IJCRR - 4(20), October, 2012
Date of Publication: 20-Oct-2012
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ANTIMICROBIAL POTENTIAL AND PHYTOCHEMICAL ANALYSIS OF PLANT EXTRACTS OF ANETHUM SOWA
Author: Rashmi Mathur
Category: General Sciences
Abstract:Anethum sowa are widely used traditional medicinal plant to treat various ailments. To provide a scientific basis for traditional uses of Anethum sowa,their ethanolic, aqueous,n-Butanol and petroleum ether extracts of various plant parts were tested against human pathogenic microorganisms (Pseudomonas aeruginosa , Escherichia coli, Cogulase positive staphylococci, Cogulase negative staphylococci , Enterococcus species, Candida albicans and Candida parapsilosis).The antimicrobial potential of Anethum sowa against human pathogenic microorganisms was investigated. Their isolated phytoconstituents were evaluated for their antimicrobial potential. Antimicrobial activity of various plants extracts was compared with commercially available antibiotics. The antimicrobial potential of the above plant extracts was seen against the test organism using agar gel diffusion susceptibility test by standard technique of Opara and Anasa(1993) . The resulting zones of inhibition were measured. The MIC (Minimum inhibitory concentration), MBC (Minimum Bactericidal Concentration) and MFC (Minimum Fungicidal Concentration) of the plant extracts was determined according to the Micro broth dilution technique (Murray et.al.1999). Phytochemical estimation was carried out according to the methods described by Trease and Evans (1989) .Ethanolic and n-Butanol extracts showed considerably good antibacterial activity against all bacteria and fungi. Among all solvents used ethanol extract gave the highest zone of inhibition. With Escherichia coli zone of inhibition was 23.8mm, with Cogulase (+) Staphylococci it was 24.5 mm., with Cogulase (-) Staphylococci 23.0 mm., with Pseudomonas aeruginosa 19.6 mm.,with Enterococcus sp. 15.3 mm., with Candida albicans and Candida parapsilosis it was 26.5 mm and 25.2 mm respectively. Phytochemical analysis showed the presence of alkaloids, flavonoids, tannins, saponins, steroids, triterpenoids, terpenoids, phenol and glycosides.
Keywords: Antimicrobial activity, phytochemical analysis, Anethum sowa
Anethum sowa belongs to family Apiaceae, commonly known as sowa. The genus name Anethum is derived from Greek word aneeson or aneeton, which means strong smelling. Its common use in ayurvedic medicine is in abdominal discomfort, colic and for promoting digestion (Pullaiah, 2002). The collection were made from different area of Ajmer district i.e. Kishangarh (Site1), Beawar (Site2), Kekri (Site3), Pushkar (Site4)and Ajmer(Site5) for the study of antibacterial and antifungal activity. Medicinal plants have been used for the treatment of various human ailments since long. A revolution came in the medicinal world with the discovery of antibiotics, for treatment of various bacterial infections. However, their indiscriminate use has led to an alarming increase in antibiotic resistance among microorganisms, giving rise to multiresistant strains, which has become a global concern (Shariff, 2001) . Thus, there is a renewed interest in exploring natural resources for such compounds. The need of the hour is to screen a number of new medicinal plants for promising biological activity and there in vitro propagation to conserve the biodiversity (Mathur et al., 2008; Shekhawat et al., 2009, 2002) . Various plants have been documented in the development of novel drugs, by evaluating their antimicrobial activity, studying active phytochemical constituents and bioactive compounds by various modern analytical techniques. It is believed that crude extract from plants are more effective than isolated components due to their synergistic effect. From the safety point of view, spices and medicinal herbs are mostly targeted to meet the therapeutic demands. Since then efficacy of many medicinal plants in the treatment of many diseases have been put to test in many laboratories (Shajahan and Ramesh, 2004). MATERIAL AND METHODS Method of extraction:- The fresh plant parts were collected, properly washed in tap water, rinsed in sterile distilled water and then air dried in the hot air oven to remove moisture and to dry them. They were then grounded soxhlet extracted using 70% ethanol. The extraction lasted for 24 hours. Antimicrobial susceptibility testing: - The antimicrobial potential of the above plant extracts was seen against the test organisms using the agar-gel diffusion susceptibility test. Sterile Mueller – Hinton plates were taken one plate/organism tested. Three wells of about 3.0 mm diameter were aseptically punched on each agar plate using a sterile cork borer, with at least 30 mm distance between adjacent wells and the periphery. According to the standard technique of Opara and Anasa (1993) - 2-4 colonies of the test organisms were inoculated in sterile broth and these inoculums was swabbed using sterile swab on the surface of above punched Mueller - Hinton agar plates. A fixed volume (0.1 ml) of the plant extract was then introduced into the wells