Anticaries potential of ethnomedicinal plants used by Malayali ... - NOPR

Indian Journal of Traditional Knowledge Vol. 15(1), January 2016, pp. 109-115

Anticaries potential of ethnomedicinal plants used by Malayali tribals from Kolli Hills, India Kuppusamy Sekar1, Kasi Murugan2*, Perumal Pandikumar3, Saleh Al-Sohaibani2 & Savarimuthu Ignacimuthu3* 1

Research and Development Center, Bharathiar University, Coimbatore–641046, TN, India;

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Department of Botany and Microbiology, College of Science, PO Box 2455, King Saud University, Riyadh–11451, Saudi Arabia; 3

Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai–600034, Tamil Nadu E-mail: [email protected] Received 05 May 2015, revised 18 August 2015

Malayali tribals of Kolli hills, Tamil Nadu, India use many plants for oral healthcare. The present study documents the dental caries preventing medicinal plants used by them and attempts to validate their claim. An ethnomedicinal survey comprising field visits, collection of information using specific questionnaire was done to collect the medicinal plants used by them. The plants cited by the informants were collected and successively extracted with hexane, ethyl acetate and methanol and evaluated for their antimicrobial efficacy against four cariogenic clinical isolates. The MIC, anti-biofilm efficacy and GC-MS phytoconstituent identification were also done. This study identified 15 species that were commonly used by the Malayali tribals to maintain oral health and hygiene. Among the tested extracts, the methanol extract of Tephrosia purpurea (L.) Pers. Fabaceae showed highest inhibitory activity against the cariogenic isolates. At 1 mg/ml concentration, it inhibited the biofilm formation by 92.0%, 77.6%, 74.1% and 94.9% against L. casei, S. mutans, S. aureus and K. pneumoniae, respectively. The active methanol extract’s GC-MS analysis resulted in the identification of eleven major compounds. The rich traditional knowledge of tribal people has immense potential for caries and other oral health management along with pharmacological lead compound studies. Keywords: Medicinal plants, Anticaries effect, Antibiofilm effect, Malayali tribe IPC Int. Cl.8: A61K 36/00, A01D 5/00, A01D 5/21

Oral health is fundamental to the general wellbeing of individuals and potentially affects both the quality and length of life apart from an aesthetic standpoint1. Since, it is an integral part of health; when neglected, it results in various types of ailments such as dental caries and periodontal diseases2. Dental caries are one of major oral health problem for people of all ages all over the world. According to the Global Burden of Disease 2010 study, there is a predictable increase in the burden of untreated caries due to population growth and longevity. The most prevalent permanent teeth’s untreated caries affected 2.4 billion people whereas the 10th-most prevalent deciduous teeth’s untreated caries affected 621 million children worldwide during 20103. It has been well documented that several traditional medicinal plants confer extensive antibacterial activity against various microorganisms, including dental caries causatives. People all around the world use many plants and their —————— *Corresponding author

products for oral health maintenance. Even though the toothbrushes and toothpastes are used widely, natural methods of tooth cleaning using chewing sticks selected and prepared from the twigs, stems or roots from a variety of plant species have been practiced for thousands of years in Asia, Africa, the Middle East and the Americas4. Many earlier studies had shown the way for the acceptance of traditional medicine and natural products as an alternative form of oral healthcare5. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used in traditional medicine are considered as suitable alternatives to synthetic chemicals6. Years of knowledge on medicinal plants in traditional systems of medicine for oral hygiene and treatment of dental diseases could channelize the search for effective oral hygiene products and anticaries agents. WHO also advises the researchers to explore the possible use of natural products such as herb and plant extracts with the aim of overcoming the side effects7 and reducing

