ANTIBACTERIAL EVALUATION OF MEDICINAL PLANTS USED BY KORKUS IN MELGHAT FOREST AGAINST GASTROINTESTINAL INFECTIONS

ANTIBACTERIAL EVALUATION OF MEDICINAL PLANTS USED BY KORKUS IN MELGHAT FOREST AGAINST GASTROINTESTINAL INFECTIONS

1. H. Tambekar* and B. S. Khante

Post Graduate Department of Microbiology, S. G.B. Amravati University, Amravati, Maharashtra, India

ABSTRACT

Use of plants as a source of traditional healing systems around the world that utilize herbal remedies is an important source for the discovery of new antimicrobials against resistant strains of bacteria. Medicinal plants have been a major source of therapeutic agents in Korkus from Melghat since time immemorial. Incredible knowledge of phytomedicine is acquired in non-coded form by these tribals and rural community is clear from evidences related to folklore medicines.  Acacia leucopholia (Bark), Butea monosperma (Seed, Flowers), Woodfordia fruticosa (Root, Flowers), Sphaeranthus indicus (Fruits, Whole plant), Maytenus emerginata (Root, Leaves), Acacia arabica (Leaves), Caesalpinia bonducella (Seeds), Gardenia gummifera (Resin) were selected. Dried powders of plant parts were extracted in different solvents like water, ethanol, methanol and acetone. Antibacterial activity was tested by disc diffusion method against standard cultures of Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri. Methanol extract of Woodfordia fruticosa, Acacia leucopholia, ethanol extracts of Sphaeranthus indicus, Butea monosperma, Maytenus emerginata were active against the test pathogens. Phytochemical analysis of the extracts showed presence of cardiac glycosides, anthraquinone, flavonoids, tannins phenolics in the methanol extracts of Woodfordia fruticosa, Acacia leucopholia.

Enteric infections

INTRODUCTION: The large Melghat tract In Maharashtra State is a hilly terrain of Satpura ranges, which is highly dense forest and occupies an area of about 4000 Sq. km.  Melghat with dominant population of Korkus and Gond tribes still depends on knowledge of local “vaidus” and “bhagats” for primary health care. These local doctors depend on seasonally available local flora for treating human diseases and store herbal material available in hilly areas as dried roots, rhizomes, fruits, seeds, etc ¹.

Charaka and Sushruta had recommended sweetened decoction of flowers of Woodfordia fruticosa for fever, haemothermia, persistent dysentery. Dried flowers are sprinkled over ulcers and wounds, for diminishing their discharge. Conflictions of flowers are used as stimulant and astringent, given in dysentery to check hemorrhages and leucorrhoea. Kumaraswamy et al., ² has studied extracts of Woodfordia fruticosa in various solvents and concluded that petroleum ether followed by methanol extract were highly potent antibacterial agent. Bark of Acacia leucopholea is used for cholera and diarrhea ³. Flowers of Butea monosperma are boiled in water to obtain a dye and also used as an antiseptic, against infections of round worms, thread worms and Giardiasis Badhe and Pande ⁴.

Roots of Maytenus emerginata are used in gastrointestinal troubles, especially dysentery ^{5, 6}. Pulverized leaves are given in milk to children as a vermifuge. Leaf ash with ghee is applied on sores as ointment ⁷. The juice of Sphaeranthus indicus an aromatic herb, the plant is styptic and useful in liver and gastric disorders ⁸. Sticks of Acacia arabica are well known remedies for dental caries ⁴.   Dwivedi et al ⁹ studied antibacterial, antimalarial activity of Seed of Caesalpinia bonducella. Resinous exudation of leaf buds and shoots of Gardenia gummifera is used to stop diarrhea due to dentition ³.

Traditional medicines are cheaper, with minimum side reactions and safe. To ensure the safety of its products and practices standardization is of vital importance. The knowledge of medicinal plants came from our ancient literature i.e. Vedas, with time and fast developmental changes had lead pathogens to gain resistance against new generation antibiotics. Hence search for new antibacterial sources that are of green origin has multiple advantages. In the Indian system of medicine, most practitioners formulate and dispense their own recipes; this requires proper documentation and research.

