Narender, T., Puri, A., Shweta, Khaliq T., Saxena, R., Bhatia, G. and Chandra, R., 2006. ... Tripathi, S.N., Chandola, H:M., Singh, B.C. and Chadha, AN., 1990.
Phytochemicals A THERAPEUTANT FOR CRITICAL DISEASE MANAGEMENT
D.R. Khanna A.K. Chopra G. Prasad D.S. Malik R. Bhutiani
emicals: A Therapeutant for Critical Disease Management (2008) . D.R. Khanna, A.K. Chopra, G. Prasad, D.S. Malik and R. Bhutiani
Pages 99-106
by: DAYA PUBLISHING HOUSE
Chapter 20
sessment of Hypoglycemic Activity of Indigenous Herbs Rahul Gupta and A.M. Saxena* Department of Zoology, University of Lucknow, Lucknow,India-226007
ABSTRACT Diabetes has become the global metabolic disorder conspiring socio-economic and health hazards. India has a glorious history of medicinal plants and herbs. Diabetes has been treated with plant medicines since antiquity. Even today for healthy life people prefer herbal medicines from indigenous source. Recent scientific investigation has confirmed the efficacy of many of these preparations. Herbal remediation could be relatively cheap, effective and safe in complete cure of diabetes. It also revives our traditional culture. Current write-up provides fruitful guidelines and unified methodology for search of antidiabetic herbs.
Keywo1·ds: Ayurveda, Diabetes melliius, Indigenous herbs, Experimental models, Extraction.
Introduction Diabetes is emerging as the epidemic metabolic disorder of world, imposing considerable socioeconomic and health burden in almost every age group. Diabetes is characterized by presence of excessive sugar in blood and urine due to deficiency in production of insulin by pancreas or presence of ineffective insulin. Diabetes mellitus because of its frequency affects almost every cell in body and • Corresponding Author: E-mail: anandmsaxenassredlffmail.corn
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Phytochemicals: A Therapeutant for Critical Disease Management
essential biochemical activities. The productive' atmosphere to diabetes is provided by heredity, obesity, severe and continued mental stress, endocrine imbalance, dietary imprudence, after effects of infection, big differences in social structure and re.duction in physical labour etc. For treatment of diabetes, the services of insulin limited to those who could afford them, at the same time their repeated dose induce insulin resistance in patients. Studies reveal that the uses of synthetic oral hypoglycemic agents have adverse health effects other than blood sugar lowering. India has a rich flora includes a vast variety of medicinal plants; almost all of them are native to the soil. Therefore the role of anti-diabetic herbs from indigenous source has become more important. Even a number of plants and herbs have been successfully tested but an authentic herbal antidiabetic remedy for all is yet to be traced out. Great opportunities are still open for scientific investigations of herbal medicine for diabetes mellitus. For search of genuine antidiabetic herbs from indigenous source it is necessary to use proper methodology in generating correct and pure scientific data. In this writeup an attempt is made to provide a unified methodology and fruitful guidelines for proper collection - ...ojplant material, their extraction and preliminary screening of blood sugar lowering activity.
Indian History of Herbs Footprints of herbal medicines against various maladies have been made during the days of Vedic glory. Aryans compiled their work related to herbs in holy Vedas when they came to north India. References about a number of herbal remedies have been mentioned in 'Rig-Veda' (about 200 B.C.). It was further pronounced distinctly in' Atharva-veda' ( about 200 B.C.) in which description of medicinal plants has also been made under separate chapter Ayurveda'. I
Sushruta (about 400 B.C.) was one of the students of Divodasa Dhanvantari, who compiled the teaching of his preceptor in 'Sushruta Samhita' (A compendium of ancient Indian surgery). Sushruta lays emphasis on the treatment of disease first and then advises the preservation of health. He carefully classified 700 drugs in 37 classes. It was Charak (about 600 B.C.) who made the scientific classification of herbal drugs based on remedial properties; he also compiled the information obtained from the work of hi~ Guru (Master) Agnivesa in his renowned treatise 'Charaka Samhita' (A compendium of general medicine) previously ·· it was known as 'Agnivesa Sarnhita'. In this he described 50 ganas (classes) of herbal remedies comprising 500 crude .drugs. In this process Nagarjuna further editing 'Sushruta Sarnhita', described the presence of active pharmacological materials of plants in bark, leaf, flower, fruit and root etc. Similar pharmacognostic classification was followed by Vagbhata 1 in his compilation 'Astanga Samhita'. Vagbhata 2 also made references about the effect of different types of plants in various diseases. Later on, Srangadhar (about 1400 AD.) mentioned the efficacy of herbal drugs and Dhanvantari (about 1500 A.O.) further classified the medicinal plants to avoid the confusion in identification. Bhavmishra (about 1600 A.O.) also wrote his monumental work entitled 'Bhavaprakash' and provided descriptive names of medicinal plants. In recent past from the accumulated knowledge of our ancient literature useful remedies and herbal drugs against Madhurneha have been identified and compiled by V.L. Sen (1923), Chopra et al. (1955), Nadkarni et al. (1976), Satyavati et al. (1987), Rahman et al. (1989), KR. Kirtikar and B.D. Basu (1993), S.K. Mukherjee et al. (1988), AM. Saxena et al. (2002), Grover et al. (2002), Mukherjee et al. (2006) and other workers. These workers made remarkable contribution in assessment and rearrangement of indigenous hypoglycemic herbs.