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the resistance development against the antimicrobials already in use. The Kolli hills in Namakkal district of Tamil Nadu are a ‘Naturalists Heaven’, a treasure trove of medicinal plants, and the native home of traditional hill country and people. The forest types of Kolli hills range from evergreen to moist deciduous and dry deciduous. This study was aimed to document and scientifically validate the local knowledge on medicinal plants used to maintain oral hygiene by the Malayali’s of Kolli hills. Materials and methods Study area and people Namakkal district is located in Tamil Nadu, between the geocoordinates 11º00'00"- 11º34'8"N and 77º40'15" and 78º29'30"E. The study area Kolli Hills (Kollimalai in Tamil) is located in the extreme eastern part of Namakkal district (Fig. 1). The area falls within the latitudes 11º55'05" to 11º21'10"N and 78º17'05" to 78º27'45"E. Kolli Hills contain about 68.34% of the total tribal population of the Namakkal district and Malayalis are dominant among them8. The resident Malayalis (meaning people of the hills) are friendly, sturdy and hardworking people, who generally keep to themselves. Interviews Field visits were undertaken between January 2011 and May 2011. This study was conducted among the Malayali tribes living in the villages such as Semmedu, Keel Solakadu, Vasular patty, Villaram, Parriyur, Arikkal, Valavanthi Nadu, Valluppur nadu, Arriyur Nadu and Arappalliswarar kovil. The information regarding the medicinal plants used for oral healthcare were collected from a total of 13 informants who were chosen based on their traditional medicinal practice, age and botanical knowledge by the methodology as suggested by Somashekara Achar 9. Collection of plants and preparation of extracts The plant specimens were collected, their selection was carried out on the basis of preference ranking exercise, and their botanical identity was confirmed by Botanical Survey of India, Coimbatore. The prepared voucher specimens were deposited at the herbarium, Entomology Research Institute, Loyola College, Chennai, India. The collected plant materials were shade dried at room temperature and coarsely powdered in a manual mill. The powder was successively extracted with hexane, ethyl acetate and methanol, using cold percolation method for 48 hrs.

Fig.1—Map showing the location of Kolli hills.

The extract was filtered through a Buchner funnel with Whatman No.1 filter paper. The filtrate was evaporated to dryness under reduced pressure using rotary evaporator. The extracts were stored at 4 °C, until used. Collection and recovery of caries sample Dental caries clinical samples were collected from patients attending the outpatient ward of Government Headquarters Hospital, Erode, Tamil Nadu following the methodology of Smiline et al.10. Samples were dispersed, inoculated onto selective media including Mitis Salivarius Bacitracin Agar, Lactic Bacteria Differential Agar, Manitol Salt Agar, and Blood agar (HiMedia, Mumbai, India) for selective isolation of organisms like Streptococcus sp., Lactobacillus sp., Staphylococcus sp., Klebsiella pneumoniae. The isolates were identified following standard methods 11. The surface attachment, biofilm build-up, responsible slime and extracellular polysaccharide (EPS) producing cariogenic isolates were determined using tube adherence test12 Congo red plate method12 and confirmed using microtitre plate assay12. Anticaries susceptibility test The in vitro anticaries efficacies of the plants were evaluated using Mueller Hinton agar (MHA) disc-diffusion method12. Briefly, 6 mm sterile disc loaded with 5 or 2.5 mg/disc of each extract were placed on the surface of the previously inoculated medium plates and incubated at 37 °C for 24 hrs. Respective solvents and nalidixic acid (30 µg/ disc)

