Abstract
Cyperus rotundus, Caesalpinia bonducella, Tinospora cordifolia, Gardenia gummifera, Ailanthus excelsa, Acacia arabica, Embelia ribes and Ventilago maderspatana from Melghat forest were screened for their antibacterial potential against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhi, Shigella flexneri, Salmonella paratyphi, Salmonella typhimurium, Pseudomonas aeruginosa, Enterobacter aerogenes by disc diffusion method. Out of these medicinal plants Caesalpinia bonducella, Gardenia gummifera and Acacia arabica showed remarkable antibacterial potential. The phytochemical analysis had showed the presence of Cardiac glycosides in all extracts (aqueous, acetone, ethanol and methanol) of Acacia arabica, Gardenia gummifera and ethanol, methanol extracts of Caesalpinia bonducella. Flavonoids were present in Gardenia gummifera, Ailanthus excelsa and acetone, methanol extracts of Acacia Arabica. Tannins and phenolic were present in Cyperus rotundus, Embelia ribes, and organic extracts of Ventilago maderspatana.
Keywords: Antibacterial activity, Melghat, Medicinal Plants, Phytochemical
Introduction
Melghat forest is part of Amravati district of Maharashtra State (India) and it preserves innumerable valuable medicinal plants. The knowledge of these medicinal plants was passed traditionally from one generation to other without documentation (according to Mr. R. B. Giri, 1983). Korkus or Bhumka or Bhagats traditionally used plants for the treatments of diarrhea, dysentery, stomachache, and any other enteric disorder but their antibacterial potential were not documented (Badhe and Pande, 1988; Tambekar and Saratkar, 2005). Almas (2001) demonstrated antibacterial potential of Babul. Lavhale and Mishra, (2007) claimed antitumor, antileukemic antifeedant activities of quassinoids in Ailanthus excelsa; Rani and Khullar (2004) showed moderate antibacterial activity of aqueous and methanol extracts of Embelia ribes, Caesalpinia bonducella. Dwivedi, et al. (2006) studied antibacterial, antimalerial activity of Caesalpinia bonducella seeds. The aqueous, ethanol and methanol extracts of Nut grass were studied by Jha et al. (2006) while Shivkumar et al. (2007) demonstrated its anticonvalscent activity. Loizzo et al. (2006), Dell'Agli et al. (2008) and Shemali et al. (2001) studied the antihypertensive, antimalerial and antibacterial (ethyl acetate extract, 6mg/disc) properties of bark of Ailanthus excelsa. Jain et al. (2007) studied the antimicrobial activities of Embelia ribes in Piper longum. Diarex, an herbal formulation against non-specific diarrhea, containing Tinospora cordifolia is an effective drug (Irfan et al., 2001).
Despite the numerous advances in medicine, the prevalence of infectious diseases continues to rise due to emergence of antibiotic resistant pathogens, which are attributed to the widespread use of antibiotics. Search for new antibacterial agents from plants has now a day gained an importance. The interest primarily has arisen from the belief that green medicine is safe and dependable, compared with costly synthetic drugs that can have adverse effects. Therefore, the objective of this study was to screen medicinal plants from Melghat forest for antibacterial potential against different enteric pathogens by scientific experimentation.
Materials and methods
Selection of Medicinal plants and preparation of extracts
With help of traditional herbal healer (Korkus or Bhumka or Bhagats of Melghat forest), we identified 8 medicinal plants, Acacia arabica (leaves), Caesalpinia bonducella (seeds), Cyperus rotundus (rhizomes), Embelia ribes (seeds), Gardenia gummifera (resinous exudation of leaf buds and shoots), Tinospora cordifolia (stem) and Ventilago maderspatana (stem, bark) from Melghat forest (Table 1), which are used by these people against diarrhoeal or abdominal discomforts or intestinal infections. R. B. Giri, Range Forest Officer, Maharashtra Forest Rangers College, Chikhaldara identified these plants. Selected parts of plants were collected, cleaned and disinfected with water and mercuric chlorides (0.5%), dried in shadow and ground to powder in grinder mixer. A 10 g of powder was soaked in 100 mL of solvent (water, ethanol, methanol, and acetone), refluxed in soxlet apparatus, filtered and filtrate was evaporated in controlled conditions of temperature to avoid destruction of dissolved phytochemicals.
