|Year : 2012 | Volume
| Issue : 1 | Page : 41-45
Insignificant antimicrobial activities and phytochemical screening in different extracts of indian Ginseng
Premlata Singariya1, Padma Kumar1, Krishan Kumar Mourya2
1 Laboratory of Tissue Culture and Secondary Metabolites, Department of Botany University of Rajasthan, Jaipur, India
2 Veterinary Officer and In-charge, Goverment. Veterinary Hospital, Pahari (Bharatpur) Rajasthan, India
|Date of Web Publication||11-Aug-2012|
Dr. D. S. Kothari Post Doctoral Fellow, P.No.55, Shri Kalyan Nagar, Kartarpura, Jaipur-302015 (Rajasthan)
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of present study is to investigate the antimicrobial activity of (Flower, Ripen fruit and calyx) of Withania somnifera (RUBL20668) extracts, in order to use them as a possible source of new antimicrobial substances against important human pathogens. Materials and Methods: Crude extracts of different parts of both species of W. somnifera were evaluated against some important bacteria (two Gram +ve and four Gram-ve bacteria). Staphylococcus aureus (Gram +ve), Bacillus Subtilis (Gram +ve), Escherichia coli (Gram-ve), Raoultella planticola (Gram-ve), Pseudomonas aeruginosa (Gram-ve), Enterobactor aerogens (Gram-ve), one yeast Candida albicans and one fungus Aspergillus flavus. The dried and powdered seeds were successively extracted with hexane, toluene, iso propyl alcohol, acetone and ethanol, using the soxhlet assembly. The antimicrobial activity assay was done by both disc diffusion and serial dilution methods. Result: The results indicate that all the extracts in different polar solvents did not show any antibacterial activity against S. aureus, E. coli, R. planticola or E. aerogens or any antifungal activity against A. flavus. Conclusion: All extracts in the different polar solvents did not have or had very less antibacterial and antifungal activities.
Keywords: Aspergillus flavus , Antibacterial, Hexane, Pseudomonas aeruginosa, toluene, Withania somnifera
|How to cite this article:|
Singariya P, Kumar P, Mourya KK. Insignificant antimicrobial activities and phytochemical screening in different extracts of indian Ginseng. J Pharm Negative Results 2012;3:41-5
|How to cite this URL:|
Singariya P, Kumar P, Mourya KK. Insignificant antimicrobial activities and phytochemical screening in different extracts of indian Ginseng. J Pharm Negative Results [serial online] 2012 [cited 2019 Sep 23];3:41-5. Available from: http://www.pnrjournal.com/text.asp?2012/3/1/41/99665
| Introduction|| |
W. somnifera (Ashwagandha) has anti inflammatory effect,  analgesic effect,  anti-tumor, anti-stress, antioxidant  and memory improving effects  as also rejuvenating properties.
C. albicans is notorious for causing candidiasis, Candida infections can affect the esophagus with the potential of becoming systemic disease, causing a much more serious condition, afungemia called candidemia. , E. coli is the culprits for human urinary tract infections.  P. aeruginosa is involved in respiratory tract, urinary tract  , bloodstream, and central nervous system infections of nosocomial origin  and this pathogen is becoming resistant against gentamycin, ciprofloxacin,  tetracycline, chloramphenicol, and Norfloxacin  . The major causative agent of nosocomial infections is S. aureus along with E. coli. It has been determined that Raoultella planticola has caused severe pancreatitis in one case.  Bacillus Subtilis can contaminate food; however, it seldom results in food poisoning. E. aerogens is a nosocomial and pathogenic bacterium that causes opportunistic infections, which include most types of infections.
| Materials and Methods|| |
Crude extracts of different parts of W. somnifera (RUBL20668) were prepared with a series of non-polar to polar solvents by hot extraction method  in a soxhlet assembly. Different extracts were then screened for antimicrobial activity by disc diffusion assay  against a few medically important bacteria and fungi. The fraction that showed the best activity was then used for determining the minimum inhibitory concentration (MIC) by the tube dilution method  as also the minimum bactericidal / fungicidal concentration (MBC / MFC).
