malo,” something bad. We present for first time a classification of “cimoras,” magical-curative plants for which previous studies have not performed the botanical identification. In fact, Davis (1983) stated that “an outstanding ethnobotanical problem associated with the Huancabamba cult yet adequately to be resolved concerns the botanical meaning of the term cimora.” In previous reports, Cruz-Sanchez (1948) suggested that cimora was the term applied to a particular intoxicating blend of plants in addition to San Pedro. Friedberg (1960) disagreed with Cruz-Sanchez and stated that “cimora is not a drink with a cactus, but is a plant of Amaranthaceae, in the genus Iresine.” The anthropologist Sharon (1972) and Dobkin de Rios (1968, 1969, 1977) simply repeated Friedberg’s early determinations without reference to new voucher speci-

mens. Schultes and Hofmann (1992) consider cimoras as a beverage made up of a number of plants. In the northern Peruvian Andes, “cimora oso” is used to cure “hot pain,” mixed with vinegar, alcohol and tobacco. Schultes and Hofmann (1973) reported two Coleus species as hallucinogens. “Cimora macanche” (Sanchezia sp.) is considered a powerful “cimora” and young Peruvian people use Sanchezia species as inebriants; in literature a report is available on an unidentified species of Sanchezia supposed to have hallucinogenic effects when smoked (Schultes and Raffauf, 1990). Possible central nervous system effects are reported also for several species of Alternanthera (Girault, 1984; Bianchi and Samorini, 1993). I. herbstii was reported as an additive of ayahuasca (Bianchi and Samorini, 1993), as an ingredient of San Pedro decoction, with possible hallucinogenic properties (Schultes and Hofmann, 1973) and as specific remedies for mental illnesses (Friedberg, 1960). The “cimorilla dominadora” is used in black magic. The effects of “cimoras” are to be interrupted by the “arranque.” The “purgas” constitute another group of species, used for their ritual and magical properties. These plants, acting as drastic purges, are claimed to be useful in expelling “bad spirits” from the patient body. Among the three “hornamo” species used by Huancabamba curanderos (Polia, 1988), we identified “hornamo amarillo” as S. elatus HBK. (Asteraceae), and “hornamo morado,” as V. adscendens Trel. (Valerianaceae). “Hornamo morado” was reported by Davis (1983) as a one of the most important curative plant of the lagoons area, sometimes added to San Pedro preparation. “Hornamo amarillo” is claimed to have powerful magic effects; the plant is reported as an additive to the San Pedro preparation, and other Senecio species are known for their hallucinogenic properties (Schultes and Hofmann, 1973) and used as peyote (Schultes, 1937; Diaz, 1979). The genus contains pyrrolizidine alkaloids (Raffauf, 1970; Diaz, 1979), active on the central nervous system (Robins, 1991). We identified a “huaminga” species, of seven present in Huancabamba area, as a Huperzia sp. (Lycopodiaceae); Friedberg (1959) identified the plant as Lycopodium sp. and Girault (1984) reported L. reflexum Law. as a magic plant, used against ritual witchcrafts. The literature reports that huperzine A, an alkaloid isolated from a Chinese species of Huperzia was found to be active on central nervous system, improving short- and long-term memory in patients of cerebral arteriosclerosis with memory impairment (Zhu, 1981). Our research group, at Salerno and Naples Universities, have carried out some phytochemical and pharmacological studies on these plants to evaluate their traditional uses. From S. elatus we isolated some pyrrolizidine alkaloids (Aquino et al., 1996) that are known to possess deliriant properties (Schultes and Hofmann, 1973; Diaz, 1979). The aqueous extract of V. adscendens showed a marked CNS depressant activity (Capasso et al., 1996; Capasso and De Feo, 2003) and a significant effect in inhibiting the GABA uptake and in decreasing the intracellular content of amino acid neurotransmitter in crude synaptosome of rat (De Feo and Faro,


2003). The water extract of I. herbstii acts as a neurosedative (De Feo et al., 1996; Capasso and De Feo, 2003). Methanolic and water extracts of “huaminga” (Huperzia sp.) exerted a marked diarrhoea promoting activity (Autore et al., 1994). The data recorded on the uses of the “mishas” (Brugmansia spp.) are very important. The folk and ritual uses of two Brugmansia species (B. insignis and B. suaveolens) have been documented in several zones of the Amazonic forest (Rivier and Lindgren, 1972; Schultes and Hofmann, 1973; Hunziker, 1979; Lockwood, 1979; Schultes, 1979; McKenna et al., 1986; Cabieses Molina, 1990; Bianchi and Samorini, 1993). In Andean zones, the ritual use of B. sanguinea and B. aurea was reported by Schultes (1955, 1979) and Bristol (1969), in the Sibundoy Valley, Colombia. All Brugmansias used in Andean zone appear to be cultigens and their ritual use has been documented in Mochica, Moche and Nazca ceramics (Cabieses Molina, 1990). The plants show great variability in their morphology and some difficulties in their identification, due to the fact that Brugmansia trees cross-breed very easily; on the other hand “curanderos” search for specimens with larger or stranger leaves (Friedberg, 1963; Bristol, 1969; Schultes, 1979). The rarer cultivars are owned by and cultivated in magical gardens of “curanderos.” Brugmansia trees contain tropane alkaloids (Evans, 1979), with well known activities on central and peripheric nervous systems (Diaz, 1979; Schultes, 1979). Our data showed a real ethnobotanical culture developed by the traditional healers of the northern Peruvian Andes about the use and the properties of the “mishas.” They know the various species of Brugmansia, their morphology, their chemical and pharmacological activities. In their folk systematics, the shamans associate the potency of each “misha” with names and virtues of animals (“oso” = bear; “león” = lion; “colambo” = snake; “galga” = hunting dog; “toro” = bull) that best feature the myths of pre-Colombian cultures, and/or with therapeutic/magic properties (“rastrera,” from “rastreo” = to see things lost in the space and in time; “curandera,” from “curo” = to treat ailments). It is important to note that, due to the strong toxic properties of “mishas,” its use is reserved only to “curandero.” Generally, the “mishas” are therapeutically used as topic antinflammatories and antirheumatics, but their major use is “to dream” (induce hallucinations). Moreover, the leaves of Brugmansia species are tied on forehead “para ver” (to see). The obtention of altered states of consciousness is often obtained by topical application of the leaves of “mishas;” in recent literature it is possible to found such effects following transdermal applications of tropane alkaloids (Wilkinson, 1987; Ziskind, 1988). It is interesting to underline that in the European Middle Ages, the witches used Solanaceae species, rubbing these plants on their broomsticks before climbing upon them, thus achieving in their peculiar way a sensation of flight. Some Brugmansia species are used in black magic (Polia, 1988). The use of “mishas” is strictly reserved to

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“curanderos,” due to their strong “hot” properties and the idea that Brugmansias are bad or dangerous is still prevalent. Only in particular cases, the “curandero” add “mishas” to the San Pedro decoction, in order to enhance its hallucinogenic effects. As for all hallucinogenic species the use of Brugmansia imposes a ritual diet; the effects of “mishas” can be interrupted by the “corte.” We recorded only information about the most potent “misha:” the “misha del Inga Rey” and about other “mishas,” but it was impossible to collect these species, because of the ritual fear that “curanderos” have for these species. We have chemically and pharmacologically studied the leaves of B. arborea and isolated three tropane alkaloids with a significant spasmolytic activity (Capasso et al., 1997; Capasso and De Feo, 2003) and a significative reduction of morphine withdrawal in vitro (Capasso and De Feo, 2002). Among hallucinating plants, different species of tobacco (Nicotiana spp.) are used in curative and magical rituals; very often N. glauca Graham is used, known for its neurostimulant and hallucinating properties (Cabieses Molina, 1990). Peruvian curanderos used “tabaco moro” (N. rustica L.) to cure, “tabaco blanco” (N. tabacum L.) to perform a propitiatory “shingada,” and “tabaco cimarrón” (N. paniculata L.) for its stronger effects. In the New World tobacco was employed in sacred magical and medicinal context and its use has been documented in tribal and curative ceremonies. The plant is considered to be trance-inducing amongst many Latino-American tribes (Wilbert, 1987) and the use for its hallucinogenic effects has been documented (Schultes, 1972a,b,c,d; Wilbert, 1972; Furst, 1976; Janiger and Dobkin de Rios, 1976; Plotkin et al., 1980). The ayahuasca sessions are always accompanied by its use. In the leaves nicotine, pyridine and pyrrolidine alkaloids, and, in low quantities and particularly in the smoke, ␤-carboline alkaloids (Poindexter and Carpenter, 1962; Janiger and Dobkin de Rios, 1976) are present (Bianchi and Samorini, 1993). The administration of an alcoholic infusion of tobacco leaves through nasal mucosa (“shingada”) or as a snuff powder is documented for South America (Friedberg, 1963; Wassen, 1972). The literature yields convincing clinical evidence that nicotine and scopolamine are effective following nasal applications (De Smet, 1985; Elferink, 1983). Probably, the “shingada” is used in order to avoid the vomiting caused by the oral administration of the tincture; furthermore in this way the active principles probably arrive more rapidly to the central nervous system. Sometimes, perfumes and a decoction of San Pedro are added to the infusion on tobacco leaves. Generally, the effects of hallucinogenic remedies are stopped by using the “arranque” or “corte” (“to cortar” = to cut), a preparation made by perfumes, magic plants and white flowers; it will be very interesting to understand the real pharmacological effectiveness of this preparation. Among the other species, without magic (=psychodysleptic?) properties, some species are also known and used in