SEKAR et al.: ORAL HYGIENE MAINTAINING ANTICARIES PLANTS OF KOLLI HILLS BY MALAYALIS

were used as negative and positive controls, respectively. After incubation, diameters of zone of inhibitions were measured. Determination of MIC, antibiofilm activity and phytocontituents of Tephrosia purpurea methanol extract The MIC value of highly active Tephrosia purpurea (L.) Pers. Fabaceae methanol extract against 4 oral bacteria were determined by micro dilution technique as described13. The MIC, the lowest concentration of the compound inhibiting the visual growth upon inoculation 5 µl of above culture on the MHA agar plates was determined. The action of T. purpurea methanol extract on bacterial exopolysaccharide (EPS) production was determined by a modified gradient plate technique13. Their antibiofilm activity was determined by microtiter plate analysis as described22 using a microplate reader (model 680, Bio-Rad). The absorbance was used to calculate the standard deviation and coefficient of variation to determine the stability of the organism and the percentage of inhibitions were calculated12,13. Their GC-MS phytoconstituent analysis using Clarus 500 Mass Spectrometer (PerkinElmer, Waltham, MA, USA) coupled to a Clarus 500 Mass Spectrometer mass detector with Elite 5ms (5% phenyl 95% dimethylpolysiloxane; size, 250 mm; and ID, 0.25 mm 630 m) was carried out. The interface temperature was 280 °C and the scan range was 40 - 450 atomic mass units (AMU). The oven temperature was initially held at 70 °C for 2 min, and then programmed to increase from 70 – 280 °C at 10 °C min-1 where it was held constant for 5 min. Helium was used as carrier gas injected at a constant flow rate of 1 ml min-1. The total run time was 40 min. The injection volume was 1 ml. The solvent delay was 2 min and injected in a split ratio of 1:10. NIST library based peak analysis was performed. Results The results revealed that the Malayali tribes depended on medicinal plants for oral healthcare. The medicinal plants used traditionally by the local people for maintaining oral healthcare is given (Table 1). In total, 15 medicinal plant species belonging to 12 families have been traditionally used by this tribal community for oral health maintenance. The most frequently used plants belong to the family Fabaceae and the least cited family was Solanaceae. Among reported plants parts, leaves were dominant

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(32%), followed by chewing sticks (23%) (Fig. 2a). Chewing plant parts were the dominant mode of usage (39%) followed by using as tooth powders (22%) (Fig. 2b). A major portion of the formulations was administered in the form of tooth brush or powder. A total of 45 extracts obtained from 15 highly cited medicinal plants were screened against caries isolates. The hexane extract of all the selected plants failed to show significant activity against any of the bacteria tested. The anticaries activity was found to be in the order of Azadirachta indica > Lawsonia innermis > Tephrosia purpurea > Mimusops elengi > Psidium gujaya > Achyranthes aspera > Acacia arabica > Argemone mexicana > Ficus bengalensis > Solanum melongena > Albizia lebbeck > Mentha arvensis > Mangifera indica > Solanum nigrum > Mimosa pudica (Table 2). This order was based on the number of bacteria controlled as well as the zone of inhibition. Among these, the methanol extract of T. purpurea was taken for further studies since the antibacterial efficacy of A. indica and L. innermis were well established. Among the solvents tested, methanol exhibited higher activity indicating the potential for extracting the anticariogenic compounds from T. purpurea. The MIC of methanol extract of T. purpurea against L. casei and S. mutans was 1mg/ml; while for S. aureus and K. pneumoniae it was 2 mg/ml. The standard chlorhexidine showed MIC of 25 µg/ ml. The modified original gradient-plate method performed using CRA indicated that methanol extract of T. purpurea had a strong growth inhibitory selective action on the cariogenic isolates biofilm formation. The growth of cariogenic isolates produced typical black color colonies at concentrated areas (Fig. 3a). The colonies became pinkish red, white and smooth along with the increasing concentration gradient indicative of biofilm formation in a concentration-dependent manner (Fig. 3b). The T. purpurea (Fig. 4a-c) methanol extract was very effective in inhibiting the biofilm formation of the tested organisms. At 1 mg/ml concentration, it inhibited the biofilm formation by 92.0%, 77.6%, 74.1% and 94.9% for L. casei, S. mutans, S. aureus and K. pneumoniae, respectively. The GC–MS chromatogram of the methanol extract of T. purpurea resulted in the identification of 11 components belonging to different groups (Fig. 5). The relative percentage of identified and isolated


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Table 1—Ethnomedicinal plant species, plant parts used and ailments cured by the Malayali Tribes in Kolli Hills. Local name Karuvaelam

Naayuruvi

Vagai

Botanical name and family

Parts used

Mode of uses Oral diseases

Ethnomedicinal uses

Twigs, leaves, resin, ash Achyranthes aspera Whole L.; Amaranthaceae plant

Twigs are used as tooth brush. Common salt mixed ash is used to massage the gum and tooth area for relieving toothache. Stem is used as tooth brush. Whole plant ash is used as tooth powder to cure pyorrhea, toothache and gum bleeding.

