SCREENING OF SOME HERBAL PREPARATIONS USED IN INDIAN AYURVEDIC MEDICINE FOR ITS ANTIBACTERIAL POTENTIALS

SCREENING OF SOME HERBAL PREPARATIONS USED IN INDIAN AYURVEDIC MEDICINE FOR ITS ANTIBACTERIAL POTENTIALS

D. H. Tambekar* and S. B. Dahikar

Post Graduate Department of Microbiology, Sant Gadge Baba Amravati University,
Amravati, Maharashtra, India

ABSTRACT:Ayurvedic medicine plays a crucial role in healthcare and serves the health need of a vast majority of people in developing countries. Some clinically used ayurvedic herbal preparations were investigated (solvent extract preparation) for antibacterial potential against enteric bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumoniae, Salmonella typhi, Staphylococcus epidermidis, Salmonella typhimurium and Proteus vulgaris. In present study Shatawaryadi churna, Panchasakar churna, Talisadi churna, Dashmula churna and Manjistadi churna were potent antibacterial against Staphylococcus epidermidis, Proteus vulgaris, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. Study suggested thatthese herbal preparations may be useful as an alternative medicine in the treatment of enteric bacterial infection.

INTRODUCTION: Herbal medicines are plant derived material or preparations, which contain raw or processed ingredient from one or more plants or its parts, with therapeutic value and used as dietary supplements to fight or prevent common disease in various systems of medicine such as Ayurveda, Unani and Siddha ¹. Plant derived natural products have received considerable attention in recent years due to their diverse pharmacological activities.

The traditional herbal combinations and extracts are known to improve health by combating or preventing microbial infections and curing various ailments and diseases ^{2, 3}. The plant-derived medicines are relatively safer than synthetic drugs and offering profound therapeutic benefits by providing alternative and effective treatment for chronic disorders and various diseases ^{4, 5}. More than 1500 herbal preparations are sold in India as dietary supplements or ethnic traditional medicine to treat the diseases but only a few of them have been scientifically explored for its antibacterial potentials ^{6, 7}.

The most frequently used herbal preparations is churna; a preparations comprising of fine powders of medicinal plants and may be single or in combination. Combinations of medicinal plants may increases the antimicrobial spectrum and potency of activity. Enteric or diarrhoeal infections account for high proportion of health problems in the developing countries and contribute to the death of 3.3 to 6.0 million children annually. Enteric bacteria such as Salmonellasp., Shigella sp., Proteus sp., Klebsiella sp., E. coli, Pseudomonas sp., Vibrio cholerae, and S. aureus are major etiologic agents of sporadic and epidemic diarrhea in both children and adults ⁸. Recently, it has been demonstrated that many human pathogenic bacteria have developed resistance against several synthetic drugs. Available reports on lesser efficacy and more side effects of synthetic drugs need to search an alternative medicine ⁹.

There are several reports on antimicrobial activity of crude extracts prepared from plants that inhibit various bacterial pathogens, but a limited numbers of in vitro studies on herbal preparations have been published. Therefore, there is need of the hour to identify antibacterial potential of herbal products based on diseases for which no medicine or only palliative therapy is available ¹⁰. At this juncture, it is of interest to determine the scientific basis for the traditional use of these herbal medicines and evaluate the antibacterial potential in significance to prevention of enteric bacterial infection.

Materials and Methods:  The commercial herbal preparations as given in Table 1 were purchased from the local market of Amravati. These herbal preparations have multiple botanical ingredients in addition to some chemical substances.

TABLE 1: HERBAL PREPARATION WITH ITS INGREDIENTS

Preparation of extracts: The aqueous extract was prepared by adding 20 g of herbal preparations in 200 mL distilled water, heated at  60^°C  for 2 h, filtered through cloth and the  filtrate was evaporated on sand bath. The dry mass was then stored at 4^°C. The organic solvent extract was prepared by adding 20 g herbal preparation (powder) in 200 mL of organic solvent (acetone, ethanol, and methanol) in screw-capped bottles; shake at 190-220 rpm on a rotary shaker. After 24h of shaking, the extract was filtered, evaporated in vacuum, and dried by rotary evaporator at 60^°C ¹¹. Dried extracts were stored in labeled sterile screw capped bottles at 4^°C and later used for the in vitro study.

Bacterial cultures: The standard pathogenic bacterial cultures procured from IMTECH, Chandigarh, India and used in the present study (Table 1). The bacterial cultures rejuvenated in Mueller- Hinton broth (Hi-media laboratories, Mumbai, India) at 37^°C for 18 h and then stocked at 4⁰ C in Mueller-Hinton Agar. The inoculum size of the bacterial culture standardized according to the National committee for Clinical Laboratory Standards guideline ¹². The pathogenic bacterial culture inoculated into sterile Nutrient broth and incubated at 37^°C for 3 h until the culture attained a turbidity of 0.5 McFarland units. The final inoculum size standardized to 10⁵ CFU/mL with the help of SPC and Nephlo-turbidometer.