MATERIALS AND METHODS: Frequent field visits were conducted in study area throughout year March to October for collection of specimen samples at flowering and fruiting stages. Specimen samples were identified by Mr. R.B. Giri, Ranger forest officer, Maharashtra forest Rangers college, Chikhaldara (Table 1). Selected parts of plants were collected, cleaned and disinfected with water and mercuric chloride (0.5%), dried in shadow and ground to powder in mixer grinder. For preparation of extracts, 10 g of powder was soaked in100 ml of solvents (water, ethanol, methanol, and acetone) refluxed after 24 h in Soxhlet apparatus, filtered and filtrate was evaporated in controlled conditions of temperatures to avoid destruction of dissolved phytochemicals.

Bacterial inoculums preparation: The standard pathogenic bacterial strains were procured from IMTECH, Chandigarh, India. The bacteria were rejuvenated in nutrient broth medium. Subcultures were prepared from the stock for bioassay. A loopful of culture was inoculated in 10 ml of sterile nutrient broth and incubated at 37⁰ C for 3h turbidity of the culture was standardized to 10⁵ CFU with the help of SPC and Nephlo-turbidometer.

TABLE 1: PLNT PARTS SCREENED FOR ANTIBACTERIAL STUDY

Preparation of disc for antibacterial activities: Sterile blotting paper disc (10mm) were soaked in the solution in such concentration that the amount of solution absorbed by each disc was 2, 4, 6, 8, 10 mg of each extracts of Acacia leucopholia (Bark), Butea monosperma (Seed and Flowers), Woodfordia fruticosa (Root, Flowers), Maytenus emerginata (Leaves and Root), Sphaeranthus indicus (Fruits and Whole plant), Acacia arabica (Leaves), Caesalpinia bonducella (Seed), Gardenia gummifera (Resins). The prepared discs were dried in controlled temperature and used for study.

Agar disc diffusion for antibacterial activities: For antibacterial properties, 0.1mL bacterial suspension of 10⁵ CFU ml ^-1 was uniformly   spread on Mueller-Hinton agar (MHA) plate to form lawn cultures. The dried discs (dried at 37⁰C over night) were applied to the surface of MHA plates seeded with 3h broth culture of test bacterium. The plates were incubated for 18 h at 37⁰C. Antibiotic susceptibility discs, Ciproflxacin10 µg, were used as positive control while disc soaked in various organic solvents and dried were placed on lawns as negative control. The antibacterial activity was evaluated for 10mg/disc and diameter of inhibition zones were measured.

Phytochemical analysis: The presence of saponins, tannins, anthraquinones, alkaloids, triterpenes, flavonoids, glycosides, reduced sugar and phenols were detected by qualitative methods given by    Khandelwal ¹⁰. Antibacterial response of the medicinal plants against various pathogens was calculated as:

Results and Discussion: The present study was conducted to investigate antibacterial properties of selected plants from Melghat forest and used in Indian Folkloric Medicine. Total 11 parts of eight plants were used in 44 extract preparations (Table 2). Leaves of Acacia arabica proved strongest antibacterial among the selected plants for study, followed by flowers of Woodfordia fruticosa, Butea monosperma, whole plant of Sphaeranthus indicus and so on (fig. 1). Methanol extract of flowers of Woodfordia fruticosa and bark of Acacia leucopholia were effective against all the test pathogens followed by ethanol extracts. S. aureus was most sensitive test pathogen. It was sensitive to ethanol extracts of Acacia arabica, Acacia leucopholia, Sphaeranthus indicus, Caesalpinia bonducella, Gardenia gummifera and Woodfordia fruticosa. Strong sensitivity was observed in S. aureus, E. aerogenes, K. pneumoniae, Pr. vulgaris, Sh. flexneri, E. coli, S. typhimurium, S. typhi, Ps. aeruginosa and S. paratyphi in decreasing order (fig. 2).

FIG. 1: COMPARISON OF ANTIBACTERIAL RESPONSE IN VARIOUS EXTRACTS OF SELECTED MEDICINAL PLANTS

FIG. 2: COMPARITIVE ANTIBACTERIAL SENSTIVITY OF PATHOGENS AGAINST VARIOUS MEDICINAL PLANTS

TABLE 2: ANTIBACTERIAL ACTIVITY OF MEDICINAL PLANTS (DIAMETER OF ZONE OF INHIBITION MEASURED IN mm. (ZOI)

Note: Aqueous - aq, Ethanol - e, Methanol -m, Acetone –ac

Presence of steroids cardiac glycosides, anthraquinone, flavonoids and phenolics in both extracts suggested that solubility of different phytochemicals in different solvents must be responsible to show the difference in their antibacterial effects. Moderate sensitivity of Acacia leucopholia in ethanol and Methanol extracts in most of test pathogens must be due to its tannin contents found in Acacia species ¹¹. Methanol extracts when tested by Dabur et al., ¹², against clinical isolates and standard strains found that MIC was 300µg/ml for E. coli, B. cereus and Klebsiella sp.