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other areas of Andes, with different therapeutic purposes, but most of the plants considered in this research have been noted for the first time for their ethnomedical uses. In fact, a comparison with ethnobotanical data available for Peru showed that only for few plants have reported therapeutic uses (Herrera, 1940; Girault, 1984; Soukup, 1987; Alarco de Zandra, 1988; De Feo, 1992; Velasco-Negueruela et al., 1995). The vernacular names of the plants reported in this study are generally new: some names, recorded in this study, indicated other plants in different parts of Andes. There are no phytochemical and/or pharmacological studies for most of these species. We have carried out phytochemical and pharmacological researches on some of these plants. From G. oleifolia Muschler we have isolated kauranoid diterpenes (Catalano et al., 1993); A. nitidum (HBK.) Schlecht contains a series of substances with antibacterial activity (De Feo et al., 1992); the non-polar constituents of D. foliosissimum Blake have also been characterized (Senatore et al., 1991); from B. cinnamomea Lindl. were isolated a set of flavonoid compounds, some of which displayed in vitro anti-HIV activity (Aquino et al., 1996). On the other hand, a survey from Chemical Abstracts reveals that only few among the reported species have been studied phytochemically and/or pharmacologically. Very interesting is the fact that many plants are used to cure the respiratory diseases that are common in populations leaving at high altitudes. Plants with antiseptic and anthelmintic properties are also common. Many of properties ascribed to the reported plants are primarily superstitious or magical, and one can find some similarities with the Doctrine of Signatures of the European Middle Age. For example, the “cabello del bosque” [Huperzia sp.], an epiphytic species, is claimed to act in hair growth; the “huayme-huayme” [S. scandens (L.) DC.], whose leaves are similar to female genital external organs, is claimed to promote the fertility in women and is used in love magic (“guayanche”). The dosage and the prescription of the vegetal remedies are very specific, showing a complete knowledge of the possible toxic activity of the medicinal species used. It is interesting to note that many plants with hallucinating activity are assumed by absorption through the nasal mucous (“shingada”). In my experience, the “shingada” was very painful. It constitutes a very ancient ritual practice, usually made by using tobacco leaves soaked in cane alcohol and has been documented in some Peruvian civilizations (Schultes and Hofmann, 1973; Wilbert, 1987; Cabieses Molina, 1990; Torres et al., 1991). Through observations made during the field study, one can hypothesize a synergism and potentiation deriving by the use of cane alcohol, very much used by the shamans during the rituals. Very particular is the prescription of soaked plants in woman’s and lamb’s milk, as in the case described for Chuguiraga jussieui. All other prescribed formulations (decoction, infusion, tincture) are usual.

The “hot” and “cold” classification for plants and diseases seems never to have been studied before, and it appears important to probe into this subject as well as into the use of vegetal remedies to psychosomatic discomforts, for example the fright or “susto,” that really affects the Andean people (Polia, 1988; De Feo, 1992). This ethnobotanical research surely showed the deep knowledge by healers and people of northern Peruvian Andes about medicinal plants, particularly about those with hallucinating properties and the linkage between the Andean people and the nature. Hallucinogenic species are associated with supernatural and the dissociation of body and spirit, experienced under the influence of hallucinogens, is a basic concept in religious system. Thus, psychoactive plants are used in rituals intimately associated to religion. Further studies are needed to save a cultural heritage that nowadays risks being lost forever.

References Agurell, S., 1969. Cactaceae alkaloids. Lloydia 32, 206–216. Alarco de Zandra, A., 1988. Perú, el libro de las plantas mágicas. Concytec, Lima, p. 152. Aquino, R., De Feo, V., De Tommasi, N., De Simone, F., Pizza, C., 1996. Flora Officinale dell’America Latina—Contributo allo Studio della Flora Andina. Edizioni Gutemberg, Fisciano. Autore, G., Capasso, F., De Feo, V., De Simone, F., Mascolo, N., 1994. Attività gastrointestinale di Huperzia sp. (Lycopodiaceae). Atti del IV Congresso Italo-Latinoamericano di Etnomedicina, Roma, 19–22 settembre 1994, p. 33. Bianchi, A., Samorini, G., 1993. Plants in association with ayahuasca. Jahrbuch für Etnomedizin, 21–42. Bristol, M.L., 1969. Tree Datura drugs of the Colombian Sibundoy. Botanical Museum Leaflets, Harvard University 22, 165–227. Cabieses Molina, F., 1990. The Magic Plants of Ancient Peru. Atti del V Congresso Nazionale della Società Italiana di Fitochimica, Ischia, 30 maggio—2 giugno 1990, LP2. Capasso, A., De Feo, V., 2003. Alkaloids of Brugmansia arborea (L.) Lagerheim reduce morphine withdrawal in vitro. Phytotherapy Research, in press. Capasso, A., De Feo, V., 2002. Central nervous system pharmacological effects of plants from northern Peruvian Andes: Valeriana adscendens, Iresine herbstii and Brugmansia arborea. Pharmaceutical Biology 40, 274–293. Capasso, A., De Feo, V., De Simone, F., Sorrentino, L., 1996. Pharmacological effects of aqueous extract from Valeriana adscendens. Phytotherapy Research 10, 309–312. Capasso, A., De Feo, V., De Simone, F., Sorrentino, L., 1997. Activitydirected isolation of Spasmolytic (anti-cholinergic) alkaloids from Brugmansia arborea (L.) Lagerheim. International Journal of Pharmacognosy 35, 43–48. Catalano, S., Cioni, P.L., Menichini, A., Bilia, A.R., Morelli, I., De Feo, V., 1993. Kauranoid diterpenes in Gynoxys oleifolia. Planta Medica 59, 278–280. Chiappe, M., Lemlij, M., Millones, L., 1985. Alucinogenos y Shamanismo en el Peru contemporaneo. El Virrey, Lima. Crosby, D.M., McLaughlin, J.L., 1973. Cactus alkaloids. XIX. Crystallization of mescaline HCl and 3-methoxytyramine HCl from Tricocerues pachanoi. Lloydia 36, 416–418. Cruz Sánchez, G., 1948. Informe sobre las aplicaciones populares de la cimora en el norte del Perú. Revista de Farmacologia y Medicina Experimental, Lima 1, 253–258.