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t Status .ing an ample scope and obvious advantage for research and manufacturing of herbal products, even then the indigenous herbs have yet not been able in its commercial exploitation for cure of diabetes mellitus. Many times the mere chance and unsystematic approaches by t workers led to mutually inconsistent results and it become very difficult to arrive at final ion about blood sugar lowering effect of herbs. Some times these experiments leading to · g results generated with same plant. This was due to interplay of alterable like plant species, · plant used, extract preparation, experimental models used, dose and the route of administration
mmended Methodology o avoid above mentioned contentions here a unified guideline proposed for proper collection, ion of plant material and screening of its hypoglycemic activity. This may be useful in exploration ploitation of antidiabetic property of indigenous herbs and meet the various demands of a ard experimental set up. ·
per Collection of Plant Material First of all the collecting personal must have complete knowledge about identification of required t material. It enables to distinguish between required medicinal plant and other native species, ds unwanted mingling. He should possess information about environmental requirement and th of plant, harvesting conditions, best collection time, drying and processing techniques etc. ecting personal should also posses awareness of conservation process, sustainability and personal The plants from which test material required should be healthy and free from pest etc. Collection uld take place during early morning and under dry condition. The collecting equipments should cleaned before every campaign. Over harvesting and unnecessary damage to plant must be avoided. hanical damage to collected plant material should avoid preventing loss of active ingredient. uce the time of transportation of freshly collected plant material and take special care to avoid desirable quality changes of plant material. Maintain appropriate labeling and documentation of - llected plant material during each campaign.
For the drying and processing of plant material place and equipments .must be clean and · infected properly. The place should be protected from pests and domestic animals. The plant material should be air dried by spreading it in a thin layer to provide good air circulation and uniform ;trying. Direct exposur~ to sun light and directly drying on ground must be avoided for best preservation f active ingredient of plant material. The drying plant material should be regularly screened and discoloured, damaged or contaminated pieces removed (Figure 20.1). · Dried material should be packed properly in plastic bags or containers as early as possible to void pest attack. Whole process should be properly documented and monitored along with duly ecked by a responsible expert (Figure 20.2). · ..
reparation of Herbal Extract For extraction purpose many workers followed different procedures but study of literature suggest at Soxhlet extraction process provided standard results. The Soxhlet extractor was invented by
Phytochemicals: A Therapeuiani for Critical Disease Management
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Franz Von Soxhlet in 1879. It is a type of laboratory glassware designed to extract any compound fro a solid test material. The extraction assembly includes Soxhlet extraction chamber, vertical condenser, round botto boiling flask, water bath and water supply to condenser. For extraction, mechanically grinded co powder of dried plant material placed in porous cellulose 'thimble' made from filter paper, no thimble is placed in to extraction chamber. This extraction chamber is attached above a flask cont · a solvent (95 per cent ethanol or hexane) and below a condenser. Solvent in flask is heated to boil' in water bath, solvent evaporates and moves up in to the condenser, here it cool and drips down plant material. Now the chamber slowly fills with dripping solvent. The extraction chamber is desi in such a way that when solvent surrounding test material in 'thimble' exceeds a certain level overflows by siphon action back down in to boiling flask. This cycle of repeated washings percolations allowed many times. In every cycle a portion of compound from test material dissolves · solvent, once the compound reaches the boiling solvent flask, it stay there and does not participate· cycle. Only distilled solvent rises in extraction chamber (Figure 20.3). The extraction usually complete when solution in chamber is of same colour as pure solvent., the end of extraction the excess solvent may be recovered by using horizontal condenser or removed evaporator, leaving behind only extracted compound. This extract is in crude form and ready pharmacological screening. The prepared extract can be further purified as per requirement.
Importan t Steps in Extraction 1. The ratio of test material and solvent should be 1:10.
