Antibacterial activity of some Indian Ayurvedic preparations against enteric bacterial pathogens.

In Ayurveda, various herbal preparations are clinically used to prevent or cure infectious diseases. Herbal preparations such as Triphala churna, Hareetaki churna, Dashmula churna, Manjistadi churna, Sukhsarak churna, Ajmodadi churna, Shivkshar pachan churna, Mahasudarshan churna, Swadist Virechan churna and Pipramool churna were investigated by preparing their organic solvent extract 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, respectively. In the present study, Triphala churna, Hareetaki churna, Dashmula churna were potent antibacterial agents against S. epidermidis, P. vulgaris, S. aureus, E. coli, P. aeruginosa and S. typhi. The study supports the use of these herbal preparations not only as dietary supplements but also as agents to prevent or control enteric bacterial infections.

INTRODUCTION

In Ayurvedic medicine, many medicinal plants are useful in strengthening human health care system and the formulations based on such medicinal plants play an important role in modern medicine.[1] Ayurvedic practitioners also identified a number of herbal preparations for curing various ailments and diseases.[2] The primary benefits of using plant-derived medicine are relatively safer than synthetic drugs and offer profound therapeutic benefits.[3] Single and polyherbal preparations have diverse range of bioactive molecules and play a dominant role in the maintenance of human health since ancient times.[4] More than 1500 herbal preparations are sold as dietary supplements or ethnic traditional medicines.[5] The most frequently used type of herbal preparations are churnas. Churnas are preparations comprising of fine powders of medicinal plants and may be single or in combination. Combinations of medicinal plants may increase the antimicrobial spectrum and potency of the preparations. Enteric or diarrheal infections are major public health problems in developing countries and contribute to the death of 3.3-6.0 million children annually. Enteric bacteria comprised of Salmonella sp., Shigella spp., Proteus sp., Klebsiella sp., Escherichia coli, Pseudomonas sp., Vibrio cholerae and Staphylococcus aureus are the major etiological agents of sporadic and epidemic diarrhea both in children and adults.[6] Recently, it has been demonstrated that many human pathogenic bacteria have developed resistance against several synthetic drugs.[7-9] 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. It 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.[1011] Hence an attempt was made to screen the antibacterial potential of herbal preparations in the control 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 tested for antibacterial potential

Preparation of Extracts

The aqueous extract was prepared by adding 20 g of herbal preparations in 200 mL distilled water, boiled on low heat for 2 h, filtered through cloth and filtrate was evaporated to dry on sand bath. The organicsolvent extracts were separately prepared by adding 20 g herbal preparation (powder) in 200 mL of respective organic solvent (acetone, ethanol and methanol) in screw-capped bottles, shaked at 190-220 rpm on a rotary shaker. After 24 h of shaking, it was filtrated, evaporated in vacuum and dried by rotary evaporator at 60°C.[12] Dried extracts were stored in labeled sterile screw capped bottles at 4°C and later used in vitro study.

Bacterial Cultures

The standard pathogenic bacterial cultures were procured from IMTECH, Chandigarh, India and used in the present study. The bacteria rejuvenated in Mueller-Hinton broth (Hi-media laboratories, Mumbai, India) at 37°C for 18 h and then stocked at 4°C in Mueller-Hinton Agar. Subcultures were prepared from the stock for bioassay. The inoculum size of the bacterial culture was standardized according to the National committee for Clinical Laboratory Standards[13] guideline. The pathogenic bacterial culture was 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 was 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 5 mg of each extracts of herbal preparations. The prepared disc were dried in controlled temperature to remove excess of solvent and used for study.

Antibacterial Activity Using Disc Diffusion Method

The modified paper disc diffusion[13] was employed to determine the antibacterial activity of both aqueous and organic extract of the herbal preparations. In each test tube turbidity of inoculum was matched with McFarland turbidity standard. Inoculum was spread over the nutrient agar plate using a sterile cotton swab in order to obtain uniform microbial growth. Then the prepared antibacterial discs were kept over the lawn and pressed slightly along with positive and negative control. Ampicillin 10 mcg/disc (Hi-Media) were used as positive control while disc soaked in sterile distilled water and various organic solvents and dried were placed on lawns as negative control. The plates were incubated for 18 h at 37°C. The antibacterial activity was evaluated for 5 mg/disc and diameter of inhibition zones were measured. Experiment was carried out in triplicate and the averages diameter of zone of inhibition was recorded. The antibacterial activity was classified as highly active (>20 mm), mild active (15-20 mm) and slightly active (12-15 mm) and less than 12 mm was taken as inactive. Antimicrobial Sensitivity Index (ASI) was calculated by following formula:

RESULTS AND DISCUSSION

In the present study, the samples namely Triphala churna, Hareetaki churna, Dashmula churna, Manjistadi churna, Sukhsarak churna, Ajmodadi churna, Shivkshar pachan churna, Mahasudarshan churna, Swadist Virechan churna and Pipramool churna were tested for their antibacterial properties against the various bacterial pathogens [Figure 1].