V. De Feo / Journal of Ethnopharmacology 85 (2003) 243–256 Davis, E.W., 1983. Sacred plants in the San Pedro cult. Botanical Museum Leaflets, Harvard University 29, 367–382. De Feo, V., 1992. Medicinal and magical plants on northern Peruvian Andes. Fitoterapia 63, 417–440. De Feo, V., Faro, C., 2003. Pharmacological effects of extracts from Valeriana adscendens Trel. II. Effects on GABA uptake and amino acids. Phytotherapy Research, in press. De Feo, V., della Valle, C., De Simone, F., Pizza, C., 1992. Chemical constituents and antimicrobial activity of Arcytophyllum nitidum HBK. Annali di Chimica 82, 476–486. De Feo, V., Capasso, A., De Simone, F., Sorrentino, L., 1996. CNS pharmacological effects of aqueous extract from Iresine herbstii. International Journal of Pharmacognosy 34, 184–188. De Smet, P.A.G.M., 1985. A multidisciplinary overview of intoxicating snuff rituals in the western hemisphere. Journal of Ethnopharmacology 13, 3–49. Diaz, J.L., 1979. Ethnoparmacology and ethnopharmacognosy of Mexican psychodysleptic plants. Journal of Psychedelic Drugs 11, 71–101. Dobkin de Rios, M., 1968. Trichocereus pachanoi: a mescaline cactus used in folk healing uses in Peru. Economic Botany 22, 191–194. Dobkin de Rios, M., 1969. Folk curing with a psychedelic cactus in North Coast Peru. International Journal of Social Psychiatry 15, 23–32. Dobkin de Rios, M., 1977. Plant hallucinogens and the religion of the Mochica, an ancient Peruvian people. Economic Botany 31, 189–203. Dobkin de Rios, M., Cardenas, M., 1980. Plant hallucinogen, shamanism and Nazca ceramics. Journal of Ethnopharmacology 2, 233–246. Elferink, J.G., 1983. The narcotic and hallucinogenic use of tobacco in pre-Columbian Central America. Journal of Ethnopharmacology 7, 111–122. Evans, W.C., 1979. Tropane alkaloids of the Solanaceae. In: Hawkes, J.G., Lester, R.N., Shelding, A.D. (Eds.), The Biology and Taxonomy of Solanaceae. Linnean Society Symposia Series no. 7. Academic Press, London, pp. 245–254. Ferreyra, R., 1960. Algunos Aspectos Fitogeográficos del Peru. Publicaciones del Instituto Geográfico, Universidad Nacional Mayor de San Marcos, Lima, 1960, pp. 41–48. Ferreyra, R., 1986. Flora del Peru—Dicotiledoneas. Imprenta Editorial Suramericana, Lima. Friedberg, C., 1959. Rapport sommaire d’une mission au Pérou. Journal d’Agricolture Tropicale et de Botanique Appliquée 6, 439–450. Friedberg, C., 1960. Utilization d’un cactus à mescaline au nord du Pérou. Journal d’Agriculture Tropicale et Botanique Appliquée 6, 439–450. Friedberg, C., 1963. Mission au Pérou—Mai 1961–Mars 1962. Journal d’Agriculture Tropicale et Botanique Appliquée 10, pages 32–52, 245–258, 344–386. Furst, P.T., 1976. Hallucinogens and Culture. Chandler and Sharp, San Francisco. Gennaro, M.C., Gioannini, E., Giacosa, D., Siccardi, D., 1996. Determination of mescaline in hallucinogenic Cactaceae by ion-interaction HPLC. Analytical Letters 29, 2399–2409. Girault, L., 1984. Kallawaya, Guérisseurs Itinérants des Andes. Editions de l’Orstom, Paris. Goodman, Gilman, A., Hardman, J.G., Limbird, L.E., Gilman, A., Goodman, S.L., 1996. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill, New York. Herrera, F.L., 1940. Plantas que curan y plantas que matan de la Flora del Cusco. Revista del Museo Nacional, Lima IX (1), pp. 74–127. Hunziker, T.A., 1979. South American Solanaceae: a synoptic survey. In: Hawkes, J.G., Lester, R.N. Shelding, A.D. (Eds.), The Biology and Taxonomy of Solanaceae. Linnean Society Symposia Series no. 7. Academic Press, London, pp. 49–85. Janiger, O., Dobkin de Rios, M., 1976. Nicotiana, an hallucinogen? Economic Botany 30, 149–151. Jimenez, A.C., 1973. Ethnobotany and phytochemistry. Boletino de la Sociedad Quimica del Peru 39, 252-257, via Chemical Abstracts 82:13924 (1975).

255

Klüver, H., 1966. Mescal and Mechanisms of Hallucinations. Phoenix Books. The University of Chicago Press, Chicago, pp. 65–80. Lockwood, T.E., 1979. The ethnobotany of Brugmansia. Journal of Ethnopharmacology 1, 147–164. McBride, J.F. (Ed.), 1938–1981. Flora of Peru. Fieldiana: Botany. Field Museum of Natural History, Chicago. McKenna, D.J., Luna, L.E., Towers, C.H.N., 1986. Ingredientes biodinamicos en las plantas que se mezclan al ayahuasca. Una farmacopea tradicional no identificada. America Indigena 46, 73–98. McLaughlin, J.L., 1973. Peyote: an introduction. Lloydia 36, 1–8. Pardanani, J.H., McLaughlin, J.L., Kondrat, R.W., Cooks, R.G., 1977. Cactus alkaloids. XXXVI. Mescaline and related compounds from Trichocereus peruvianus. Lloydia 40, 585–590. Plotkin, M.J., Mittermeier, R.A., Costable, I., 1980. Psychotomimetic use of tobacco in Surinam and French Guiana. Journal of Ethnopharmacology 2, 295–297. Poindexter, E.H., Carpenter, R.D., 1962. Isolation of harmane and nor-harmane from cigarette smoke. Chemistry and Industry 26 (1), 176. Poisson, J., 1960. The presence of mescaline in a Peruvian cactus. Annales Farmaceutiques Françaises 18, 764–765. Polia, M., 1988. Las Lagunas de los Encantos—Medicina Tradicional Andina en el Peru septentrional. Club Grau, Ce.Pe.Cer., Lima. Pummangura, S., McLaughlin, J.L., Schiffendecker, R.C., 1982. Cactus alkaloids. LI. Lack of mescaline traslocation in grafted Trichocereus. Journal of Natural Products 45, 215–216. Raffauf, R.F., 1970. A Handbook of Alkaloids and Alkaloid-containing Plants. Wiley Interscience, New York. Rivier, L., Lindgren, J.E., 1972. Ayahuasca, the South American Hallucinogenic drink: an ethnobotanical and chemical investigation. Economic Botany 26, 101–129. Robins, D.J., 1991. Pyrrolizidine alkaloids. Natural Product Reports 8, 213–221. Schnell, R., 1987. La Flore et la Végétation de l’Amérique Tropicale, vol. 2. Masson, Paris, p. 127. Schultes, R.E., 1937. Peyote (Lophophora williamsii) and plants confused with it. Botanical Museum Leaflets, Harvard University 5, 61–88. Schultes, R.E., 1955. A new narcotic genus from the Amazon slope in the Colombian Andes. Botanical Museum Leaflets, Harvard University 17, 1–11. Schultes, R.E., 1972a. An overview of hallucinogens in western hemisphere. In: Furst, P. (Ed.), Flesh of the Gods: The Ritual Use of Hallucinogens. Praeger, New York, pp. 3–54. Schultes, R.E., 1972b. De Plantis Toxicariis e Mundo Novo Commentationes. X. New data on the Malpighiaceous narcotics of South America. Botanical Museum Leaflets, Harvard University 23, pp. 137–147. Schultes, R.E., 1972c. De Plantis toxicariis de Mundo Novo commentationes. XI. The ethnobotanical significance of additives to New World hallucinogens. Plant Science Bulletin 18, 34–41. Schultes, R.E., 1972d. The utilization of hallucinogens in primitive societies, use, misure or abuse? In: Keup, W. (Ed.), Drug Abuse. Charles C. Thomas, Springfield, pp. 17–27. Schultes, R.E., 1979. Solanaceous hallucinogens and their role in the development of the New World cultures. In: Hawkes, J.G., Lester, R.N., Shelding, A.D. (Eds.), The Biology and Taxonomy of Solanaceae. Linnean Society Symposia Series no. 7. Academic Press, London, pp. 137–160, and references cited therein. Schultes, R.E., Hofmann, A., 1973. The Botany and Chemistry of Hallucinogens. Charles C. Thompson, Springfield. Schultes, R.E., Hofmann, A., 1979. Plants of the Gods. Mc Graw-Hill, New York. Schultes, R.E., Hofmann, A., 1992. Plants of the Gods: Their Sacred, Healing, and Hallucinogenic Powers. Healing Arts Press, Rochester. Schultes, R.E., Raffauf, R.F., 1990. The Healing Forest: Medicinal and Toxic Plants of the Northwest Amazonia. Dioscorides Press, Portland. Senatore, F., De Feo, V., Zhou, Z.L., 1991. Non polar constituents of two Peruvian Asteraceae. La Rivista Italiana delle Sostanze Grasse 68, 493–495.

256


Sharon, D., 1972. The San Pedro cactus in Peruvian folk healing. In: Furst, P. (Ed.), Flesh of the Gods: The Ritual Use of Hallucinogens. Praeger, New York, pp. 114–135. Shulgin, A.T., 1973. Mescaline: the chemistry and pharmacology of its analogs. Lloydia 36, 45–58. Shulgin, A.T., 1979. Chemistry of phenethylamines related to mescaline. Journal of Psychedelic Drugs 11, 41–52. Smith, T.A., 1977. Phenethylamine and related compounds in plants. Phytochemistry 16, 9–18. Soukup, J., 1987. Vocabulario de los Nombres tradicionales de la Flora Peruana y catalogo de los generos. Editorial Salesiana, Lima. Torres, C.M., Repke, D.B., Chan, K., McKenna, D., Llagostera, A., Schultes, R.E., 1991. Snuff powders from pre-Hispanic San Pedro de Atacama: chemical and contestual analysis. Current Anthropology 32, 640–649. Tosi, J., 1960. Zonas de vida natural en el Peru. Memoria explicativa sobre el mapa ecologico del Perú y Mapa ecologica del Perú. Turrialba, Instituto Interamericano de Ciencias Agricolas, Boletin Tecnico no. 5, 271 pp. Velasco-Negueruela, A., Pérez-Alonso, M.J., Esenarro-Abarca, G., 1995. Medicinal plants from Pampallakta: an Andean community in Cuzco. Fitoterapia 66, 447–461.