Table 1.
Plants selected for study
| Botanical name | Local name | Vernacular name | Plants parts used | Medicinal use by Korkus |
|
Acacia arabica (Mimosaceae) |
Babul, Acacia |
Babul | Leaves | Astringent, diarrhea, dysentery |
|
Ailanthus excelsa (Simaroubaceae) |
Maharukh | Tree of heaven | Leaves | Decoction of leaves in pectoral lesions, diarrhea |
|
Caesalpinia bonducella (Caesalpiniaceae) |
Sagargoti | Fever Nut, Nikkar nut, Bonduc nut, |
Seeds | Digestive problems, dysentery, vomiting |
|
Cyperus rotundus (Cyperaceae) |
Nagarmotha | Nut grass | Rhizomes | Diarrhea, dysentery, indigestion |
|
Embelia ribes (Myrsinaceae) |
Vavdinga | Babreng, embelia | Seeds | Dyspepsia, colic pain, cough, asthma |
|
Gardenia gummifera (Rubiaceae) |
Dikamali | Gummy cape jasmine |
Resinous exudates of leaf buds and shoots |
Nervous disorders, diarrhea due to dentition |
|
Tinospora cordifolia (menispermaceae) |
Gulvel | Gulancha tinospora |
Stem | Chronic fever, polyuria diabetes |
|
Ventilago maderspatana (Rhamnaceae) |
Raktavalli raktapapadi |
-- | Stem, bark | Stomachic, tonic and stimulant |
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 18hr and then stocked at 4°C in Mueller-Hinton Agar. Subcultures were prepared from the stock for bioassay. A loopful of culture was inoculated in 10 mL of sterile nutrient broth and incubated at 37°C for 3hr. Turbidity of the culture was standardized to 105 CFU with the help of SPC and Nephlo-turbidometer.
Preparation of Disc for antibacterial activities
Sterile Whatman filter paper discs (10 mm) were soaked in the solution in such concentration that, the amount of solution absorbed by each disc contain 2, 4, 6, 8,10 mg of extract of each aqueous and organic extracts of Acacia arabica (leaves), Caesalpinia bonducella (seeds), Cyperus rotundus (rhizomes), Embelia ribes (seed), Gardenia gummifera (resinous exudation of leaf buds and shoots), Tinospora cordifolia (stem) and Ventilago maderspatana (stem, bark). These prepared discs were dried in controlled temperature and used for the study.
Agar gel diffusion antibacterial activities
For antibacterial properties, 0.1 ml 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 37°C overnight) were applied to the surface of MHA plates seeded with 3hr broth culture of the test bacterium. The plates were then incubated for 18hr at 37°C. Antibiotic susceptibility discs, ampicillin 10µg, 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 by measuring the diameter of inhibition zone. The experiment was performed in duplicate and the mean of the diameter of the inhibition zones was calculated.
Phytochemical analysis
The presence of saponins, tannins, anthraquinones, alkaloids, triterpenes, flvonoids, glycosides, reduced sugar, and phlobatannins were detected by simple qualitative methods (Khandelwal, 2001).
Results and discussion
During the past decades, traditional systems of medicine have become increasingly important in view of their safety. A current estimate suggests that, in many developing countries, a large proportion of population relies heavily on traditional practitioners and medicinal plants to meet primary health care needs. The present study was conducted to investigate antibacterial properties of 8 selected plants from Melghat forest, which is less studied and used in Indian Folkloric Medicine. Herbal remedies play a fundamental role in traditional medicine in rural areas of India where the therapeutic treatment of choice as antiseptic, anti-inflammatory and in treatment of infectious diseases including diarrhea. In present study, attempt was made to correlate traditional herbal medicinal knowledge held by the Indian native people with modern scientific laboratory-based assay.