Methanol extracts of the East India globe thistle was potent antibacterial against S. aureus and Sh. flexneri. Deshpande et al., ¹³ studied acetone and methanol extracts of fruit of Sphaeranthus indicus exhibited activity against Gram +ve and resistant to Gram –ve bacteria. Similar results were also observed in organic solvents extracts in discs with highest concentration of 10 mg in present work. Khare ³, stated that contents of Alkaloids, Tannins and volatile oil posse’s antibacterial properties against Vibrio cholera and Micrococcus sp. Flavonoids are synthesized by the plants in response to microbial infections, in vitro they were effective antimicrobial substances against wide array of microorganisms ¹⁴. Kumar et al., ¹⁵ found that extracts of Sphaeranthus indicus were highly antibacterial. Antibacterial activity of Woodfordia fruticosa flowers was recorded ¹². The results were parallel to our findings for the highest contents in the discs i.e.10 mg. Sensitivity of S. aureus, Pseudomonas supported flower’s use as an antiseptic by the Korkus. Parekh and Chanda¹⁶ recorded that crude methanol extracts of Woodfordia fruticosa was good antibacterial at against all tested microorganisms as it is rich in tannins.

Ethanol extract of Butea monosperma was able to produce sensitivity in S. typhi, E. coli, Pseudomonas and S. paratyphi. Shigella flexneri was strongly inhibited by the same extract. Moderate sensitivity was observed in S. aureus, E. aerogenes, S. typhimurium, Pr. vulgaris and Klebsiella sp. Flowers of Butea monosperma were reported as active against worms, piles, colic pains. Tannins in Butea monosperma were active in diarrhea, dysentery when new formulations in herbal medicines were designed and studied for their antibacterial phytochemicals ³. Ethanol extract of Maytenus emerginata (leaves) was antibacterial to Klebsiella sp. and Pr. Vulgaris proved by Nair et al., ¹⁷ and Tambekar and Khante ¹⁸.  E. coli, S. aureus, E. aerogenes, S. typhi, S. typhimurium and Sh. flexneri were mild sensitive. Pr. vulgaris was strongly sensitive to ethanol extract of root of Maytenus emerginata. Pseudomonas was also strongly inhibited but Sh. flexneri; S. paratyphi and E. coli were resistant to ethanol extract at highest conc. of 10 mg of the root. While others were mild sensitive to the same extract.

Methanol extract of seeds of Caesalpinia bonducella proved antibacterial against S. aureus, Sh. flexneri and E. aerogenes ¹⁹. Organic extracts of Gardenia gummifera were active against S. aureus, K. pneumoniae, and E. aerogenes. Sh. flexneri a causative agent of bacterial dysentery was resistant to aqueous extracts of all plants but sensitive to methanol extract of Caesalpinia bonducella (2 mg/disc). Parekh and Chanda ²⁰ and    Moon et al., ⁸, also demonstrated similar antibacterial properties of these plants. Acacia arabica proved its antibacterial against all test pathogens. S. typhi was inhibited by all three organic extracts of babul while positive control was inefficient to inhibit the pathogen. Methanol extract of babul showed maximum inhibition of E. coli, S. aureus, S. typhi, K. pneumoniae, Sh. flexneri and E. aerogenes. Almas ²¹ and bioassay studies of Dabur et al., ¹² also reported such antibacterial potentials (Table 2). Preliminary phytochemical analysis of the extracts of these plants showed presence of anthraquinones, flavonoids, cardiac glycosides, tannins, and phenolics.

SUMMARY AND CONCLUSION: Further study of phytochemicals present in these medicinal plants may reveal some new antimicrobials. Alkaloids from Butea monosperma, essential oils in Sphaeranthus indicus, and tannins in Acacia leucopholia, Acacia arabica must be studied in detail. From the results we can conclude that the crude extracts of Woodfordia fruticosa, Sphaeranthus indicus, Butea monosperma, Acacia leucopholia and Maytenus emerginata exhibited significant antimicrobial activity and properties that support folkloric use in the treatment of some diseases as broad spectrum antimicrobial agent. This probably explains the use of these plants by the indigenous people in Melghat against a number of enteric infections since generations.   