Wassen, G.H., 1972. The anthropological outlook for Amerindian medicinal plants. In: Swain, T. (Ed.), Plants in the Development of Modern Medicine. Harvard University Press, Cambridge, MA, pp. 2–65. Weber, H., 1969. Zur Natüralischen vegetation-gliederung von Südamerica. In: Fittau, E.J., Illes, J., Klinger, H., Schwabe, G.H., Sioli, H. (Eds.), Biogeography and Ecology in South America. W. Yunk V. N., Le Hague, pp. 474–518. Weberbauer, A., 1911. Die Pflanzenweet der peruanischen Anden. Verlag von Wilhelm Engelmann, Liepzig, pages 118–121, 215–217. Wilbert, J., 1972. Tobacco and shamanistic ecstasy among the Warrao Indians of Venezuela. In: Furst, P. (Ed.), Flesh of the Gods: The Ritual Use of Hallucinogens. Praeger, New York, pp. 53–83. Wilbert, J., 1987. Tobacco and Shamanism in South America. Psychoactive Plants of the World, vol. 1. Yale University Press, New Haven and London. Wilkinson, J.A., 1987. Side effects of transdermal scopolamine. Journal of Emergency Medicine 5, 387–392. Zhu, X.Z., 1981. Development of natural products as drug acting on central nervous system. Memorias del Instituto Oswaldo Cruz 86 (Suppl. 2), 173–175. Ziskind, A.A., 1988. Transdermal scopolamine-induced psychosis. Postgraduate Medicine 84, 73–76.


Tribulus terrestris: preliminary study of its diuretic and contractile effects and comparison with Zea mays Muneer Al-Ali a,∗ , Salman Wahbi b , Husni Twaij c , Ahmad Al-Badr c a

c

Department of Urology, St. Bartholomew’s, The Royal London & Homerton University Hospitals, 224B East End Road, East Finchley, London N2 8AX, UK b Department of Laboratory, Al-Rasheed Military Hospital, Baghdad, Iraq Department of Pharmacognosy & Pharmacology, Biological Research Centre, Scientific Research Council, Baghdad, Iraq Received 9 August 2002; received in revised form 20 December 2002; accepted 20 December 2002

Abstract Objectives: Tribulus terrestris L. (Zygophyllaceae) which is called Al-Gutub (in Iraqi dialect) or Qutˆıba (in classical Arabic medicine), and Zea mays were both used alone or in combination by Iraqi herbalists to propel urinary stones. We studied the aqueous extract of the leaves and fruits of T. terrestris and the hair of Z. mays, to determine their diuretic activity and the contractile effect of T. terrestris. Methods: The aqueous extract was filtered and the solvent was evaporated to produce a dry crude extract. The dry extract was then dissolved in physiological saline to make the required concentrations. Wistar male rats were used for the diuresis test and strips of isolated Guinea pig ileum were used for the contractility test. Results: The aqueous extract of T. terrestris, in oral dose of 5 g/kg elicited a positive diuresis, which was slightly more than that of furosemide. Z. mays aqueous extract did not result in significant diuresis when given alone in oral dose of 5 g/kg, while combination of Z. mays and T. terrestris extracts produced the same extent of diuresis as that produced by T. terrestris alone. Na+ , K+ and Cl+ concentrations in the urine had also much increased. In addition to its diuretic activity T. terrestris had evoked a contractile activity on Guinea pig ileum. Conclusion: T. terrestris has long been used empirically to propel urinary stones. The diuretic and contractile effects of T. terrestris indicate that it has the potential of propelling urinary stones and merits further pharmacological studies. © 2003 Published by Elsevier Science Ireland Ltd. Keywords: Diuresis; Smooth muscles; Tribulus terrestris; Ureter; Urinary stones; Zea mays

1. Introduction Tribulus terrestris L. (Zygophyllaceae), Arabic names, “Al-Gutub” or “Qutˆıba,” is a widely distributed plant in Iraq and all the Mediterranian region. It is a herbal remedy used for various medicinal purposes including the treatment of kidney troubles, particularly stones. For this purpose it is either used alone or in combination with Zea mays. It is made into concoction with empirical posology. It is also used as a cough remedy and as an insect repellent. Different organs of this plant were used to treat various ailments. A literature survey revealed hypotensive and, cardiac depressant effects and contractile activities on smooth muscles (Mossa et al., 1983). It has been found to be effective in treating angina pectoris by dilating the coronary arteries and improving the cardiac circulation (Wag et al., 1990). T. terrestris has been commonly used in folk medicine in

∗ Corresponding

author. E-mail address: [email protected] (M. Al-Ali).

Turkey as diuretic and against colicky pains, hypertension and hypercholesterolemia (Arcasoy et al., 1998). It has been shown to increase the free serum testosterone (Brown et al., 2001) and to be effective in the treatment of sexual and erectile dysfunction by conversion of its phytochemical derivative, protodioscine to De Hydro Epi Androsterone (DHEA) (Adiomoelja, 2000). It has protective effect on genetic damage (Liu et al., 1995) and stimulates melanocyte proliferation in the treatment of vitiligo (Lin et al., 1999) and it has an antibacterial and cytotoxic activity (Ali et al., 2001). A nematocidal activity has also been reported (Nandal and Bhatti, 1983). With regard to urinary stones, it has been shown to reduce the amount of urinary oxalate in rats (Sangeeta et al., 1994), and it has an antiurolithic activity in experimentally induced urolithiasis in rats too (Anand et al., 1994). This plant is given by Iraqi herbalists in the form of beverages (bush teas) prepared by soaking the leaves and fruits in water or boiling them. Our study aims at evaluating the diuretic activities and other related pharmacological effects of T. terrestris, and

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comparing it to the diuretic activity of the aqueous extract of Z. mays. This study has been prompted by the observation made by one of the authors (MAA) that patients had passed renal and/or ureteric stones on taking this herbal medicine either alone or together with Z. mays. To our knowledge the diuretic effect of T. terrestris has not been previously studied.

2. Material and methods 2.1. Plant material and preparation of the extract The leaves and fruits of T. terrestris and the hair of Z. mays were collected. The herbs were identified and authenticated by the National Herbarium of Iraq. The dried plant material (500 g) was ground into a coarse powder and extracted at room temperature with distilled water by shaking. The extract then filtered and the solvent was evaporated under vacuum at 40 ◦ C to produce a dry crude extract (147.060 g = 29.41% for T. terrestris and 55.25 g = 11.05% for Z. mays). The extract was dissolved in physiological saline solution to make the required concentrations. 2.2. Pharmacological and biochemical studies The doses of T. terrestris were 5 g/kg p.os and 3.4– 54.4 mg/ml of organ bath and Z. mays dose was 5 g/kg p.os. Doses were expressed in gram of crude plant material per kilogram of body weight. 2.3. Diuretic test Thirty Wistar male rats (250–320 g) were used for each test, placed in metabolic cages. They were allocated to 5 groups of 6 animals each, and treated orally with one of the following materials: 1. 2. 3. 4. 5.

Control dose of solvent (saline). Furosemide 120 mg/kg as a standard diuretic. T. terrestris extract 5 g/kg. Z. mays extract 5 g/kg. A combination of both T. terrestris and Z. mays, 5 g/kg of each.

Urine was collected over a period of 24 h. Urine sodium and potassium levels were determined by using Corning EEL flame photometer model 450. The Chloridemeter model 92C Corning EEL has been used for the estimation of chloride in urine. The instruments were calibrated with standard solutions (containing different concentrations of sodium, potassium and chloride). 2.4. Statistical analysis Statistical significance was calculated following the assessment method adopted by Cummings (Cummings et al.,

1960). The criterion for acceptance or rejection in both directions (diuresis and antidiuresis) for the 24 h test, is a change of 75% or more in either direction in the volume of urine caused by the extract as compared to the control, which was accepted as significant. 2.5. Effect on smooth muscle Terminal strips of isolated Guinea pig ileum (2–3 cm in length) were suspended in an isolated organ bath containing Krebs solution at 37 ◦ C (Perry, 1979). Isometric contractions of the ileum were recorded by force transducer (UFI) coupled to Lectromed recorder. 2.6. Behavioral effect and toxicity 2.6.1. Gross behavioral effects (mice) These studies were carried out along with the toxicity studies. The animals were observed continuously for 1 h, intermittently for 4 h and after 24 and 48 h (Turner, 1965). 2.6.2. Determination of LD50 (Litchfield and Wilcoxon, 1949) Graded doses of the aqueous extract of T. terrestris and Z. mays in 0.5 ml of saline were administered i.p. to six groups of six albino mice (25–30 g). They were kept in transparent plastic cages at 24 ◦ C. Mortality was recorded after 24 h. 2.7. Drugs Furosemide (Lasix-Hoechst) was used as a standard diuretic agent.