Collection of plant material
Different parts of W. somnifera were collected in the month of January, from the Jaipur district of Rajasthan. Plants samples were identified and deposited in the herbarium, department of botany, university of Rajasthan, Jaipur. The collected plant materials were transferred immediately to the laboratory cleaned with water and the selected plant parts were separately shade-dried for one week. Each shade-dried plant part was powdered with the help of a grinder. Fine powder of each sample was stored in a clean container to be used for Soxhlet extraction following the method of Subramanian and Nagarjan  in the different polar solvents.
Each plant part (10 gm) was sequentially extracted with different solvents (250 ml) according to their increasing polarity (Petroleum ether < ethyl acetate < glacial acetic acid) by using the Soxhlet apparatus for 18 hours at a temperature not exceeding the boiling point of the respective solvent. The obtained extracts were filtered by using Whatman No. 1 filter paper and then concentrated at 40 0 C by using an evaporator. The residual extracts were stored in a refrigerator at 4 0 C in small and sterile glass bottles. The Percent extractive values were calculated by the following formula.
Drugs and chemicals used
Gentamycin and Ketoconazole were used as standard antibiotics for bacteria and fungi respectively.
Petroleum ether, ethyl acetate, glacial acetic acid, Nutrient Agar (for bacteria) and Sabouraud Dextrose Agar (for fungi).
Test pathogenic microorganisms were procured from the Microbial Type Culture Collection, IMTECH, Chandigarh, India [Table 1]. The reference strains of the bacteria were maintained on nutrient agar slants, subcultured regularly (after every 30 days) and stored at 4 o C as well as at -80 o C by preparing suspensions in 10% glycerol.
The initial screening of different stem extracts for their antibacterial activity, was carried out using Mueller Hinton and Nutrient agar media, which did not reveal any significant difference. Thus further studies were carried out using nutrient agar medium only.  Bacterial strains were grown and maintained on the Nutrient Agar (NA) medium, while and fungi were maintained on Sabouraud Dextrose Agar (SDA) medium. Disc diffusion assay , was performed for screening. The NA and SDA base plates were seeded with the bacterial and fungal inoculum, respectively (inoculum size 1×10 8 CFU / ml for bacteria and 1×10 7 cell / ml for fungi). Sterile filters paper discs (Whatman no. 1, 5mm in diameter) were impregnated with 100 μl of each of the extracts (100 mg / ml) to give a final concentration of 1 mg/disc and left to dry in vacuo so as to remove the residual solvent, which might interfere with the determination. The zones of inhibition (ZOI) were measured and compared with the standard reference antibiotics.  The activity index for each extract was calculated [Table 2],[Table 3].
|Table 2: Zone of Inhibition (mm) and activity index of different extracts of Withania somnifera in different polar solvents against tested pathogens|
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|Table 3: Minimum inhibitory concentration and (MBC / MFC) of different extracts of Withania somnifera in different polar solvents against tested pathogens|
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Determination of minimum inhibitory concentration by the broth dilution method
Minimum inhibitory concentration (MIC) was determined for each plant extract showing antimicrobial activity against the test pathogens. Bacterial and fungal suspensions were used as the negative control, while broth containing a standard drug was used as the positive control. Each extract was assayed in duplicate and each time two sets of tubes were prepared (serial dilution method), one was kept for incubation, while the other set was kept at 4°C for comparing the turbidity in the test tubes.  The MIC values were taken as the lowest concentration of the extracts in the test tubes that showed no turbidity after incubation. The turbidity of the test tube was interpreted as a visible growth of microorganisms.
Determination of minimum bactericidal / fungicidal concentration
The minimum bactericidal concentration (MBC) was determined by sub culturing the test dilution on Mueller Hinton Agar and further incubating it for 24 hours. The highest dilution that yielded no single bacterial colony was taken as the minimum bactericidal concentration.  MBC was calculated for some of the extracts that showed high antimicrobial activity against highly sensitive organisms.