Figure 1

Antibacterial potential of herbal preparations

Results showed that Triphala churna possess strong antibacterial activity against Staphylococcus epidermidis, S. aureus while moderate against Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and weak against Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Bacillus subtilis. Acetone and methanol extracts of Hareetaki churna was strong antibacterial agents against S. epidermidis, S. aureus at 5 mg/disc while moderate against B. subtilis, P. vulgaris, S. typhi, P. aeruginosa and mild against K. pneumoniae, E. aerogenes, S. typhimurium and E. coli. Hareetaki churna is used as mild laxative, detoxifier and improve digestive functions. Herbs used in Hareetaki churna possess antibacterial properties; Terminalia chebula antibacterial against E. coli, S. aureus, P. aeruginosa, P. vulgaris, S. epidermidis, S. typhi, S. typhimurium [14-16] and methicilin-resistant S. aureus.[17] Mahasudarshan churna is an excellent remedy for the complication associated with fever, liver enlargement, spleen, fatigue and nausea, etc. Holarrhena antidysenterica is one of the important ingredients of Mahasudarshan churna has reported antibacterial activityagainst S. aureus, S. flexneri, S. typhosa, B. subtilis.[1819] Studies have indicated the antibacterial potential of Mahasudarshan churna against S. typhi, S. epidermidis, E. coli, S. aureus, K. pneumoniae, P. vulgaris and P. aeruginosa [Table 2]. Mahasudarshan churna is useful to control infectious diseases such as typhoid fever, intestinal infection, urinary tract infections and respiratory infection which are associated with the above-mentioned bacterial pathogens.

Table 2

Antibacterial activities of herbal preparation against various bacterial pathogens

Methanol and acetone extracts of Ajmodadi churna were strong antibacterial agents against S. aureus, S. epidermidis, S. typhi, B. subtilis, E. coli and P. vulgaris while aqueous extract was less antibacterial. Ajmodadi churna is rich formulation of carminative and antispasmodic herbal ingredients. All the ingredients of Ajmodadi churna such as Trachyspermum ammi is carminative, antispasmodic and antibacterial against E. coli, S. aureus and P. aeruginosa.[2021] Plumbago zeylanica is antibacterial against E. coli and P. aeruginosa.[22] Sukhsarak churna is a laxative and blood purifier, useful in digestive and liver disorder. In present study it was found strong antibacterial against S. aureus, S. epidermidis, B. subtilis, S. typhimurium, E. coli, K. pneumoniae, S. typhi and P. vulgaris. Methanol extract of Swadist Virechan churna was a strong antibacterial agent against S. aureus, B. subtilis, P. vulgaris, P. aeruginosa and B. subtilis [Figure 1]. The use of Swadist Virechan churna may be helpful to reduce bacterial infections caused by above test pathogens. Manjistadi churna indicated as alterative and blood purifier, useful in fever. Study indicated that Manjistadi churna was antibacterial against S. epidermidis, S. aureus, B. subtilis, S. typhimurium, E. coli, P. aeruginosa, K. pneumonia, P. vulgaris, S. typhi and E. aerogenes. Dashmula churna is a herbal preparation of 10 important medicinal plants [Table 1]. Study also proved the antibacterial potential of Dashmula churna against S. aureus, S. epidermidis, P. vulgaris, S. typhi, B. subtilis, E. coli, K. pneumonia, E. aerogenes and P. aeruginosa and useful in treatment of the bacterial infections [Table 2]. Almost all the ingredients of Dashmula churna were reported as potent antibacterial agent against various bacterial pathogens. Aegle marmelos, an important ingredient of Dashmula churna root, have antibacterial properties against V. cholerae, E. coli and Shigella sp.[23] Pipramool churna was also showed significant antibacterial activity against S. aureus, S. epidermidis, E. coli, B. subtilis and P. vulgaris. Shivkshar Pachan churna is the fortified form of Hingwashtak churna. Ferula asafetida is an important ingredient of Shivkshar Pachan churna and reported as antibacterial agent against Bacillus megaterium, B. subtilis, L. acidophilus, M. luteus, S. epidermidis, S. aureus, E. coli, S. typhi and S. flexneri.[2425] Present study indicated that both the aqueous and organic extracts of Shivkshar Pachan churna is potent antibacterial agent against S. aureus, S. epidermidis, B. subtilis, P. aeruginosa and P. vulgaris. Thus clinically used herbal preparations proved to posses antibacterial potential against S. aureus, S. epidermidis, P. aeruginosa, E. coli, P. vulgaris, K. pneumoniae and S. typhimurium. Hence diseases caused by test organism such as skin infections (impetigo, folliculitis), invasive diseases (wound infections, osteomyelitis, bacteremia), wound infection, urinary tract infections, endocarditis, septicemia, respiratory tract infection, eye infections, etc. may be prevented or controlled by the use of above tested ayurvedic preparations.

CONCLUSIONS

Our findings suggested that, ayurvedic herbal preparations extracts have great potential as antimicrobial activity against enteric bacterial pathogens and they can be used in the treatment of infectious diseases. The data obtained in these studies justify the use of these ayurvedic herbal preparations in medical practice by majority of the populations in India. The study also 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.