Preparation of disc for antibacterial activities: The aqueous, ethanol, methanol and acetone extracts were prepared in their respective solvents and the sterile blotting paper disc (10 mm) were soaked in the diluted extract in such concentration that the amount of solution absorbed by each disc was 5 mg of each extracts of herbal preparations. The prepared discs dried in controlled temperature to remove excess of solvent and used in study.

Antibacterial Activity using disc diffusion method: The modified paper disc diffusion employed to determine the antibacterial activity of both aqueous and organic solvents extract of herbal preparations. Turbidity of inoculums matched with McFarland turbidity standard. Inoculums spread over the Nutrient agar plate using a sterile cotton swab in order to get a uniform microbial growth. Then the prepared antibacterial disc placed over the lawn and pressed slightly along with positive and negative controls. Ampicillin 10 µg/disc (Hi-Media, Mumbai) used as positive control while disc soaked in sterile distilled water or various organic solvents and dried placed on lawns as negative control.

The plates incubated for 18h at 37^°C. The antibacterial activity evaluated for 5 mg/disc and diameter of inhibition zones measured. Experiment carried out in triplicate and the averages diameter of zone of inhibition recorded. The antibacterial activity was classified as strong (>20 mm), moderate (15-20 mm) and mild (12-15 mm) and less than 12 mm was taken as inactive. Antimicrobial Sensitivity Index (ASI) calculated by following formula ¹³.

RESULTS AND DISCUSSION: In the present study, herbal preparations such as Shatawaryadi churna, Panchasakar churna, Talisadi churna, Dashmula churna, Manjistadi churna, Bilwadi churna, Jatiphaladi churna, Gokharu churna, and Hingwashtak churna were screened for antibacterial potential against enteric bacterial pathogens. They exhibited significant antibacterial activity against S. epidermidis, S. aureus, P. vulgaris, E. coli, K. pneumoniae, P. aeruginosa, B. subtilis, E. aerogenes, S. typhimurium and S. typhi.

As per antibacterial sensitivity index of herbal preparations (ASI) as shown in fig. 1, it was observed that the Shatwaryadi churna, Panchsakar churna, Talisadi churna, Dashmula churna and Manjistadu churna were potent antibacterial where as Bilwadi churna, Jatiphaladi churna, sukhsarak churna, Gokharu churna and Hingwashtak churna showed significant antibacterial activity against enteric bacterial pathogens.  The Shatawaryadi churna, Panchasakar churna and Talisadi churna are potent antibacterial against S. aureus, S. epidermidis, P. aeruginosa, S. typhimurium, K. pneumoniae, E. aerogenes P. vulgaris, B. subtilis S. typhi and E. coli (Table 2).

FIG. 1: ANTIBACTERIAL POTENTIAL OF HERBAL DRUGS (ASI)

Asparagus racemosus is important ingredients (Table 1) of Shatawaryadi churna is aphrodisiac, demulcent, nutritive, coolant, diuretic, alterative, useful in piles, eye infection, uterine disorder etc ¹⁴. Uma et al, ¹⁵ reported antimicrobial properties of A. racemosus against enterotoxigenic E. coli, enteropathogenic E. coli, S. typhimurium, S. entertidis, S. dysenteriae, S. flexineri, K. pneumoniae and P. aeruginosa. Emblica officinalis exhibited potent antibacterial activity against E. coli. K. pneumoniae, P. mirabilis, P. aeruginosa, S. typhi, S. paratyphi A, S. paratyphi B and S. marcescens ¹⁶.

All the herbs used in Panchasakar churna possess antibacterial properties;  Lo-Cantore  et al, ¹⁷reported that Foeniculum vulgare and Zingiber officinale was antibacterial against E. coli and B. megaterium. Terminalia chebula exhibited antibacterial activity against enteric bacterial pathogens such as E. coli, S. aureus, P. aeruginosa, P. vulgaris, S. epidermidis, S. typhi, S. typhimurium ^18,19. Talisadi churna is very useful respiratory discomfort and cough, acute exacerbation of asthma chronic and allergic bronchitis and content shunthi and pippali. Shunthi is known as Vishvabhaishjya (an herb useful in all diseases) and pippali are rejuvenator for the respiratory and digestive system. Piper longum is antibacterial against E. coli, P. aeruginosa, B. cereus, K. pneumoniae, S. typhi, S. marcescens, S. dysenteriae and S. aureus. Taxus baccata (Talispatra)is antibacterial against S. typhimurium and P. syringae ²⁰.