Albizia lebbeck Benth.; Fabaceae

Twig is used as tooth brush to cure pyorrhea and diseases of the gum.

Tender leaves paste is used for controlling dysentery. Gum mixed with egg white yolk is applied on burns or scalds. Leaves and seeds find use in diabetes treatment. Leaf paste and castor oil boiled juice with pepper and turmeric is applied topically for wound treatment. Plant ash is used for wheezing and coughs treatment. Root paste is applied over dog bite. Seed oil is smeared on wounds for healing or on the lesions of leper’s. Leaf and bark decoctions wheezing protectants. Astringent barks and seeds are used for hemorrhoids and diarrhea treatment. Buffalo dung pounded seed powder is applied externally 4-5 days for skin diseases. Leaf paste finds application in wound curing. Tender leaves and barks are consumed for helminthiasis. Leafy twigs are hanged in front of the house entrance to keep away evil spirits. Leaf paste is applied topically to treat skin diseases. Leaf powder is mixed with coconut oil and applied topically to treat cuts, wounds, heel cracks and felon pain in legs. Dry seed powder is mixed with warm water and taken orally to treat cholera. Decoction is used to treat vomiting.

Acacia arabica W.; Fabaceae

Twigs, seeds, leaves

Brahma thandu Argemone mexicana Leaves, Smoke is inhaled to kill tooth L;. Papaveraceae seeds worms. Veppamaram

Azadirachta.indica A.Juss.; Meliaceae

Marudhaani

Lawsonia innermis L;. Lythraceae.

Maamaram

Mangifera indica L.; Anacardiaceae Mentha arvensis. L.; Lamiaceae Mimosa pudica L.; Fabaceae Mimusops elengi L.; Sapotaceae

Pudina Thottal sinungi Magilam

Twigs, leaves

Twigs are used as toothbrush to develop strong teeth and also considered good for dental caries and gum infection. Leaves Leaf power is boiled and used as mouth rinse against toothache. Leaves, seeds Aerial parts Whole plant Leaves, fruit, bark

Leaves are rolled and used as tooth brush. Decoction of the aerial parts is used for mouth rinse. Decoction of whole plant is used to gargle for toothache. Dried leaf powder is used as tooth powder for scouring teeth, gum inflammation and caries. Leaf decoction is used as mouth freshener. Leaves, Leaves are pounded and used as stem tooth paste for scouring teeth.

Guajya

Psidium gaujava L.; Myrtaceae

Kantankattiri

Solanum melongena Whole L.; Solanaceae plant

Manathakkali

Solanum nigrum L.; Solanaceae

Fruit smoke is inhaled through mouth for relieving tooth ache. Seed paste is used for gum inflammation. Leaves Leaves are cooked and consumed and to treat mouth ulcers. Fruits are fruits eaten as raw to treat mouth ulcers.

phytochemicals and their contributory important functional groups revealed more bioactive compounds and validated the use of T. purpurea as a natural tooth brush. Among the phytochemicals, 3H-Pyrazol3-one, 2,4-dihydro-2,4,5-trimethyl, dl-Glyceraldehyde dimer, 2-Hexanol, 2,5-dimethyl-, (S)-, 4-Hydroxy-3methylacetophenone, Sucrose, 3',5'-Dimethoxy–