3. Results Results are shown in Table 1. The aqueous extract of the leaves and fruits of T. terrestris resulted in a marked diuresis during the 24 h of the test. The urine volume had increased by 189% of the control, which was slightly more than that produced by furosemide (179%). Sodium, potassium and chloride concentrations in the urine had also much increased. Z. mays hair aqueous extract failed to show significant diuresis when given alone (22% increase) and failed to potentiate the diuretic activity of T. terrestris. There was much more increase in urine volume and potassium excretion when T. terrestris was given alone compared to that caused by combining it with Z. mays (189 and 170% versus 100 and 70%, respectively). However, Z. mays caused significant increase in the amount of urinary sodium, potassium and chloride when administered alone, although less pronounced than that of T. terrestris alone or in combination. It had particularly produced pronounced potentiation of the amount of sodium excreted by T. terrestris extract (117% versus 49%).

Table 1 Effect of T. terrestris and Z. mays aqueous extracts on the urine volume, Na+ , K+ , and Cl− concentration of the urine in normal fed male rats over a period of 24 h (mean ± S.E.M.) Agents and dose

Post-treatment

Urine volume (ml)

Na+

K+

Cl−

Urine volume (ml)

Na+

K+

Cl−

5.0 ± 0.38 6.14 ± 0.63

0.35 ± 0.04 0.38 ± 0.09

0.58 ± 0.04 0.73 ± 0.08

0.98 ± 0.07 1.17 ± 0.12

5.6 ± 0.41 15.6 ± 1.02 (179%)∗∗∗

0.47 ± 0.07 1.3 ± 0.12 (177%)∗∗∗

0.67 ± 0.05 1.4 ± 0.16 (109%)∗∗∗

1.19 ± 0.08 2.85 ± 0.19 (140%)∗∗∗

6.0 ± 0.56

0.38 ± 0.03

0.45 ± 0.03

0.87 ± 0.03

16.2 ± 1.2 (189%)∗∗∗

0.7 ± 0.05 (49%)∗∗∗

1.81 ± 0.13 (170%)∗∗∗

2.58 ± 0.16 (117%)∗∗∗

4.3 ± 0.56

0.24 ± 0.04

0.37 ± 0.03

0.71 ± 0.05

11.2 ± 1.17 (100%)∗∗∗

1.02 ± 0.16 (117%)∗

1.14 ± 0.16 (70%)∗∗∗

2.19 ± 0.26 (85%)∗∗∗

4.5 ± 0.43

0.34 ± 0.07

0.46 ± 0.06

0.91 ± 0.13

6.83 ± 0.79 (22%)+

0.67 ± 0.13 (42%)+

1.08 ± 0.17 (61%)∗∗

1.85 ± 0.28 (56%)∗∗∗

n = 6 per group; b.w.: body weight; numbers in parentheses represent the percentages of increase of urine volume, sodium, potassium and chloride as compared with the control (saline); concentrations of the above mentioned electrolytes are expressed as mEq./l. + P = 0.1. ∗ P = 0.05. ∗∗ P = 0.01. ∗∗∗ P = 0.025.

Tension (g) (mean ± S.E.M.)

0.02 0.02 0.04 0.06 0.12

259

Table 2 Muscular contractions in the Guinea pig ileum induced by aqueous extracts of T. terrestris

Extract concentration (mg crude/ml organ bath)

0.085 0.18 0.29 0.35 0.44 ± ± ± ± ±

3.4 6.8 13.6 27.2 54.4

The above contractile responses of the extract were prevented by prior treatment with atropine sulfate 0.6 ␮g/ml bath.

In addition to the diuretic activity of T. terrestris extract, it evoked contractile activity in Guinea pig ileum as shown in Table 2 and Fig. 1. T. terrestris and Z. mays extracts had no significant effect on the spontaneous motor activity and rectal temperature. They also produced no change in behaviour, food and water intake, and morphology of viscera. The computed LD50 is 19.8 g/kg body weight expressed as crude plant for T. terrestris and 14.5 g/kg body weight for Z. mays.

Fig. 1. Effect of graded concentrations (mg crude/ml organ bath) of T. terrestris on Guinea pig ileum.


Saline Furosemide 120 mg/kg b.w. T. terrestris 5 g crude/kg b.w. T. terrestris and Z. mays 5 g crude b.w. Z. mays 5 g crude/kg b.w.

Pre-treatment

260


4. Discussion Guinea pig ileum is a useful preparation to study the effect of drugs on smooth muscles and the results obtained might be considered for the induction of the effect on other smooth muscles such as the ureter. Thus, the increased tonicity of the smooth muscles which was produced by T. terrestris extract together with its diuretic activity had helped in the propulsion of stones along the urinary tract. Other workers (Sangeeta et al., 1994; Anand et al., 1994) have also shown its preventive effect on urinary stones. We have not investigated this aspect of its action, which is worth studying. However, Arcasoy et al. have shown that T. terrestris L. saponin mixture caused a significant decrease in the peristaltic movements of the isolated sheep ureter and rabbit jejunum preparations in a dose-dependent manner, and there was no effect on the isolated rabbit aorta and its contractile response to KCl or noradrenaline (Arcasoy et al., 1998). T. terrestris has been found safe without any side effects in a large cohort of patients treated for ischaemic heart disease (Wag et al., 1990). However, Bourke has reported that it caused some hepatopathy in sheep (Bourke, 1983). The diuretic action of this plant makes it useful as an anti-hypertensive agent, and its hypotensive activity has been previously reported (Mossa et al., 1983). Its other reported activities merit further investigation. We might have used high furosemide and plant doses and feel that further investigation should test lower doses on a larger number of animals by dose. We conclude that our findings had demonstrated that T. terrestris has significant diuretic and contractile effects which makes it useful in the propulsion of urinary stones. Nevertheless, it merits further pharmacological investigation before embarking on its use as a remedy for any disease. References Adiomoelja, A., 2000. Phytochemicals and the breakthrough of traditional herbs in the management of sexual dysfunction. International Journal of Andrology 23 (Suppl. 2), 82–84.

Ali, N.A., Julich, W.D., Kusnick, C., 2001. Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. Journal of Ethnopharmacology 74 (2), 173–179. Anand, R., Patnek, G.K., Kulshreshtha, D.K., Dawan, B.N., 1994. Activity of certain fractions of Tribulus terrestris fruits against experimentally induced urolithiasis in rats. Indian Journal of Experimental Biology 32 (8), 548–552. Arcasoy, H.B., Erenmemisoglu, A., Tekol, Y., Kurucu, S., Kartal, M., 1998. Effect of Tribulus terrestris L. saponin mixture on some smooth muscle preparations: a preliminary study. Bollettino Chimico Farmaceutico 137, 473–475. Bourke, C.A., 1983. Hepatopathy in sheep associated with Tribulus terrestris. Australian Veterinary Journal 60 (6), 189. Brown, G.A., Vukovich, M.D., Martini, E.R., Kohut, M.L., Frank, W.D., Jackson, D.A., 2001. Endocrine and lipid responses to chronic androstenendiol-herbal supplementation in 30–58 year old men. Journal of the American College of Nutrition 20 (5), 520–528. Cummings, J.R., Haynes, J.D., Lipchuck, L.M., Rosenberg, M.A., 1960. The Journal of Pharmacology and Experimental Therapeutics 128, 914. Quoted from Turner, R.A., 1965. Screening Methods in Pharmacology. Academic Press, New York. Lin, Z.X., Hoult, J.R., Raman, A., 1999. Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo. Journal of Ethnopharmacology 66 (2), 142–150. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluation of dose-effect experiments. The Journal of Pharmacology and Experimental Therapeutics 96, 99–113. Liu, Q., Chen, Y., Wang, J., Chen, X., Han, Y., 1995. Preventive effect of Tribulus terrestris L. on genetic damage. Zhonggno Zhong Yao Za Zhi 20 (7), 477–479. Mossa, J.S., Al-Yahya, M.A., Al-Meshal, I.A., Tariq, M., 1983. Phytochemical and biological screening of Saudi medical plants. Fitoterapia 54 (4), 147–152. Nandal, S.N., Bhatti, D.S., 1983. Preliminary screening of some weed shrubs for their nematocidal activity against Meloidogyne Javanica. Indian Journal of Nematology 13 (1), 123–127. Perry, W.L.W., 1979. Pharmacological experiments on isolated preparations by the staff of Department of Pharmacology, University of Edinburgh, Churchill Livingstone, Edinburgh. Sangeeta, D., Sidhu, H., Thiud, S.K., Nath, R., 1994. Effect of Tribulus terrestris on oxalate metabolism in rats. Journal of Ethnopharmacology 44 (3), 61–66. Turner, R.A., 1965. Screening Methods in Pharmacology. Academic Press, New York. Wag, B., Ma, L., Liu, T., 1990. 406 cases of angina pectoris in coronary heart disease treated with saponin of Tribulus terrestris. Zhong Xi Yi Jie He Za Zhi 10 (2), 85–87.