in the increasing concentration and then incubated at 370C for 24 hours. The resulting zones of inhibition were measured. Determination of minimum inhibitory concentration (MIC):- The MIC of the plant extracts was determined according to the micro broth dilution technique (Murray et al., 1999) . It was performed in 96-well microtiter plates for determining the minimum inhibitory concentration (MIC). Standardized suspensions of the test organisms(Pseudomonas aeruginosa , Escherichia coli, Cogulase positive staphylococci, Cogulase negative staphylococci , Enterococcus species, Candida albicans and Candida parapsilosis) were inoculated into a series of 96-well microtiter plate, including one growth and one sterility control .Brain Heart Infusion(BHI) and Sabouraud dextrose broth containing plant extracts in increasing concentration viz 2.5,5,7.5,10,12.5,15,17.5,20 mg/ml. and incubated at 37? C for 24 hours. After overnight incubation these tubes were observed for turbidity. The microtiter plate showing the minimum turbidity was noted for MIC. Minimum Bactericidal Concentration (MBC) The MBCs were determined by first selecting tubes that showed no growth during MIC determination; a loopful from each tube was subcultured onto extract free agar plates, incubated for further 24 hours at 37oC. The least concentration, at which no growth was observed, was noted as the MBC. Minimum Fungicidal Concentration (MFC) The MFC were determined by first selecting tubes that showed no growth during MIC determination; a loopful from each tube was subcultured onto extract free agar plates, incubated for further 24 hours at 37oC. The least concentration, at which no growth was observed, was noted as the MFC. Phytochemical estimation: - This was carried out according to the methods described by Trease and Evans , (1989) RESULTS All the bacterial and fungal pathogens used in this work demonstrated susceptibility to the Ethanol, n-Butanol, Chloroform, Distilled water and Petroleum ether using extracts of Anethum sowa. Ethanol extract gave the highest zone of inhibition (23.8mm) on Escherichia coli. Cogulase (+) Staphylococci showed the highest zone of inhibition i.e. 24.5 mm in diameter. Cogulase (-) Staphylococci showed the highest zone of inhibition i.e. 23.0 mm in diameter. With Pseudomonas aeruginosa the highest zone of inhibition was 19.6 mm in diameter. With Enterococcus sp. highest zone of inhibition recorded was 15.3 mm in diameter. With Candida albicans and Candida parapsilosis it showed the highest zone of inhibition i.e. 25.0 mm and 24.2 mm in diameter respectively. The observation that Anethum sowa has good inhibition against Escherichia coli, Pseudomonas aeruginosa, Enterococcus sp., Cogulase (+) Staphylococcus sp., Cogulase (-) Staphylococcus sp., Candida albicans and Candida parapsilosis tends to prove worthy remedy to the problem of drug resistance against these pathogens which are already known to be resistant to most of the standard antibiotics (Penicillin, Tetracycline, Erythromycin, Streptomycin, Fluconazole and Amphotericin B) Tetracycline showed no zone of inhibition against all tested microorganisms. E.coli developed resistance against most of the antibiotics except Streptomycin. Pseudomonas aeruginosa and Enterococci showed resistance against all tested antibiotics. Anethum sowa showed effective results against most of the resistant organisms. Bacteria developed resistance against most of the tested antibiotics. Flower and Stem showed very effective results as compared to Leaf and Root of Anethum sowa. Graph 1,2,3,4 and 5 shows comparison of ethanolic, distilled water, nButanol, petroleum ether and chloroform plant parts extracts of Anethum sowa effects on test bacteria and fungi. Graph shows that ethanol solvent shows appreciable results in comparison to all solvents. Graph 6 showed Comparison of commercially available antibiotics with maximum zone found in Anethum sowa. This graph shows that antibiotics against Candida albicans and Candida parapsilosis show low level of inhibition in comparison to Anethum sowa. Table 1 showed Preliminary phytochemical screening of plant parts of Anethum sowa. During phytochemical estimation with Petroleum ether as a solvent in comparison to all plant parts root showed absence of Alkaloid where as Tannin was not found in all the plant parts except stem. In phytochemical estimation using all solvents Flavonoids, Triterpenoids, Steroids and Terpenoids were found in all plant parts. During phytochemical estimation with respect to Reducing Sugar all solvents and all plant parts showed absence of reducing sugar except Flower part with solvents petroleum ether and chloroform.Table2 showed Minimum Inhibitory Concentration of different extracts of Anethum sowa. With respect to Petroleum ether MIC and MBC was observed against all the tested organisms with all plant parts except stem with organism Enterococci and Root with organisms Cogulase (+) Staphylococci and Candida parapsilosis showed no zone of inhibition. With respect to n-Butanol extracts leaves part showed no MIC and MBC values against Candida albicans. Stem part showed no MIC and MBC values against