Total activity determination
Total activity (TA) is the volume to which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated, and is expressed in ml / g. 
Mean value and standard deviation were calculated for each test bacteria and fungi. Data were analyzed by one way ANOVA and p values were considered significant at P > 0.005. 
| Results and Discussion|| |
The preliminary phyto profiling for the different parts of W. somnifera were carried out according to Bokhari.  The precent yield of the extracts was also analyzed wherein the highest yield was recorded for the calyx extract in ethanol (15.71%) followed by ripened fruit extract (12.99%) in iso propyl alcohol [Table 4].
|Table 4: Total activity and % Yield of different extracts of Withania somnifera in different polar solvents against tested pathogens|
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Antimicrobial activity [assessed in terms of zone of inhibition (ZOI) in mm and activity index] of the different parts of W. somnifera extracts in different polar solvents, tested against selected microorganisms were recorded [Table 2]. In the present study, a total of eight extracts of selected plant were tested for their bioactivity, among which all these extracts showed insignificant antimicrobial potential against the test microbes. However, all these extracts showed no activity against S. aureus, E. coli, R. planticola or E. aerogens and antifungal activity against A. flavus, at the tested concentration. The highest antibacterial as well as antifungal activities were recorded for ripened fruit extract in iso propyl alcohol (ZOI-8.67 ± 0.27 and AI-0.867) and (ZOI-8.17 ± 0.64 and AI-1.671) against P. aeruginosa and C. albicans respectively.
Minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration
MIC and MBC / MFC values were evaluated for those plant extracts, that showed activity in the diffusion assay. The range of MIC and MBC / MFC of the extracts recorded was 3.75-15 mg / ml. In the present investigation, the lowest MIC value 3.75 mg / ml was recorded in the ripened fruit extract (against P. aeruginosa and C. albicans) in iso propyl alcohol [Table 3].
Total activity indicates the volume at which an extract can be diluted with still having the ability to kill microorganisms. The calyx and ripened fruit extracts showed high values of TA 41.9 ml against P. aeruginosa and 34.64 ml against C. albicans [Table 4].
| Conclusion|| |
W. somnifera extracts in different polar solvents at a tested concentration did not show ZOI against the selected pathogens [Table 1], although in the traditional (folk) medicine system, the root and leaf extracts were used to treat the various aliments. However,, in this study, the lack of antimicrobial activity of these extracts will be useful in avoiding any study repeated in this direction, in the future.
| Acknowledgment|| |
The authors expressed their thanks to UGC for providing financial assistance under the Dr. D. S. Kothari, Post Doctoral fellowship scheme.
| References|| |
|1.||Al Hindawi MK, Al Khafaji SH, Abdul Nabi MH. Anti-granuloma activity of Iraqi Withania somnifera. J Ethnopharmacol. 1992; 37: 113-6. |
|2.||Kulkarni SK, Ninan I. Inhibition of morphine tolerance and dependence by Withania somnifera in mice. J. EthnoPharmacology. 1997; 57(3): 213-7 |
|3.||Bhattacharya SK, Satyan KS, Ghosal S. Antioxidant activity of glycowithanolides from W somnifera in rat brain frontal cortex and striatum. Indian J. Experimental Biology. 1997; 35: 236-9. |