TABLE 2:  ANTIBACTERIAL POTENTIAL OF HERBAL PREPARATIONS

(Zone of inhibition of growth in mm, Average of 3 reading)

Dashmula churna found antibacterial against S. aureus, S. epidermidis, P. vulgaris, S. typhi, B. subtilis, E. coli, K. pneumoniae, E. aerogenes and P. aeruginosa (fig. 2).Ingredients of Dashmula churna possess antibacterial properties. Mazumderet al, ²¹ reported anti-diarrhoeal properties of Aegle marmelos root against V. cholerae, E. coli and Shigella sp. Manjistadi churna and Bilwadi churna was antibacterial against all tested bacterial pathogens viz. S. epidermidis, S. aureus, B. subtilis, S. typhimurium, E. coli, P. aeruginosa, K. pneumoniae, P. vulgaris, S. typhi, and E. aerogenes. Manjistadi churna might be useful in prevent the bacterial infections such as staphylococcal infections, enteric infections, urinary tract infections etc. All the ingredients of these churna possess antibacterial properties; Rubia cordifolia is clinically used for the purification of blood by the physicians of the Indian System of Medicine. It possesses antiinflammatory, immuneregulator, anticancer, anticlotting properties.

Aegle marmelos antibacterial against E. coli, Klebsiella sp, Citrobacter sp, Enterobacter sp, S. typhi, S. paratyphi, Shigella sp, P. vulgaris, P. mirabilis and Pseudomonas sp ²². Jatiphaladi churna is antibacterial against S. epidermidis, S. aureus, E. coli, S. typhimurium, P. aeruginosa, S. typhi, K. pneumoniae, P. vulgaris and E. aerogenes. Myristica fragrans is important ingredients of Jatiphaladi churna and it is antibacterial against multi-drug resistant S. typhi, E. coli, serotypes of Salmonella, L. monocytogenes and A. hydrophila ¹⁸. Gokharu churna is strong antibacterial against S. aureus, S. epidermidis, P. vulgaris, E. coli, P. aeruginosa, K. pneumoniae and S. typhi. Gokharu churna is single plant preparation, (Tribulus terrestris), used in urinary tract infection, urinary calculosis, burning in micturation, urinary affections, gout, and kidney diseases and also have aphrodisiac, diuretic, demulcent properties. Tribulus terrestris is reported antibacterial against E. coli, S. aureus, B. cereus, P. aeruginosa and antifungal against C. albicans ²³. Hingwashtak churna was antibacterial against S. aureus, S. epidermidis, P. vulgaris, B. subtilis, S. typhi, K. pneumoniae, P. aeruginosa and E. coli (Fig. 2). An ingredient of Hingwashtak churna possesses antibacterial activity. Rahman et al,²⁴ reported antimicrobial activities of Ferula assafoetida against B. megaterium, B. subtilis, L. acidophilus, M. luteus, S. epidermidis, S. aureus, V. cholerae, E. coli, S. typhi and S. flexneri.

According to antibacterial sensitivity index of herbal preparations (ASI) as shown in fig 1, it was observed that the Shatwaryadi churna, Panchsakar churna, Talisadi churna, Dashmula churna and Manjistadu churna were potent antibacterial where as Bilwadi churna, Jatiphaladi churna, sukhsarak churna, Gokharu churna and Hingwashtak churna showed significant antibacterial activity against enteric bacterial pathogens. From the study, it was concluded that, the herbal preparation studied were strong antibacterial agent against bacteria associated with dysentery, diarrhoea, gastroenteritis, urinary tract infection, invasive disease, skin infections (impetigo, folliculitis), endocarditis, septicemia, respiratory tract infection, eye infections etc. Scientific evaluation of these herbal preparations gives better information regarding the antibacterial efficacy of herbal medicine available in India. This study supports the use of these herbal preparations in combating or controlling the bacterial infection.

FIG. 2: PHOTO PLATE SHOWING ANTIBACTERIAL ACTIVITY OF HERBAL PREPARATION AGAINST BACTERIAL PATHOGENS

CONCLUSION: Our findings suggest that,ayurvedic herbal preparations extracts have great potential as antimicrobial agent against many enteric bacterial pathogens and can be used in the treatment of infectious diseases. Scientific evaluation of these herbal preparations gives better information regarding the antibacterial efficacy of herbal medicine available in India. This study supports the use of these herbal preparations not only as the dietary supplement but also as agent to prevent or control the enteric bacterial infections. Further research is underway to isolate the compounds responsible for the antibacterial activity.