A total of 32 extracts of 8 medicinal plants were tested for antibacterial activity. Out of these, 18 extracts were with antibacterial potential. Escherichia coli, Salmonella typhi, Proteus vulgaris, Salmonella paratyphi, Salmonella typhimurium and Pseudomonas aeruginosa were resistant to Cyperus rotundus, Caesalpinia bonducella, Tinospora cordifolia, Ailanthus excelsa, Embelia ribes and Ventilago maderspatana with 10mg/disc. Proteus vulgaris was sensitive to acetone extract (6mg/disc) of Cyperus rotundus. Methanol extract of Caesalpinia bonducella proved antibacterial to S.aureus, S.flexneri, and E.aerogenes. Organic extracts of Gardenia gummifera was active against S.aureus, K. pneumoniae, and E.aerogenes. Ventilago maderspatana was antibacterial against S.aureus, K.pneumoniae, P.vulgaris, S. flexneri and E.aerogene. (Parekh and Chanda, 2006) and Moon et al. (2006) also demonstrated similar antibacterial properties of these plants. S. flexneri a causative agent of bacterial dysentery was resistant to aqueous extracts of all plants but sensitive to methanol extract of Caesalpinia bonducella (2mg/disc), which was also observed by Jha et al. (2006). 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, S. flexneri and E. aerogenes. Almas, (2001) and bioassay studies of Dabur et al. (2007) 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 (Table 3).
Table 2.
Zone of inhibition (including the diameter of disc in mm) of different extracts of selected plants with test pathogens.
| Plants |
Cyperus rotundus |
Caesalpinia bonducella |
Tinospora cordifolia |
Gardenia gummifera |
Ailanthus excelsa |
Acacia arabica |
Embelia ribes |
Ventilago madraspatana |
Amoxicillin | Negative Control |
|||||||||||||||
| Extract mg/disc → | 8 | 10 | 2 | 4 | 6 | 8 | 10 | 10 | 2 | 4 | 6 | 8 | 10 | 10 | 2 | 4 | 6 | 8 | 10 | 10 | 8 | 10 | 10 | - | |
|
Escherichia coli (MTCC443) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 50 | - |
| Acetone | 13 | 15 | 16 | 17 | 19 | 20 | 21 | 13 | 16 | 17 | 18 | 19 | 20 | - | - | 15 | 16 | 18 | 21 | - | - | - | 11 | ||
| Ethanol | 13 | 16 | 18 | 18 | 19 | 19 | 20 | 12 | 15 | 18 | 19 | 19 | 21 | - | - | - | 14 | 15 | 17 | - | - | - | 11 | ||
| Methanol | - | - | - | - | - | - | 15 | - | - | - | - | - | - | - | 17 | 18 | 20 | 22 | - | - | - | - | - | ||
|
Staphylococcus aureus (MTCC96) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 12 | 40 | - |
| Acetone | 13 | 17 | - | - | - | - | 13 | 16 | 18 | 18 | 19 | 20 | 20 | - | 14 | 16 | 20 | 22 | 24 | - | - | 14 | - | ||
| Ethanol | 14 | 18 | - | - | - | 13 | 14 | 13 | 15 | 17 | 17 | 18 | 19 | - | - | 14 | 15 | 16 | 17 | 14 | 14 | 16 | 12 | ||
| Methanol | 14 | 15 | 25 | 27 | 31 | 33 | 34 | 12 | 14 | 18 | 19 | 21 | 22 | - | 14 | 20 | 20 | 21 | 25 | - | 13 | 16 | - | ||
|
Enterobacter aerogenes (MTCC111) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | 14 | - | - | - | - | - | - | - | - | - | 38 | - |
| Acetone | - | - | - | - | - | - | 16 | - | - | - | 16 | 18 | 20 | - | - | 14 | 15 | 16 | 18 | 13 | 16 | 19 | 11 | ||
| Ethanol | 13 | 14 | - | - | - | 16 | 18 | 13 | - | 14 | 15 | 17 | 20 | - | - | - | 14 | 16 | 18 | 14 | 14 | 16 | 12 | ||
| Methanol | 14 | 15 | - | 15 | 24 | 28 | 33 | - | - | 12 | 14 | 16 | 20 | - | - | - | 13 | 18 | 22 | - | 14 | 16 | - | ||
|
Pseudomonas aeruginosa (MTCC424) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 29 | 12 |