Anti-inflammatory and analgesic activity in the polyherbal formulation Maharasnadhi Quathar M. Ira Thabrew a,∗ , M.G. Dharmasiri b , L. Senaratne c a

Department of Biochemistry, Faculty of Medicine, University of Kelaniya, 6, Talagolla Road, Ragama, Sri Lanka b Department of Zoology, Faculty of Science, University of Colombo, 03 Colombo, Sri Lanka c Bandaranayake Memorial Ayurvedic Research Institute, Nawinna, Sri Lanka Received 9 August 2002; received in revised form 20 December 2002; accepted 20 December 2002

Abstract Maharasnadhi Quathar (MRQ) is a polyherbal preparation recommended by Ayurvedic medical practitioners for treatment of arthritic conditions. An investigation has been carried out with rats and human rheumatoid arthritis (RA) patients, to determine the anti-inflammatory and analgesic potential of MRQ. Results obtained demonstrate that MRQ can significantly and dose-dependently inhibit carrageenan-induced rat paw oedema (the inhibition at 3 h was greater than at 1 h after induction of oedema). MRQ could also increase the reaction time of rats in the hot-plate test (by 57% after the first hour of treatment), although it had no effect on the reaction time in the tail-flick test, indicating that MRQ possesses analgesic activity that is probably mediated via a supra-spinal effect. MRQ also exerted a dose-dependent (a) protective effect on heat-induced erythrocyte lysis, and (b) inhibition of 5-lipoxygenase activity. In RA patients, after 3 months of MRQ treatment, there was a marked improvement in the pain and inflammation experienced by the patients as well as in the mobility of the affected joints. From the overall results obtained, it may be concluded that MRQ possesses significant anti-inflammatory and analgesic activities. Alteration in synthesis of prostaglandins and leukotrienes, membrane stabilization and anti-oxidant activity are some of the possible mechanisms through which MRQ mediates its anti-arthritic effects. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Maharasnadhi Quathar; Anti-inflammatory; Analgesic; Carrageenan; Anti-oxidant; Arthritis

1. Introduction In Sri Lanka, Ayurvedic and other traditional medical practitioners use many herbal preparations for the treatment of arthritis (Attygalle, 1952; Jayaweera, 1982). However, the claimed pharmacological activities of many of these formulations have not been proven nor refuted by controlled studies. According to the Kashaya Sangrahaya (1927) and Ayurveda Pharmacopoeia (1975), Maharasnadhi Quathar (MRQ) is a polyherbal formulation believed to have the potential for providing relief to rheumatoid arthritis (RA) patients. This formulation is prepared from parts of 26 different plants (Thabrew et al., 2001) that are used in traditional medicine for a variety of purposes (Ayurveda Pharmacopoeia, 1975; Jayaweera, 1982) such as (a) reduc∗ Corresponding

author. E-mail address: [email protected] (M.I. Thabrew).

tion of pain (e.g. Alpinia calcarata, Sida cordifolia, Cedrus deodara, Adhatoda vasica, Boerhaavia diffusa, Tragia involucrate and Piper chaba hunter), (b) reduction of inflammation (e.g. Barleria prionitis, A. vasica, C. deodara, B. diffusa, and Solanum xanthocarpum), and (c) anti-pyretic activity ( e.g. Coriandrum sativum, Zingiber officinale, Ricinus communis, A. calcarata and C. deodara). Included are also plants able to (a) improve appetite and digestion (e.g. Cyperus rotundus, Kaempteria galanga, C. deodara, Acorus calamus and Z. officinale), (b) act as immunostimulants (e.g. Tinospora cordifolia and S. cordifolia), and (c) act as diuretics (e.g. B. prionitis and C. deodara) and laxatives (e.g. Cedrus ricinus, Cassia fistula, R. communis and Terminalia chebula). The anti-arthritic properties in this formulation have, however, not been subject to any scientifically controlled investigations so far. Investigations have therefore been carried out using RA patients and rats as experimental models, to assess the analgesic and anti-inflammatory potential of MRQ.


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2. Materials and methods

2.5. Preparation and dosage of MRQ

2.1. Materials

The parts of plants used in the preparation of the MRQ were collected, sun dried and mixed in the required proportions by the Pharmacist at the BMARI according to instructions in the Ayurveda Pharmacopoeia (1975). The preparation of the drug formulation for patient treatment and the daily dosage were in accordance with information in the Ayurveda Pharmacopoeia (1975). The plant mixture containing 80 g of A. calcarata plus 40 g of a mixture of parts of 25 other plants (1.6 g of each) was boiled in 1.6 l water until the volume was reduced to 400 ml. Half this volume was administered orally at 6 a.m. while the remainder was given 12 h later. When the drug was to be administered to rats, the extract was freeze dried and stored at −20 ◦ C until required (the yield of the freeze-dried extract was 19.8%). During experimentation, the required amount of freeze-dried extract was weighed and reconstituted in distilled water. Since for ethical reasons, an allopathic drug (e.g. NSAID) used in the treatment of RA patients could not be administered as a positive control to patients attending the RA clinics at the BMARI, the effects produced by MRQ treatment were compared with those produced by another herbal preparation Weldehi Choornaya (WC) considered to be beneficial for the treatment of RA patients according to the Ayurveda Pharmacopoeia (1975) and several Ayurvedic practitioners in Sri Lanka. Thirty female patients with uncomplicated RA (age range 30–68 years) were included in this group. WC was prepared by the Pharmacist, BMARI, by grinding to a fine powder, a sun-dried plant material mixture containing Smilax glabra tuber (480 g), Myristica fragrans seeds (40 g), Piper nigrum seeds (30 g), together with 20 g of a mixture of Eugenia caryophyllus (dried flower buds) and mace plus 1.6 g each of Triticum sativum seeds, Hyoseyamus reticulates seeds, Nigella sativa seeds, Azima tetracantha roots, Terminalia catappa seeds, T. cordifolia stem, Glycyrhiza glabra stem, Melochia corchorifolia root and Gymnema lactiferum root. Half a teaspoon of the powder mixed with a little bees honey was administered orally to patients twice a day, as recommended in the Ayurveda Pharmacopoeia (1975).

All chemicals were purchased from Sigma Chemical Co. Ltd., St. Louis, USA. MRQ was obtained by kind courtesy of the Director, Bandaranayake Ayurvedic Research Institute (BMARI), Nawinna, Sri Lanka. 2.2. Animals Male, Wistar rats (150–250 g) from a colony maintained at the Department of Zoology, University of Colombo were used. The rats were housed under standardised animal house conditions and fed with pelleted rat chow purchased from Vet House Ltd., Colombo, Sri Lanka. All animals had free access to water at all times. 2.3. Patient selection Patients for the study were selected from individuals attending the routine RA clinics at the BMARI. A total of 50 patients with uncomplicated RA were selected. All patients who participated in the study were females whose ages ranged from 35 to 65 years. When choosing patients for the study, preference was given to those who were not on any medication at the time of selection and were free of any other systemic disease, which required specific treatment. A 2-week washout period was advised for patients who had been on other forms of treatment for RA prior to selection for the present study. 2.4. The composition of MRQ The composition of this formulation is as shown below: Alpinia calcarata (rhizome) Ricinus communis (roots) Kempteria galanga (rhizome) Adhatoda vasica (root) Terminalia chebula (dried fruit) Cyperus rotundus (tubers) Tinospora cordifolia (stem) Finiculum vulgare (seeds) Withania somnifera (rhizome) Cassia fistula (bark) Nigella sativa (seeds) Coriandrum sativum (seeds) Solanum xanthocarpum (whole plant)

Sida cordifolia (root) Cedrus deodara (bark) Acorus calamus (rhizome) Zingiber officinale (rhizome) Piper chaba hunter (roots)

2.6. Anti-inflammatory studies in rats Boerhaavia diffusa (roots) Argyra speciosa (stem) Tribulus terrestrin (whole plant) Aconitum heterophyllum (rhizome) Asparagus racemosus (rhizome) Barleria prionitis (root) Solanum melongena (roots) Tragia involutraca (whole plant)

2.6.1. Effect of MRQ on carrageenan-induced paw oedema This was determined as described by Winter (1962). Forty-five male rats were randomly divided into five equal groups. The rats in groups 1–3 were orally treated with 250, 500 and 750 mg/kg of extract in 1 ml distilled water, respectively. Rats in groups 4 (controls) and 5 were treated with 1 ml distilled water and indomethacin (4 mg/kg), respectively. After 1 h, oedema was induced in all these rats by injecting with 0.05 ml of 1% carrageenan suspension in normal saline, into the sub-plantar surface of the left hind paw. The volume of the paw was measured 30 min before injection and at 1 and 3 h after the injection of carrageenan.