REFERENCES:

4. WHO: Summary of WHO guidelines for the assessment of herbal medicines. Herbal Gram. 1993: 28: 13014.
5. Tambekar DH, Khante BS, Dahikar SB, Banginwar YS: Antibacterial Properties of Contents of Triphala: A Traditional Indian Herbal Preparation. Continental J. Microbiol. 2007; 1(3):8-12.
6. Pandey MM, Rastogi S, Rawat AKS: Indian herbal drug for general healthcare: An overview. Int J Alter Med. 2008: 6:1-5.
7. Barrett B Kiefer D, Rabago D: Assessing the risk and benefits of herbal medicine; an overview of scientific evidence, Alter There Health Med. 1999; 5(4): 40-49.
8. Handa SS: Indian efforts for quality control and standardization of herbal drugs/products. Proceedings of the 1st joint workshop on quality control and standardization of traditional medicine Indo-China experience, 2004.
9. Tyler V: Herbal medicine: From the past to future, Pub Health Nutr. 2000; 3:447-452.
10. WHO:General guidelines for methodologies on research and evaluation of traditional medicine, World Health Organization, Geneva, 2000.
11. WHO: 5th Programme Report, Programme for control of diarrhoeal diseases, Geneva. WHO Bulletin, 1985: 63: 557-772.
12. Dawson W: Herbal medicine and the EU directive, J R Coll Physicians Edinb. 2005; 35: 25-27.
13. Patwardhan B, Warude D, Pushpangadan P, Bhatt N: Ayurveda and Traditional Chinese Medicine A Comparative Overview. eCAM. 2005; 2(4):465–473.
14. Parekh J and Chanda SV: In vitro Antimicrobial Activity and Phytochemical Analysis of Some Indian Medicinal Plants. Turk. J. Biol. 2007; 31:53058.
15. NCCLS (National Committee for Clinical Laboratory Standards): Performance Standards for antimicrobial susceptibility testing. 8th Informational Supplement. M100S12. National Committee for Clinical Laboratory Standards. Villanova, Pa, 2002.
16. Tambekar DH, Hirulkar NB, Waghmare AS: MAR indexing to discriminate the source of faecal contamination in drinking water. Nature, Environ.Poll. Technol. 2005; 4 (4): 525-528.
17. Mitra AP: The Wealth of India. CSIR Publications, New Delhi, 1985.
18. Uma B, Prabhakar K, Rajendran S and Lakshmi SY: Antimicrobial activities of some medicinal plants against extended spectrum beta lactamase producing Gram negative enteric bacterial pathogens.Pharmacology online, 2009; 1: 389-392.
19. Saeed S and Tariq P: Antibacterial activities of Emblica Officinalis and Coriandrum sativum against Gram negative urinary pathogens. Pak. J. Pharm. Sci. 2007; 20(1):32-35.
20. Lo-Cantore P, Iacobellis NS, De-Marco A, Capasso F and Senatore F: Antibacterial activity of Coriander sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils. J. Agri. Food Chem.2004; 52:7862-7866.
21. Rani P and Khullar N:Antimicrobial evaluation of some medicinal plants for their antienteric potential against multi-drug resistant Salmonella typhi. Phytotherapy Res. 2004; 18(8):670-73.
22. Tambekar DH, Dahikar SB and Lahare MD: Antibacterial potentials of some herbal preparations available in India. Res. J. Med. Medicinal Sci. 2009; 3(2):224-227.
23. Reddy P, Srinivas J, Jamil K, Madhusudan P, Anjari G and Das B: Antibacterial activity of isolates from Piper longam and Taxus baccata. Pharmaceutical Biol. 2001;39(3):236-339.
24. MazumderR, Bhattacharya S, Mazumder A, Pattnaik AK, Tiwary PM and Chaudhary S: Antidiarrhoeal evaluation of Aegle Marmelos (Correa) Linn. root extract. Phytotherapy Res. 2006; 20(1):82–84.
25. Balakrishnan N, Bhaskar VH, Jayakar B and Sangameswaran B: Antibacterial activity of Mimosa pudica, Aegle marmelos and Sida cordifolia. Phcog. Mag. 2006; 2(7):198-199.
26. Elizabeth and Williamsons: Major herbs of Ayurveda, Churchill living stone, Elsevier Science Ltd, London, 2002.
27. Rahman MU, Gul S and Ejaz AO: Antimicrobial activities of Ferula assafoetida oil against Gram positive and Gram negative bacteria. American-Eurasian J. Agri. Env. Sci. 2008; 4(2):203-206.
    

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