Leaf paste is applied topically to treat cuts and wounds. Fruit juice is used to treat diarrhea and dysentery. Bark is used to treat uterus problems. Seeds are used as aphrodisiac. The decoction of barks is used to treat fever, diarrhea and headache Stem bark decoction is used for diarrhea treatment. Leaves boiled with chilly are used controlling vomiting, indigestion, cough and cholera. Leaf paste is applied topically to treat headache. Decoction of leaves is used to treat fever and chest pain. Cooked leaves are used as food supplement to treat stomachache and cough.

acetophenone, 3-O-Methyl-d-glucose, 4-((1E)-3Hydroxy-1-propenyl)-2-methoxyphenol, 2-Allyl-1,4dimethoxy-3-methyl-benzene, 2-Phenyl-2,3-dihydro1,5-benzothiazepin-4(5H)-one, 2-[1-Hydroxy-2-(7phenyl-2,3,6,7-tetrahydro -1H-[1,4] diazepin-5-yl)vinyl]-4-methoxy-phenol were the major compounds. Most of these compounds were phenolics that have


varied biological activities, and it confirmed the antibacterial potential of T. purpurea against cariogenic organisms. Discussion Almost all cultures have used a range of plants or the materials derived from them for the prevention and treatment of diseases. Currently plant based natural therapies are increasingly accepted as complementary to mainstream healthcare. Good oral

Fig.2(a)- plant parts (b) and their mode of use by the Malayali tribes of Kolli hills for oral health care.

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hygiene maintenance and dental caries prevention can be achieved by regular removal of food deposits and dental plaque. Even though toothbrushes and toothpastes have found wide usage, natural methods of teeth-cleaning using chewing sticks are very common12. Their availability, low cost, simplicity, and religious and/or traditional associations have made them popular. The World Health Organization has even supported their use as an effective ‘tool’ for oral hygiene taking into account the historical importance of this ‘miswak’ in the field of oral hygiene and the value people attach to it since ancient times14. Usually these chewing sticks are made from a range of plant species and are used for routine tooth cleaning in Asia, Africa, South America, and the Middle East14. In Indian subcontinent various plants such as Azadirachta indica, Acacia nilotica, Juglans regia, etc., have been used as the source for chewing sticks15.

Fig.3 (a) Modified Gradient-plating technique images showing the growth of cariogenic organism on CRA plate without plant extract (b) and with plant extracts.

Table 2—Ethnomedicinal plant species, plant parts used and ailments cured by the Malayali Tribes in Kolli Hills. Values (zone in mm/ 6mm of Hi-media discs) Extract’s (5mg/ml) growth inhibitory action on cariogenic organism (Inhibitory zone diameter in mm)

Plant name

L. casei Hexane

Ethylacetate

S. mutans Methanol Hexane

Ethyl acetate

S. aureus Methanol

Hexane

K. pneumoniae

Ethyl acetate

Methanol

Hexane Ethyl acetate Methanol

Acacia arabica

7.33±0.33 11.00±0.57 11.66±0.33

-

9.66±0.33 10.66±0.33

-

9.33±0.33

10.33±0.33

-

11.66±0.33

8.33±0.33

Achyranthes aspera Albizia lebbeck Argemone mexicana Azadirachta indica Ficus bengalensis Lawsonia innermis Mangifera indica Mentha arvensis Mimosa pudica Psidium gaujava Solanum melongena var. incanum Solanum nigrum Tephrosia purpure

8.66±0.33 -

12.33±0.33 12.00±0.57 8.33±0.33 19.33±0.33 15.00±0.57 18.33±0.33 9.33±0.66 9.33±0.33 12.33±0.33 16.33±0.33 10.00±0.57

-

10.33±0.33 8.66±0.33 8.66±0.66 14.00±0.57 11.33±0.33 13.66±0.33 10.33±0.33 9.33±0.33 9.66±0.33 11.66±0.33 8.00±0.57

