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Screening of the medicinal plants and determination of minimum inhibitory concentration (MIC) against Vibrio cholerae and Vibrio parahaemolyticus.
A simple in vitro screening assay was employed for the standard strain of Vibrio cholerae, 12 isolates of Vibrio cholerae non-O1, and Vibrio parahaemolyticus. Aqueous and organic solvent extracts of different parts of the plants were investigated by using the disk diffusion method. Extracts from 16 medicinal plants were selected on account of the reported traditional uses for the treatment of cholera and gastrointestinal diseases, and they were assayed for vibriocidal activities.
The different extracts differed significantly in their vibriocidal properties with respect to different solvents. The MIC values of the plant extracts against test bacteria were found to be in the range of 2.5-20 mg/ml.
The results indicated that Lawsonia inermis, Saraca indica, Syzygium cumini, Terminalia belerica, Allium sativum, and Datura stramonium served as broad-spectrum vibriocidal agents.
Cholera is an acute intestinal disease caused by Vibrio cholerae. The organism has a short incubation period of less than a day, extending up to five days. The enterotoxin produced by this rod causes copious, painless, watery diarrhea leading to vomiting, severe dehydration, and even death if treatment is not prompt. Cholera can spread as an endemic, epidemic, or pandemic. Typically, antimicrobial agents are administered for 3-5 days; however, a single-dose therapy with tetracycline, doxycycline, furazolidone, or ciprofloxacin has been seen to be effective in reducing the duration and volume of diarrhea. However, the resistance to antimicrobial agents, driven in part by their widespread traditional use, has become an increasing problem throughout the world.
With the alarming incidence of antibiotic resistance in bacteria of medical importance, there is increasing interest in plants as a source of agents for the treatment of microbial diseases. According to the World Health Organization more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. The most significant bioactive compounds obtained from plants include alkaloids, flavonoids, tannins and phenolic substances. The vibriocidal action of different extracts from the leaves of the shrub Punica granatum was studied against various strains of Vibrio cholerae. The leaf extract of Mimusaops elengi showed antibacterial activity against V. cholerae and other bacteria. Muruganandan et al. have reported the antibacterial activity of the Syzygium cumini bark. Reddy et al. have found antibacterial constituents from the berries of Piper nigrum.
A justified belief persists that traditional medicine is a lot cheaper and more effective than modern medicine. In developing countries, people belonging to the poor socioeconomic background use folk medicine for the treatment of common infections. Research on bioactive substances could possibly lead to the discovery of new compounds, resulting in the formulation of new and more potent antibacterial drugs, to overcome the problem of resistance to currently available antibiotics. This article reports the results of a survey that was done based on folk uses by traditional practitioners in the tribal area of the Mahakoshal region of Central India, along with a bioassay test for vibriocidal activity. In the present study a total of 16 plants were studied to determine and analyze the vibriocidal activity.
The plant samples were collected from tribal regions of Mandla and Dindori (M.P.), India; based on the information provided in the enthnobotanical survey of India and from the local medicine men of tribal regions, and were screened for potential vibriocidal activity [Table 1].
The microorganisms were obtained from the culture collection center of the Bacteriology Laboratory, R. D. University, Jabalpur, and (NICED), Kolkata. A total of 12 bacterial strains of Vibrio cholerae non-O1 (BGCC#59, BGCC#60, BGCC#61, BGCC#62, BGCC#63, BGCC#64, BGCC#65, BGCC#66, BGCC#67, BGCC#68, BGCC#69, and BGCC#70), involved in the present study were obtained from the culture collection center of the Bacteriology Laboratory, R.D. University, Jabalpur (MP), India. Standard strains of Vibrio cholerae (H17004) and Vibrio parahaemolyticus (KX-V138) were obtained from the National Institute of Cholera and Enteric Diseases (NICED), Kolkata.
The method of Collins et al., was followed with certain modifications for the preparation of bacterial inoculum. Accurately 0.2 ml of overnight cultures of each organism were dispensed into 20 ml of sterile nutrient broth and incubated at 37°C for 24 hours to standardize the culture to 106 cfu/ml. A loopful of this standardized culture was used for the antibacterial assay.
Ten grams of air-dried powder was placed in distilled water and boiled for 6 hours. At intervals of 2 hours it was filtered through eight layers of muslin cloth and centrifuged at 5000 X g for 15 minutes and the supernatants were collected. The supernatant was concentrated for 6 hours to make the final volume one-fourth of the original volume.
Ten grams of air-dried powder was placed in 100 ml of organic solvent (ethanol and acetone) in a conical flask, plugged with cotton, and kept on a rotary shaker at 190-220 rpm for 24 hours. After 2 hours it was filtered through eight layers of muslin cloth and centrifuged at 5000 X g for 15 minutes. The supernatants were collected and the solvents were evaporated to make the final volume one-fourth of the original volume.
Vibriocidal activity was tested using the disk diffusion method. Petri plates containing 20 ml of Mueller Hinton agar medium were seeded with a 24-hour old culture of bacterial strains. The plant extracts were tested in a concentration of 40 mg/ml, applying 10 μl of each sample to sterile filter paper disks (5 mm in diameter) and placed on the surface of the medium. Incubation was performed at 37°C for 24 hours. The inoculum size was adjusted to achieve a final inoculum of approximately 108 cfu/ml. The assessment of vibriocidal activity was based on the size of the inhibition zone formed around the disk.