M.I. Thabrew et al. / Journal of Ethnopharmacology 85 (2003) 261–267

Oedema was expressed as the increment in paw thickness due to carrageenan administration. 2.6.2. Effect on heat-induced haemolysis The inhibitory effect of MRQ on rat erythrocyte haemolysis was assayed by a slight modification of the method described by Perez et al. (1995). Twenty microlitres of uncoagulated fresh rat blood was added to vials containing 1 ml of 0.1 M phosphate-buffered saline (PBS, pH 7.4). MRQ extract or aspirin in PBS was added to the vials (in triplicate), so as to achieve final concentrations of 200, 250, 300, 350, 400 or 450 ␮g/ml MRQ or 200, 250 or 300 ␮g/ml aspirin. PBS (15 ␮l) was added to control vials (n = 3). Prior to addition to the vials, the drug solutions were subject to centrifugation at 3000 × g on a bench centrifuged for 10 min to make sure that all the material had completely dissolved. No residues were ever observed after centrifugation indicating complete solubility of the drug preparations used. After mixing contents in vials and pre-incubating at 37 ◦ C for 15 min, the mixtures were heated for 25 min at 54 ◦ C. After spinning down the precipitate, the absorbance of the supernatant was measured at 540 nm in a spectrophotometer (JASCO V500, Jasco Corporation, Japan). The percent inhibition of haemolysis with respect to the controls was calculated as follows: A540 control − A540 sample Percent inhibition = × 100. A540 control 2.6.3. 5-Lipoxygenase inhibition assay To investigate whether the anti-inflammatory activity of MRQ could be attributed in part to an inhibition of leucotriene mediated effects, the lipoxygenase inhibitory effect of MRQ extract was assayed according to the method based on Magee (1965), as described in the assay kit purchased from Sigma Chemical Co., St. Louis, MO, USA. In the reaction mixture, MRQ extract was present in final concentrations of 50, 100 or 150 ␮g/ml; 25 ␮l distilled water was used as the control. The absorbance of each reaction mixture was measured at 234 nm at 1 min intervals from the addition of the enzyme into the reaction vessel. The change in absorbance per minute was then calculated.

263

2.8. Effects on RA patients 2.8.1. Study design Prior to being included in the study, informed, written consent was obtained from each patient, after being given an explanation of the method of treatment, objective of the trial, the need to be hospitalised during the first month of treatment, necessity for withdrawal of blood for investigation and the right to withdraw from the trial at any time during the trial. During each patient’s stay in the ward, a clinical assessment was carried out each day to assess the extent of swelling, pain, morning stiffness, grip strength and range of movement of affected joints. Swelling of large joints and digits, grip strength, and range of movement of affected joints were assessed by methods described by Ranasingha (1979). At the beginning of the trial and then at the end of every 30 days of the trial period, blood from each patient was assessed for (a) the activities of alanine amino transferase, aspartate amino transferase, and alkaline phosphatase and (b) bilirubin concentration (liver function tests), using commercially available reagent kits purchased from Randox Laboraties Ltd., UK. 2.8.2. Statistical analysis Results are presented as the means ± S.E.M. Results were analysed using one way ANOVA and linear regression analysis. A P-value 90

Ascomycota/Onygenales

OMH-FR2385

30

30

Dermatophyte

Ascomycota/Microascales

OMH-FR2625

67.5

90

Zygomycota/Mucorales Ascomycota/Onygenales

OMH-FR2874 OMH-T2379

0.01 67.5

30 67.5

Sinusitis, central nervous system colonizer Subcutaneous infections Dermatophyte

0.37 >90

>90 90 0.37 0.04

1.1 30

0.37 10

Systemic and subcutaneous infections, central nervous system and respiratory tract colonizer Systemic infections in immunocompromised patients Cutaneous infections, central nervous system colonizer Cutaneous and subcutaneous infections Subcutaneous and respiratory infections Subcutaneous infections

a N.D. Lees, IUPUI, Indianapolis, IN; OGH: Ontario General Hospital, Ottawa, ON; OMH: Ontario Ministry of Health, Toronto, ON; ATCC: American Type Culture Collection. b MICs for amphotericin B (amphB) and ketoconazole (keto) given as concentration (␮g/ml) that results in >80% growth inhibition where resistance is considered to be at concentrations of ≥8 and ≥2 ␮g/ml, respectively.

filamentous fungi were prepared for disk diffusion assays as previously described (Jones et al., 2000). Sterile 7.5 mm diameter, 3M paper discs were impregnated with 2 mg (10 ␮l) ethanolic plant extract prepared as described below, allowed to air dry, and placed face down on the inoculated agar surface. As a positive control we used 2 mg berberine, a known antifungal derived from a species of Berberis (Stermitz et al., 2000), administered in 10 ␮l of ethanol. Discs impregnated with 10 ␮l of 99% ethanol and allowed to air dry served as negative controls. Plates were then incubated at 30 ◦ C in the dark for 48 h, after which the diameter of the fungal growth inhibition zone was measured. An extract was classified as active when the diameter of the inhibition zone was equal to or larger than 8 mm, 0.5 mm larger than the diameter of the paper disc. All transfers of fungal strains were done under a biohazard level 2 laminar flow hood (Bioclone2, Microzone, Ottawa, Canada). Several of the strains used were resistant to amphotericin B and/or ketoconazole based on minimum inhibitory concentration (MIC) determinations in broth dilution assays using standardized methods (Ficker et al., 2003; Rex et al., 2001). For determining ketoconazole and amphotericin B MIC values (Table 1), sterile microtitre plates were used

with each well containing 100 ml of Sabouraud’s dextrose broth (EM Science, Gibbstown, NJ) and ∼103 colony forming units/ml. Concentration gradients ranging from 0.005 to 90 ␮g/ml for each of amphotericin B and ketoconazole (Sigma, Oakville, ON) were established in the microtitre plate rows and growth was evaluated after 2 days incubation, without shaking, at 30 ◦ C. MICs were determined at least three times for each fungal strain and recorded as the minimum concentration tested at which there was >80% reduction in fungal growth. 2.2. Study site, ethnobotanical data and plant material The Kenyah village of Long Sungai Barang, is located at the headwaters of the Kayan river in the Apo Kayan region of East Kalimantan (Indonesian Borneo). Coordinates of the site are approximately 2◦ N latitude, 115◦ E longitude. The altitude of the study area is 800 m and the mean annual rainfall in this area is between 3000 and 4000 mm with the driest months being July and August. The mean daily temperature is 20 ◦ C, fluctuating between 15 and 25 ◦ C (Leaman, 1996). The primary ethnobotanical data set used in this study was compiled by Leaman (1996). Among the documented use of

Table 2 Mean diameter (mean ± S.E.) of fungal growth inhibition zones Fungal speciesb Yeast-like C. neo (stalk)c

A. galanga A. galanga (rhizome)c A. mutica (stalk) A. mutica (rhizome) C. globosus (rhizome) C. zedoaria (rhizome) E. elatior (fruit)c E. elatior (rhizome) E. littoralis (rhizome) H. cylindricum (rhizome)c Hornstedtia sp. (stalk) Hornstedtia sp. (rhizome) Z. pachysiphon (inflourescence)c Z. pachysiphon (rhizome) Z. purpureum (rhizome) Z. officinale (rhizome)d Berberine Ethanol a

10.2 ± 0.7

Filamentous S. cer

al-1

CN1A

D10

W. der 11.9 ± 0.8 27.3 ± 7.5 8.2 ± 0.7

14.8 ± 2.1 13.3 ± 0.2 14.6 ± 1.5 13.5 ± 0.0 12.8 ± 0.7 11.5 ± 1.0

A. alt

A. fum

F. oxy

M. gyp

P. boy

Rh sp.

T. men

8.2 ± 0.7 9.5 ± 0.0 14.4 ± 1.6 11.9 ± 0.4 20.6 ± 1.6 31.1 ± 3.2 12.2 ± 1.3 16.6 ± 0.6 9.5 ± 0.0

25.3 ± 0.4 27.9 ± 0.0 9.5 ± 0.0

9.5 ± 0.0 14.2 ± 2.4 9.5 ± 0.0 8.2 ± 0.7 26.4 ± 0.9

8.8 ± 0.7

11.4 ± 0.5

9.3 ± 0.9 8.4 ± 0.9 11.3 ± 1.2 11.1 ± 0.4 14.2 ± 0.8 9.5 ± 0.0 27.3 ± 0.2 17.1 ± 0.2 11.5 ± 1.1 20.6 ± 2.3 11.5 ± 0.0 20.8 ± 3.0 12.2 ± 1.2 8.8 ± 1.1 18.2 ± 3.0 15.7 ± 0.6 12.6 ± 1.2 19.9 ± 0.4 11.1 ± 0.4 25.1 ± 0.4 16.8 ± 1.1 10.0 ± 0.2 13.9 ± 1.7 10.2 ± 0.4 19.9 ± 0.4 13.1 ± 0.4 21.3 ± 1.1

Full scientific and Kenyah names and voucher herbarium specimens are given in the text under Section 2.2. C. neo: Cryptococcus neoformans; S. cer: Saccharomyces cerevisiae; al-1, CN1A, D10: Candida albicans strains; W. der: Wangiellia dermatitidis; A. alt: Alternaria alternata; A. fum: Aspergillus fumigatus; F. oxy: Fusarium oxysporum; M. gyp: Microsporum gypseum; P. boy: Pseudallescheria; Rh sp.: Rhizopus species; T. men: Trichophyton mentagrophytes. c Used as remedy for ailments that could be attributed to fungal pathogens. d From Togo, Africa.