2. Boiling flask should be filled more than half with solvent before any extraction of test mate · 3. Before assembling extraction unit the joints of extraction chamber must be lubricated wi thin layer of white grease to avoid the risk of joint cracking. 4. Water supply to condenser should be proper. 5. Yielded crude extract or fractions should be partially standardized on the basis of yield dry p Ian t material/ unit wet plant and yield of crude extract/ unit dry test material.
Assessment of Hypoglycemia It is found that C. F. strain male albino rats of average weight of 150 gm preferred by vario workers for preparation of different experimental models and also proven very handy for screening blood sugar lowering effect. The dose of herbal extract to be used can be 250mg/kg b.w./oral SOOmg/kg b.w./ oral. On the basis of number of dose administration the experimental group can divided in to two categories.
Single Dose Administered Models Under this category following experimental models are prescribed for the assessment hypoglycemic activity of herbs: Fasted Model From the tail vein of overnight (18 hrs.) fasted rats blood sample should be collected at Ohr. extract must be administered just after. Now, at 1, 3 and 4 hrs or beyond collect the blood for su estimation by a standard method.
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(a)
(b)
(c)
(d)
Figure 20.1: Properly Collected and Dried Plant Material (a) Syzygium cumini(Bark); (b) Feronia elephantum (Bark); (c) Cinnamomum tamala (Leave); {d) Ficusbengalensis(Bark)
J
Excess amount of pellets (standard rat diet) must be putted in the cages on the previous evening, at some pellets left over in the next morning. Blood should be collected before and after · • strati on of extract as in fasted model.
From overnight (18 hrs.) fasted rats blood should be collected at Ohr for sugar estimation and extract must be administered, after half an hour 10 per cent glucose (l.Sgm/kg/ oral) should be · istered, now collected the blood for sugar estimation at 1h, 1 and 3 hrs respectively.
"" t
For the best outcomes it is necessary to use both the insulin deficient diabetic model and diabetic - with adequate insulin level as type I and type II diabetic subjects.
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Phytochemicals: A Therapeutant for Critical Disease Management
Figure20.2: Proper Storage of Plant Material
The diabetic model with insulin deficiency can be created through streptozotocin (65mg/kg b.w., i.v.) or alloxan monohydrate (40mg/kg b.w., i.v.) induced ~-cells necrosis of islets of Langerhans, for
Figure 20.3: Soxhlet ExtractionUnit
it of Hypoglycemic Activity of Indigenous Herbs
105
e can use the chilled citrate-phosphate buffer (pH 4.5) to dissolve streptozotocin or alloxan. ic animals (animals with blood sugar level above 200mg/100ml) can be selected by checking sugar with diastex. Now perform the experiments by using identical protocol as in fed model. It paratively difficult to create diabetic model of albino rats with adequate insulin level in blood. · d diabetes can be produced by using small and repeated dose of streptozotocin.
tiple Dose Administered Model this model the herbal extract should be administered in daily dose from 21-28 days. Now the edure followed in fed model must be repeated after final dose administration. By using this model can observe long term efficacy of extract. · Beside blood sugar lowering study, for preparation of perfect herbal antidiabetic drug it is necessary further study the parameters like activity of plant in different seasons, dose response relationship ucted with herbal preparation, its mode of action and its effect on different organs. At the end we t also go for the toxicity status of herbal preparation and its clinical viability.
icacy of Guidelines Proposed The correct identification, collection and extraction of plant material enable us to avoid confusion d mishap like degradation or low concentration of active material in a plant. The albino rats are se ected for experimental models because their blood sugar level are fairly stable and they have been ridely used in such studies leading to generation of standard data. The two categories of experimental group enable us to study short term and long term efficacy of t material. We proposed four different experimental models to increase the sensitivity of experiment
d spot optimum blood sugar lowering activity without fluctuation. The normal and diabetic test model will allow us to examine the efficacy of plant material against different backgrounds of blood sugar level. The study of literature suggests that oral administration of crude herbal extract or fraction - most preferable as it is in tune with the route of administration.
Conclusion There are hundreds indigenous plants and herbs still lie unidentified and unexplored, whose -irtues as described in ancient text known to few. To search some definitive herbal preventative measures against diabetes mellitus it is required to integrate the endeavors of different research personals in a definite direction on a scientific basis in which progress is most likely to be made. If we follow the proposed methodology as centralized activity, it will certainly enable us to generate reproducible results. These endeavors can provide relatively cheap, effective and safe herbal remedy tor diabetes.
Acknowledgement The authors are thankful to Prof. Nirupama Agarwal, Head, Department of Zoology, University of Lucknow, Lucknow, for providing necessary facilities.
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