The minimum inhibitory concentration (MIC) of the plant extracts were determined by the modified methods of Rios et al. and Rojas et al. The test was performed for a 2.5 mg/ml – 20 mg/ml concentration range (2.5, 5, 10, 15, 20 mg/ml) of each plant extract. The disks were impregnated with 10 μl of plant extracts, of different concentrations, placed on the Mueller Hinton inoculated agar medium and incubated at 37°C for 24 hours. The MIC was taken to be the lowest concentration inhibiting the growth of the organisms.
Medicinal plants were screened in vitro for vibriocidal activity against 12 isolates of Vibrio cholerae non-O1, and one standard strain each of Vibrio cholerae and Vibrio parahaemolyticus. The vibriocidal activity of 48 extracts (aqueous, acetone, and ethanol) belonging to 16 plant species were investigated [Table 2]. The MIC values of active extracts containing the potential bioactive compound were also determined [Table 3]. Among the plants tested Syzygium cumini, Saraca indica, Terminalia belerica, Datura stramonium, Lawsonia inermis, and Allium sativum showed high vibriocidal activity. The range of MIC values in the ethanolic extracts of Syzygium cumini, Lawsonia inermis, Terminalia belerica, and Magnifera indica were found to be 2.5–20 mg/ml, 2.5–10 mg/ml, 2.5–20 mg/ml, and 15–20 mg/ml, respectively, against test bacteria. The range of MIC values in the aqueous extracts of Allium sativum, Azadirachta indica, Tamarindus indica, Punica granatum, Eugenia caryophyllus, Mimusops elengi, and Piper nigrum were found to be 5–15 mg/ml, 5–20 mg/ml, 10–15 mg/ml, 5–20 mg/ml, 15–20 mg/ml, 15–20 mg/ml, and 20 mg/ml, respectively, against test bacteria. The range of MIC values in the acetone extracts of Saraca indica, Datura stramonium, Madhuca latifolia, Acacia catechu, and Acacia arabica were observed as 2.5–10 mg/ml, 2.5–15 mg/ml, 10–20 mg/ml, 10–20 mg/ml, and 10–20 mg/ml, respectively, against test bacteria. The MIC values in ethanolic extracts were a minimum, especially against Vibrio cholerae, Vibrio cholerae non-O1, and Vibrio parahaemolyticus (2.5 mg/ml). The MIC values in the aqueous extracts of Allium sativum were a minimum for both Vibrio cholerae and Vibrio parahaemolyticus (2.5 mg/ml) and eight isolates of Vibrio cholerae non-O1 (5 mg/ml). The MIC values in the acetone extracts of Saraca indica and Datura stramonium were low for Vibrio cholerae (2.5–5 mg/ml), Vibrio parahaemolyticus (2.5 mg/ml), and four isolates of Vibrio cholerae non-O1 (2.5–5 mg/ml). Extracts of Eugenia caryophyllus, Mimusops elengi, Piper nigrum, Punica granatum, and Tamarindus indica showed poor vibriocidal activity.
The aqueous, ethanolic, and acetone extracts exhibited potent vibriocidal activity against the bacterial strains involved in the study. The present study reports the vibriocidal activity of plant extracts used by tribals in Indian folklore medicine against cholera and gastrointestinal infections. The antibacterial activity of Lawsonia inermis has been reported against Bacillus cereus, Bacillus anthracis, Eschirichia coli, and Staphylococcus aureus. The methanolic extract of Tamarindus indica has been reported to inhibit the growth of Burkholderia pseudomallei, having a clinical origin. The MIC value of the extracts of Tamarindus indica leaves against Burkholderia pseudomallei was reported as 125 μg/ml. Similar results have been obtained in the present study. The vibriocidal action of the different extracts from the leaves of the shrub of Punica granatum against various strains of Vibrio cholerae has been reported. The presence of antibacterial activity in the leaf extract of Mimusops elengi against V. cholerae non O1 has been determined in this study. Similar results were reported by Satyanarayana et al. Mathabe et al., studied the methanolic, ethanolic, acetone, and aqueous extracts from different extracts of Indigofera daleoides, Punica granatum, Syzygium cordatum, Gymnosporia senegalensis, Ozoroa insignis, Elephantorrhiza elephantine, Elephantorrhiza burkei, Ximenia caffra, Schotia brachypetala, and Spirostachys Africana, to determine the presence of antibacterial activity against Vibrio cholerae, Escherichia coli, Staphylococcus aureus, Shigella spp., and Salmonella typhi. The extracts from the different plants showed relatively high antibacterial activity against most of the tested microorganisms with the diameter of inhibition zones ranging between 10 to 31 mm.
Modern and traditional healthcare often exist side by side, but seldom cooperate. The reasons are, lack of standardization in traditional medicine with respect to raw materials, method of production, and quality control of the finished product. The use of traditional medicine often relies on mysticism and intangible forces such as witchcraft, with some aspect based on spiritual and moral principles. While these may be valid psychologically, they cannot be rationalized scientifically. From the results it can be concluded that plant extracts have a great potential as antimicrobial compounds against microorganisms, and therefore, can be used in the treatment of infectious diseases caused by resistant microorganisms. Since the aqueous extract of A. sativum and the acetone extracts of S. indica and D. stramonium showed MIC of 2.5–5 mg/ml, these plants can be used to identify bioactive natural products, which may serve as leads for the development of new pharmaceuticals that can address the therapeutic need.
The authors are thankful to the Head of the Department of Postgraduate Studies and Research in Biological Sciences, R.D. University Jabalpur, India, for providing laboratory facilities.