|4.||Schliebs R, Liebmann A, Bhattacharya SK, Kumar A, Ghosal S, Bigl V. Systemic administration of defined extracts from Withania somnifera (Indian Ginseng) and Shilajit differentially affects cholinergic but not glutamatergic and GABAergic markers in rat brain. Neurochem Int. 1997; 30: 181-90. |
|5.||Fidel PL. Immunity to Candida. Oral Dis. 2002; 8: 69-75. |
|6.||Pappas PG. Invasive candidiasis. Infect. Dis. Clin. North Am. 2006; 20: 485-506. |
|7.||Venier AG, Talon D, Party I, Mercier-Girard D, Bertrandx. Patient and bacterial determinants involved in symptomatic urinary tract infections caused by E. coli with and without bacteraemia. Clin Microbial Infect. 2007; 13: 205-8. |
|8.||Jones TC, Hunt RD, King NW. Veterinary Pathology, 6 th Edn., Philadelphia. 1997. |
|9.||Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott's Diagnostic Microbiology. 10th Ed.; Mosby, Inc. Elsevier: USA, 2007; p. 205. |
|10.||Gailiene G, Pavilonis A, Kareiviene V. The Peculiarities of Pseudomonas aeruginosa Resistance to Antibiotics and Prevalence of Serogroups. Medicina (Kaunas). 2007; 43, 36-42. |
|11.||Li XZ, Livermore DM, Nikaido H. Role of Efflux Pump(s) in Intrinsic Resistance of Pseudomonas aeruginosa: Resistance to Tetracycline, Chloramphenicol, and Norfloxacin. Antimicrob. Agents Chemother. 1994; 38: 1732-41. |
|12.||Alvarez LF, Paloman M, Insausti J, Olaechea P, Cerda F, Sanchez GJ, et al. Staphylococcus aureus nosocomial infections in critically ill patients admitted in intensive care units. Med Clin (Barc). 2006; 126: 641-6. |
|13.||Alves MS, Riley LW, Moreira BM. "A case of severe pancreatitis complicated by Raoultella planticola infection". J. Med. Microbiol. 2007; 56: 696-8. |
|14.||Harborne JB. Phytochemical Methods, 2 nd ed. London, New York. 1984; p. 5-6. |
|15.||Cruickshank R. Medical microbiology; a guide to diagnosis and control of infection. 11 th ed., Edinburgh and London; E. & S. Livingston Ltd. 1968. |
|16.||Singariya P, Mourya KK, Kumar P. Preliminary Phyto profile & Pharmacological Evaluation of some Extracts of Cenchrus grass against Selected Pathogens. J. Pharm. Sci. & Res. 2011a; 3: 1387-93. |
|17.||Subramanian SS, Nagarjan S. Flavonoids of the seeds of Crotolaria retusa and Crotolaria striata. Current Sci. 1969; 38: 65. |
|18.||Arora DS, Kaur GJ. Antibacterial activity of some Indian medicinal plants. J Nat Med. 2007; 61: 313-7. |
|19.||Lalitha M. Manual on Antimicrobial Susceptibility testing under the auspices of Indian Association of Medical Microbiologists 2006. |
|20.||Patil V, Vadnere GP, Patel N. Absence of antimicrobial activity of alcoholic extract of Santalum album Linn. J Pharm Negative Results 2011; 2: 107-9. |
|21.||Pepeljnjak S, Kalodera Z, Zovko M. Investigation of antimicrobial activity of Pelarogarium radula (Cav.) L'Herit. Acta Pharm 2005; 55: 409-15. |
|22.||Akinyemi KO, Oladapo O, Okwara CE, Ibe CC, Fasure KA. Screening of crude extracts of six medicinal plants used in southwest Nigerian unorthodox medicine for antimethicillin resistant Staphylococcus aureus activity. BMC Complementary and Alternative Medicine. 2005; 5:6. |
|23.||Eloff JN. Quantifying the bioactivity of the plant extracts during screening and bioassay-guided fractionation. Phytomedicine. 2004; 11: 370-1. |
|24.||Gracelin D, Herin-Sheeba, Britto A, John De. Datura metel linn.- A plant with potential as Antimicrobial agent. Int. J. of applied biology and pharm. tech. 2011; 2: 429-33. |
|25.||Bokhari FM, Antifungal activity of some medicinal plants used in Jeddah Saudi Arabia. Mycopathology. 2009; 7: 51-7. |
[Table 1], [Table 2], [Table 3], [Table 4]