-

11.33±0.33 9.33±0.33 11.33±0.33 17.33±0.33 12.33±0.33 15.33±0.33 8.33±0.33

10.00±0.57 9.33±0.33 9.33±0.33 17.33±0.33 8.66±0.33 17.33±0.33 11.66±0.66

-

11.00±0.57 8.33±0.33 8.66±0.33 10.66±0.66 12.33±0.33 10.33±0.33 11.33±0.33

10.66±0.66 11.33±0.33

8.66±0.33 9.33±0.33 7.66±0.33

8.66±0.33 13.33±0.33

12.33±0.33 11.33±0.33 8.33±0.33 12.66±0.33 17.66±0.33

-

9.33±0.33 11.00±0.57 11.33±0.66 11.00±0.57 16.33±0.33 8.33±0.33 14.66±0.33 17.33±0.88

13.33±0.33 10.66±0.33 10.33±0.33 14.66±0.33 13.66±0.33 16.33±0.66 8.66±0.33 8.33±0.33 9.33±0.33 13.66±0.33 7.33±0.33

14.66±0.33 11.66±0.33 9.33±0.33 18.00±0.57 12.66±0.33 15.00±0.57 8.33±0.33 8.33±0.33 10.66±0.33 17.33±0.33 -

8.33±0.33 8.66±0.66 14.33±0.33 15.33±0.33 9.33±0.33 11.33±0.66

10.00±0.57

14.00±0.57 11.00±0.57 12.33±0.66 10.66±0.33

12.33±0.33

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Although dental caries harbors a complex microflora, the role of organisms like Streptococcus mitis, S. sanguis, S. mutans and Staphylococcuss aureus are well recognized13. An efficient dental caries control or preventive measures should target the growth and development of biofilm forming organisms. Dental caries are, however, preventable if the growth and level of the oral cavity and cariogenic microorganisms are reduced using effective practices. To the best of our knowledge, this study represents the first directed work to test the in vitro inhibitory activity of Indian

Fig. 4 (a) Tephrosia purpurea plant (b) stem (c) Root.

medicinal plants reported by the locals on cariogenic clinical pathogens. It also reports for the first time the anticaries effect of ethnomedicinal plants like Tephrosia purpurea, Acacia arabica and Argemone mexicana. Since the dental treatment usually is expensive and not readily available, especially in developing countries16, these traditional medicinal plants used to prevent dental caries could serve as an alternate or complement. The present study provides information on various traditional oral hygiene practices and herbs used for oral health and diseases. Phytochemical and pharmacological activities of these plants validate the traditional use; they also are an excellent source of newer drugs for the treatment of oral diseases. While recognizing the role of chewing sticks, WHO 2000 Consensus Report on Oral Hygiene recommends further evaluation of their effectiveness 14. The T. purpurea methanol extract GC–MS chromatogram revealed the presence of 11 bioactive phytoconstituents belonging to different groups. Studies on plant derived chemicals have supported the development of modern medicine. This fact is well recognized by today's biomedical community that continues to send modern explorers to the corners of the earth to find new plant-related cures for our maladies17.

Fig. 5—Methanol extract GC-MS chromatogram.


Conclusion The ethnobotanical investigation on medicinal plants used for oral healthcare by the Malayali tribals of Kolli hills showed the availability of plant resources for treating a wide spectrum of oral diseases. The local knowledge and the efficacy of the cited medicinal plants were justified and scientifically validated. This study also highlights the importance of gathering such ethnomedicinal information for development of valuable anticaries agent. A further detailed study on the extract of Tephrosia purpurea may yield some novel prototypes for future drug discovery with anticaries effect.

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Acknowledgement The authors acknowledge the help provided by Malayali tribals of Kolli hills by sharing their knowledge. We wish to thank Dr K Balakrishna, Emeritus Scientist, Entomology Research Institute, Loyola College, Chennai, for sharing his expertise on phytochemical analysis and discussions. The authors also extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project Nr. RGP-VPP-183.

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