REFERENCES:

1. Chopra AK., Khanna DR, Prasad G, Malik DS and Bhutiani R: Medicinal plants: Conservation, cultivation, and Utilization. Daya publishing house, Delhi, First Edition 2007.
2. Kumaraswamy MV, Kavitha HU and Satish S: Antibacterial potential of extracts Woodfordia fruticosa kurz on human pathogens. World Journal of Medical Sciences 2008; 3(2): 93-96.
3. Khare CP: Encyclopedia of Indian Medicinal Plants. Springer-Verlag, Berlin Heidelberg New York 2004.
4. Badhe PD and Pande VK: A study of medicinal and economic plants of Amravati Division, Amravati Circle, Maharashtra. Central council for research in Ayurveda and Siddha, New Delhi, BMEBR XI 1988; (1-2): 1-39.
5. Kothari MJ and Londhe AN: Ethnobotany in human health care of Chikhaldara, Amravati district in Maharashtra state, India. Ethnobotany and Medicinal plants of Indian Subcontinent. Scientific Publishers (India) Jodhpur, 2000; 273-281.
6. Tambekar DH and Khante BS: Antibacterial properties of traditionally used medicinal plants for enteric infections by adivasi’s (Bhumka) in Melghat forest (Amravati district). International Journal of Pharmaceutical Sciences and Research 2010; 3(9): 120-128.
7. Pullaiah T: Encyclopedia of world medicinal plants, Regency Publication, New Delhi, First Edition, 2006.
8. Moon A, Khan A and Wadher BJ: Evaluation of phytochemical and antibacterial properties of Medicinal plants. Journal of Current Sciences 2006; 9(1): 219-226.
9. Dwivedi SN: Evaluation of Antimalarial Herbal Drugs. Farmavita 2006. Net-Pharmaceutical Licensing Network. http://www.farmavita.net
10. Khandelwal KR: Preliminary phytochemical screening: Practical Pharmacognosy-techniques and Experiments. Nirali Publication, Pune, Eighth Edition, 2001.
11. Chopra RN, Chopra IC, Handa KL and Kapur LD: Chopra’s indigenous drugs of India. Academic Publishers, Calcutta, New Delhi, Second Edition, 1982.
12. Dabur R, Gupta A, Mandal TK, Singh DD, Bajpai V, Gurav AM and Lavekar GS: Antimicrobial activities of some Indian medicinal plants. African Journal of Traditional and Complimentary Alternative Medicine 2007; 4(3): 313-318.
13. Deshpande AR, Musaddiq SP, Rothe, Banole AM and Patil DP: Screening of some plant extracts for antibacterial activity. Asian Journal of Microbiology, Biotechnology and Environmental Sciences 2005; 7 (4):755-758.
14. Harborne JB: Phytochemical Methods. Springer (India) Pvt. Ltd, First Indian reprint, 2005.
15. Kumar PV, Chauhan NS, Padh H and Rajani M: Search for antibacterial and antifungal agents from selected Indian medicinal plants. Journal of Ethnopharmacology 2006; 107:182-188.
16. Parekh J and Chanda S: In vitro antibacterial activity of crude methanol extracts of Woodfordia fruticosa kurz flower (Lythraceae). Brazilian Journal of Microbiology 2007; 38: 204-207.
17. Nair R, Shah A, Baluja S and Chanda S: Comparison of antibacterial activity of some natural and synthetic compounds. Asian Journal of Microbiology, Biotechnology and Environmental Sciences 2006; 8 (3): 503-508.
18. Tambekar DH and Khante BS: Evaluation of antibacterial properties of ethnomedicinal herbs used by Korkus in Melghat of India against enteric pathogens. International Journal of Pharmaceutical and Biosciences 2009(b); V1 (1). www.ijpbs.net
19. Tambekar DH and Khante BS: Screening of antibacterial potentials of some medicinal plants from Melghat forest in India. African Journal of Traditional and Complimentary Alternative Medicine 2009(a); 6(3): 228-232.
20. Parekh J and Chanda S: In vitro antimicrobial activities of extracts of Launea procumbens (Roxb). (Labiatae), Vitis vinifera (Vitaceae) and Cyperus rotundus L. (Cyperaceae). African Journal of Biomedical Research 2006; 9: 89-93.
21. Almas: The antimicrobial effects of seven different types of Asian chewing sticks. Odonto- Stomatologie Tropicale 2001; 96: 17-20.