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Zingiberaceae speciesa and plant part

b

291

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240 plant species by the Kenyah people, Leaman reported on 20 species belonging to the Zingiberaceae, of which four were used as remedies for ailments that could be attributed to fungal pathogens. Such ailments included itchiness, skin infections, vitiligo, dog mange, weeping wounds and ringworm. Plant parts used in Kenyah traditional remedies, in addition to rhizomes, were collected in March 2001, for four species used to treat fungal infections and for six species used for ailments that were not likely caused by fungal pathogens (Table 2). At least three representatives of each of the 10 Zingiberaceae species were collected using their local Kenyah names, under the Indonesian National Institute of Science (LIPI) permit number 4488/V.3/KS/2001. The rhizome was the only plant part collected when it was the part used in traditional remedies. For the majority of remedies, one plant part is used and is either directly applied to the affected area as a heated poultice, ointment, infusion or body rub, or a decoction or infusion is made and ingested. Plant specimens were collected according to the method described by Burtt and Smith (1976), with ethanol used as the preserving agent, and taxonomic determinations were conducted at the Herbarium Bogoriense, Bogor, Indonesia. Voucher specimens were placed in the Herbarium Bogoriense and the University of Ottawa Herbarium, Ottawa, Canada. The collected plant species (with local Kenyah name and University of Ottawa Herbarium voucher number) included Alpinia galanga (L.) Sw. (Lia Buke’ Borak, UO-18901), Alpinia mutica Robx. (Kerapat Bai, UO-18902), Costus globosus Bl. (Pe Luan, UO-18903), Curcuma zedoaria (Berg.) Roscoe (Lia Mit Pute’, UO-18904), Etlingera elatior (Jack) R.M. Smith (Nyanding, UO-18905), Etlingera littoralis (König) (Ubut Ladyei, UO-18906), Hedychium cylindricum Ridley (Tubo Sakai, UO-18907), Hornstedtia sp. (Lamei, UO-18908), Zingiber pachysiphon Burtt and Smith (Ubut Bele Sua’, UO-18909) and Zingiber purpureum Roscoe (Lia Sukeng, UO-18910). For comparative purposes, we also included an extract of the rhizomes of Z. officinale (UO-17722) collected in Togo, Africa. 2.3. Extract preparation In total, 16 ethanol extracts of Zingiberaceae were prepared for antifungal activity testing. Extracts were prepared by blending approximately 500 g (fresh weight, consisting of ≥3 plants/species) of the preserved plant material in 99% ethanol (1:3 (w/v) ratio). The mixture was filtered using a Büchner funnel, the filtrate was retained and the residue was again blended in ethanol and processed as above. The filtrates were combined and the solvent was rotoevaporated at 45 ◦ C to near dryness, whereupon the extract was freeze-dried for 48 h to achieve complete dryness. The process yielded approximately 35–45 g of dried extract. Dried extract (200 mg) was reconstituted in 1 ml of 99% ethanol

for a final concentration of 2 mg/10 ␮l for antifungal disk assays.

3. Results and discussion The results of testing 16 ethanol extracts from species of Zingiberaceae for antifungal activity against six yeast-like and seven filamentous fungi are presented in Table 2. Several of the tested members of Zingiberaceae contain potent inhibitors of fungal growth, validating their use in traditional antifungal remedies. Extracts from rhizomes of A. galanga, C. zedoaria and Z. purpureum, in particular, had pronounced inhibitory activities against a wide range of fungi including strains resistant to the common antifungals amphotericin B and ketoconazole. Rhizome extracts from these species and Z. officinale were notable as inhibitors of the Rhizopus sp. strain used, the only strain that was not inhibited by berberine. The extracts that inhibited the filamentous fungi are of particular interest, since all but A. alternata were shown to be resistant to ketoconazole and/or amphotericin B. Interestingly, C. zedoaria inhibits a distinct group of fungi compared to A. galanga and Z. purpureum, suggesting that different antifungal agents are present in some species. Of the four species used as traditional antifungal remedies, rhizome extracts of A. galanga stood out as having significant antifungal activity in our laboratory bioassays. Clearly, the use of A. galanga rhizomes for treating ringworm is associated with these significant, broad-range antifungal activities. The antimicrobial potential of other Zingiberaceae should be further investigated since several pathogenic agents aside from fungi, including bacteria, protozoa and viruses, can cause symptoms of itchiness, vitiligo and infections of wounds. In addition, the wound healing responses to extracts of Zingiberaceae should be evaluated in the event that remedies that incorporate Zingiberaceae species also act to enhance the host natural defenses.

4. Conclusion As a group, members of the Zingiberaceae family are important in Kenyah traditional medicine for the treatment of many illnesses including infections attributable to fungi. Significant antifungal activities were evident with extracts from members of the Zingiberaceae, in particular those of A. galanga, C. zedoaria, Z. purpureum and Z. officinale. Of specific interest are those extracts that inhibited several fungi that are resistant to commonly used antifungals. Of the plant parts collected and tested, which included the rhizome, fruit, inflorescence, and stalk, the rhizomes had significantly higher activity. As species belonging to the Zingiberaceae family are generally regarded as safe for human consumption, they are excellent candidates for development as antifungal phytomedicines.


Acknowledgements The authors thank the Kenyah community of Long Sungai Barang and the Indonesian National Institute of Sciences (LIPI) for sponsorship of the field research. This work was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Research Grants to MLS and JTA and by a Canadian International Development Agency (CIDA) Innovative Research Award for field research to CEF.

References Alexander, B.D., Perfect, J.R., 1997. Antifungal resistance trends towards the year 2000. Implications for therapy and new approaches. Drugs 54, 657–678. Burtt, B.L., Smith, R.M., 1976. Notes on the collection of Zingiberaceae. Flora Malaysiana Bulletin 29, 2599–2601. Elliott, S., Brimacombe, J., 1987. The medicinal plants of Gunung Leuser National Park, Indonesia. Journal of Ethnopharmacology 19, 285–317. Ficker, C.E., Arnason, J.T., Omar, S., Sanchez-Vindas, P., Poveda Alvarez, L., Akpagana, K., Gbéassor, M., De Souza, C., Smith, M.L., 2003. Inhibition of diverse pathogenic fungi by ethnobotanically selected plant extracts. Mycoses 46, 1–9. Grosvenor, P.W., Gothard, P.K., McWilliam, N.C., Supriono, A., Gray, D.O., 1995. Medicinal plants from Riau province, Sumatra, Indonesia. Part 1. Uses. Journal of Ethnopharmacology 45, 75–95.

293

Hirschhorn, H.H., 1983. Botanical remedies of the former Dutch East Indies (Indonesia). Part I. Eumysetes, Pteridophyta, Gymnospermae, Angiospermae (monocotyledons only). Journal of Ethnopharmacology 7, 123–156. Jones, N.P., Arnason, J.T., Abou-Zaid, M., Akpagana, K., Sanchez-Vindas, P., Smith, M.L., 2000. Antifungal activity of extracts from medicinal plants used by First Nations Peoples of eastern Canada. Journal of Ethnopharmacology 73, 191–198. Leaman, D.J., 1996. The medicinal ethnobotany of the Kenyah of East Kalimantan (Indonesia). Ph.D. thesis, University of Ottawa, Ottawa, Canada. McCutcheon, A.R., Ellis, S.M., Hancock, R.E.W., Towers, G.N.H., 1992. Antibiotic screening of medicinal plants of the British Columbian native peoples. Journal of Ethnopharmacology 37, 213–223. Omar, S., Lemonnier, B., Jones, N., Ficker, C., Smith, M.L., Neema, C., Towers, G.N.H., Goel, K., Arnason, J.T., 2000. Antimicrobial activity of extracts of eastern North American hardwood trees and relation to traditional medicine. Journal of Ethnopharmacology 73, 161–170. Rex, J.H., Pfaller, M.A., Walsh, T.J., Chaturvedi, V., Espinel-Ingroff, A., Ghannoum, M.A., Gosey, L.L., Odds, F.C., Rinaldi, M.G., Sheehan, D.J., Warnock, D.W., 2001. Antifungal susceptibility testing: practical aspects and current challenges. Clinical Microbiological Reviews 14, 643–658. Stermitz, F.R., Lorenz, P., Tawara, J.N., Zenewicz, L.A., Lewis, K., 2000. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5 -methoxyhynocarpin, a multidrug inhibitor. Proceedings of the National Academy of Sciences USA 97, 1433–1437. Wu, T.L., Wu, Q.G., Chen, Z.Y., 1996. In: Proceedings of the Second Symposium on the Family Zingiberaceae. Zhongshan University Press, Guangzhou China, pp. 196–218.