| Acetone | 14 | 14 | 15 | 15 | 17 | 18 | 19 | 18 | 12 | 15 | 17 | 18 | 20 | - | - | - | - | 14 | 15 | - | - | - | 13 | ||
| Ethanol | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 13 | - | - | - | - | ||
| Methanol | 13 | 14 | 16 | 18 | 19 | 20 | 22 | 14 | 16 | 18 | 18 | 19 | 20 | - | - | - | - | 16 | 18 | - | - | - | - | ||
|
Salmonella typhi (MTCC734) |
Aqueous | 13 | 16 | 18 | 18 | 19 | 20 | 20 | 13 | 14 | 16 | 17 | 18 | 19 | - | - | - | - | - | - | - | - | 20 | 30 | - |
| Acetone | 14 | 14 | 16 | 16 | 17 | 18 | 19 | 18 | 12 | 15 | 17 | 18 | 18 | - | 15 | 16 | 18 | 20 | 23 | - | - | 17 | - | ||
| Ethanol | 15 | 17 | 15 | 17 | 18 | 19 | 19 | 19 | 19 | 21 | 15 | 17 | 19 | - | 15 | 17 | 19 | 19 | 21 | - | - | 19 | 13 | ||
| Methanol | 17 | 17 | 12 | 14 | 16 | 17 | 19 | 18 | 19 | 26 | 17 | 17 | 18 | - | 17 | 17 | 18 | 19 | 26 | - | - | 23 | 11 | ||
|
Salmonella typhimurium (MTCC98) |
Aqueous | 15 | 17 | 14 | 16 | 19 | 19 | 20 | 19 | 19 | 21 | 15 | 17 | 19 | - | - | - | - | - | - | - | - | - | 32 | 12 |
| Acetone | 14 | 14 | 16 | 16 | 17 | 19 | 19 | 18 | 12 | 15 | 17 | 18 | 18 | - | - | - | - | 13 | 15 | - | - | - | 11 | ||
| Ethanol | 13 | 14 | 13 | 15 | 17 | 17 | 20 | 17 | 15 | 17 | 19 | 19 | 22 | - | - | - | - | 12 | 15 | - | - | - | 11 | ||
| Methanol | 12 | 13 | 13 | 15 | 17 | 18 | 19 | 18 | 12 | 15 | 16 | 18 | 20 | - | - | - | - | 14 | 15 | - | - | - | - | ||
|
Proteus vulgaris (MTCC426) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 55 | 11 |
| Acetone | 17 | 23 | 14 | 19 | 20 | 21 | 22 | - | 14 | 15 | 19 | 20 | 21 | - | 11 | 14 | 16 | 16 | 21 | - | - | 13 | 11 | ||
| Ethanol | 24 | - | - | - | - | - | - | - | - | - | - | - | - | - | 14 | 16 | 19 | 20 | 20 | - | - | 15 | 12 | ||
| Methanol | - | 14 | 14 | 16 | 18 | 20 | 22 | - | 13 | 14 | 16 | 18 | 20 | - | - | 13 | 18 | 20 | 23 | - | - | 13 | 11 | ||
|
Klebsiella pneumoniae (MTCC2653) |
Aqueous | - | - | - | - | - | - | - | 13 | 14 | 16 | 18 | 20 | 21 | - | - | - | - | - | - | - | - | - | 35 | - |
| Acetone | 12 | 13 | 13 | 14 | 16 | 18 | 20 | - | - | - | 16 | 18 | 20 | - | - | - | - | 15 | 16 | 14 | 15 | 16 | 12 | ||
| Ethanol | 12 | 13 | 12 | 16 | 18 | 20 | 21 | - | - | 14 | 17 | 21 | 22 | - | - | - | - | 14 | 16 | 16 | - | 14 | 11 | ||
| Methanol | 13 | 15 | 14 | 16 | 18 | 20 | 21 | - | - | 14 | 18 | 20 | 21 | - | - | - | - | 15 | 17 | - | - | 16 | 12 | ||
|
Salmonella paratyphi (MTCC735) |
Aqueous | - | - | - | - | - | - | - | 13 | - | - | - | - | 14 | 14 | - | - | - | - | 14 | - | - | - | 32 | - |
| Acetone | - | - | - | - | - | - | 16 | 13 | - | - | - | - | - | - | - | - | - | 13 | 15 | - | - | - | 11 | ||
| Ethanol | - | 14 | 15 | 16 | 18 | 22 | 22 | 14 | 14 | 15 | 19 | 20 | 21 | 14 | - | 17 | 21 | 23 | 25 | - | - | 13 | 12 | ||
| Methanol | 13 | 13 | 13 | 16 | 18 | 18 | 22 | 13 | 14 | 16 | 18 | 20 | 21 | 15 | - | 14 | 16 | 18 | 20 | - | - | 14 | - | ||
|
Shigella flexineri (MTCC1457) |
Aqueous | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 39 | - |
| Acetone | 14 | 16 | 14 | 16 | 17 | 18 | 20 | 19 | - | 12 | 14 | 16 | 18 | - | 14 | 15 | 19 | 20 | 21 | - | - | 15 | 11 | ||
| Ethanol | 13 | 16 | 16 | 17 | 19 | 20 | 22 | 14 | 18 | 14 | 18 | 19 | 20 | - | - | 17 | 18 | 20 | 25 | - | - | 15 | 11 | ||
| Methanol | - | - | 16 | 17 | 19 | 19 | 22 | 14 | 18 | 18 | 21 | 21 | 22 | - | 15 | 16 | 18 | 22 | 24 | - | - | - | |||
Table 3.