Cogulase (-) Staphylococci ,Enterococci and Candida parapsilosis. Flower part showed against Enterococci and root part showed no MIC and MBC values against Cogulase (+) Staphylococci, Candida parapsilosis and Candida albicans. With respect to Ethanol extracts leaves part showed no MIC and MBC values against Candida albicans Stem part showed no MIC and MBC values against Cogulase (-) Staphylococci ,Enterococci and Candida parapsilosis. Flower part showed against Enterococci and root part showed no MIC and MBC values against Cogulase (+) Staphylococci and Candida parapsilosis. With respect to Aqueous extracts leaves part showed no MIC and MBC values against Candida albicans Stem part showed no MIC and MBC values against Cogulase (-) Staphylococci ,Enterococci , Pseudomonas aeruginosa and Candida parapsilosis . Flower part showed against Enterococci and root part showed no MIC and MBC values against Cogulase (+) Staphylococci, Pseudomonas aeruginosa Candida albicans and Candida parapsilosis . With respect to Chloroform extracts Stem part showed no MIC and MBC values against Cogulase (-) Staphylococci and Enterococci root part showed no MIC,MFC and MBC values against Cogulase (+) Staphylococci and Candida parapsilosis .Table3 showed Minimum Bactericidal and Fungicidal Concentrations of different extracts of Anethum sowa. The results of these investigation showed that plant extracts of Anethum sowa possess appreciable and potential antimicrobial activity against commonly encountered microorganisms in humans. DISCUSSION AND CONCLUSION The effect of Anethum sowa extracts agrees with the work of Upadhyaya and Chandrikasingh (2010) that showed antimicrobial effect of Anethum sowa over E.coli , Cogulase (+) Staphylococci , Cogulase (-), Staphylococci ,Pseudomonas aeruginosa, Candida albicans and Candida parapsilosis. The effect of Anethum sowa extracts dissagrees with the work of Bajracharya, (2008) Who showed no antibacterial effect of Anethum sowa over Escherichia coli, Klebsiella spp, Citrobacter spp,Enterobacter spp, Salmonella typhi, Salmonella paratyphi, Shigella spp, Proteus vulgaris, Proteus mirabilis and Pseudomonas spp. Kaur and Arora (2010) showed the effect of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi hot water and acetone seed extracts showed considerably good antibacterial activity against all the bacteria except Klebsiella pneumoniae and one strain of Pseudomonas aeruginosa. Anethum graveolens, Elettaria cardamomum, Foeniculum vulgare, Trachyspermum ammi and Viola odorata were found to be better/equally effective compared to standard antibiotics. V. odorata was the most effective antibacterial with minimum inhibitory concentration values ranging from 1 to 2%. Jana and Shekhawat(2010) The antimicrobial potential of the aqueous and ethanolic extracts of seeds, leaves, roots, callus and in vitro regenerated plantlets leaves of Anethum graveolens have been evaluated against important bacterial strains, Escherichia coli HB101 (MTCC-82), Bacillus subtilis (MTCC-441), Bacillus cereus (MTCC1306), Micrococcus luteus (MTCC-2452). The ethanolic extracts were found to be more potent than aqueous extracts of all parts of plant studied. The ethanolic extracts of seeds showed strong activity against all bacterial strains. In comparison to in vivo, in vitro plant extracts depicted reduced activity. The phytochemical screening of the plant parts showed that leaves, stems, roots, in vitro callus and regenerated leaves were rich in tannins, terpenoids, cardiac glycosides and flavonoids. Though, the seeds of Anethum have been used traditionally as decoctions or infusions prepared in water to treat various ailments, due to the presence of active component, hence, they show maximum activity. Escherichia coli, Pseudomonas aeruginosa, Enterococcus sp., Cogulase (+) Staphylococcus sp., Cogulase (-) Staphylococcus sp., Candida albicans and Candida parapsilosis produced appreciable susceptibility with the plant extracts. The result of this research has revealed that many active bioconstituents of Anethum sowa constitute potential qualities in its curative action. Thus it must be exploited upon by scientists in the development of human medicines and drugs. Anethum sowa research for human gastroenteritis, neonatal meningitis ,pneumonia, septicemia, urinary tract infection ,gastrointestinal infection, bactermia, bacterial endocarditis, diverticulitis , meningitis and candidiasis has thus opened a gate way for the possibility of finding cures for many strains of enteric microorganisms which are now resistant to many usable and common antibiotics in our countries. Anethum sowa is a promising cure for human diseases as have been demonstrated in this work .This work serves as an eye opener to many scientists who may utilize the result of the work in developing drugs from Anethum sowa against human pathogenic microorganisms.
Authors are grateful to the Head and Professors of Department of Microbiology J.L.N.Medical College, Ajmer for providing research facilities in their laboratory and also thankful to the Head, Department of Botany Govt.College, Ajmer.
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