Phytochemical analysis of the plant's extracts
| Constituent |
Cyperus rotundus |
Caesalpinia bonducella |
Tinospora cordeopholia |
Gardenia gumiphera |
Ailanthus excelsa |
Acacia arabica | Embelia ribes |
Ventilago madraspatna |
||||||||||||||||||||||||
| Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | Aqueous | Acetone | Ethanol | Methanol | |
| Reducing Sugars |
+ | − | + | + | − | + | − | − | − | − | + | + | − | − | − | − | − | − | − | − | + | + | + | + | − | − | − | − | + | + | + | + |
| Gums | + | − | + | + | + | − | − | − | − | + | + | + | − | + | − | − | − | − | − | − | + | + | + | + | − | − | − | − | + | + | + | + |
| Proteins | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − | − | − | + | + | − | − | − | − | − | − | − | − | + | − | − | − | − |
| Fats & Oils | − | − | + | + | + | + | − | − | − | − | + | − | + | + | + | − | − | − | + | − | + | + | − | + | − | − | − | + | − | − | − | − |
| Steroids | + | + | + | + | − | − | − | − | + | + | + | + | − | + | + | + | + | + | − | − | + | + | + | + | − | − | + | + | + | + | + | + |
| Cardiac Glycosides |
− | + | + | + | − | + | + | + | + | + | + | + | + | + | + | + | − | − | + | + | + | + | + | + | + | + | + | + | − | + | + | + |
| Anthraquinones | + | − | + | − | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Flavonoids | + | + | + | + | − | − | − | − | − | − | + | + | + | + | + | + | − | − | − | − | + | + | + | + | − | − | − | − | − | − | − | − |
| Alkaloids | − | − | + | + | + | + | + | + | + | − | − | + | + | + | + | + | + | + | + | − | + | + | + | + | + | + | + | + | + | + | + | + |
| Tannins & Phenolics |
+ | + | + | + | − | − | − | − | + | + | + | + | + | + | + | + | − | − | − | − | + | + | + | + | + | + | + | + | + | + | + | + |
The difference in the antibacterial potentials of different extracts suggested that solubility of various phytochemical in various solvents made it different from the others. Acetone extract of Ventilago maderspatana proved antibacterial to Klebsiella pneumoniae, Enterobacter aerogenes (8mg/disc) and Shigella flexneri (10 mg/disc). Ethanol extract was sensitive to Staphylococcus aureus, Shigella flexneri, and Enterobacter aerogenes (10mg/disc) and the aqueous extract had antibacterial effect on Enterobacter aerogenes only. Basu et al. (2005) also observed similar antibacterial activity in chloroform and ethanol extracts. Tinospora cordifolia did not posses bactericidal activity (Thatte et al. 1992); which is reestablished in present study. The aqueous and organic solvents extracts of Cyperus rotundus proved mild antibacterial (Grewal, 2000; Jha et al. 2006).
Conclusions
The extracts possessing high antibacterial effects should be further studied for their therapeutic use. The present study suggests that these plants extracts were antibacterial against bacterial pathogens thus supporting their folkloric usage.
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