insignificant antimicrobial activities and -...

Journal of Pharmaceutical Negative Results | Jan-Dec 2012 | Vol 3 | Issue 1 41

Address for correspondence: Dr. Premlata Singariya, Dr. D. S. Kothari Post Doctoral Fellow, P.No.55, Shri Kalyan Nagar, Kartarpura, Jaipur-302015 (Rajasthan) India. E-mail: [email protected]

Insignificant antimicrobial activities and phytochemical screening in different extracts of indian Ginseng

Original Article

Premlata Singariya, Padma Kumar,

Krishan Kumar Mourya1

Laboratory of Tissue Culture and Secondary Metabolites, Department of Botany University of Rajasthan,

Jaipur, 1Veterinary Officer and In-charge, Goverment. Veterinary

Hospital, Pahari (Bharatpur) Rajasthan

AbstractAim: 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.

Key words: Aspergillus flavus, Antibacterial, Hexane, Pseudomonas aeruginosa, toluene, Withania somnifera

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Website: www.pnrjournal.com

DOI: 10.4103/0976-9234.99665

INTRODUCTION

W. somnifera (Ashwagandha) has anti inflammatory effect,[1] analgesic effect,[2] anti-tumor, anti-stress, antioxidant[3] and memory improving effects[4] 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. [5,6] E. coli is the culprits for human urinary tract infections. [7] P. aeruginosa is involved in respiratory tract, urinary tract[8], bloodstream, and central nervous system infections of nosocomial origin[9] and this pathogen is becoming resistant against gentamycin, ciprofloxacin,[10] tetracycline, chloramphenicol, and Norfloxacin[11]. The major causative agent of nosocomial infections is S. aureus[12] along with E. coli. It has been determined that Raoultella planticola has caused severe pancreatitis in one case.[13] 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.

Journal of Pharmaceutical Negative Results | Jan-Dec 2012 | Vol 3 | Issue 142

Singariya, et al.: Insignificant antimicrobial activities of Indian Ginseng

MATERIALS AND METHODS

Experimental designCrude extracts of different parts of W. somnifera (RUBL20668) were prepared with a series of non-polar to polar solvents by hot extraction method[14] in a soxhlet assembly. Different extracts were then screened for antimicrobial activity by disc diffusion assay[15] 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[16] as also the minimum bactericidal / fungicidal concentration (MBC / MFC).

Collection of plant materialDifferent 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[17] in the different polar solvents.

Extraction procedureEach 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 400C by using an evaporator. The residual extracts were stored in a refrigerator at 40C in small and sterile glass bottles. The Percent extractive values were calculated by the following formula.

Weight of dried extractPercent Extracts = ———————————— x 100 Weight of dried plant material

Drugs and chemicals usedDrugsGentamycin and Ketoconazole were used as standard antibiotics for bacteria and fungi respectively.

ChemicalsPetroleum ether, ethyl acetate, glacial acetic acid, Nutrient Agar (for bacteria) and Sabouraud Dextrose Agar (for fungi).

Micro-organismsTest 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 4oC as well as at –80oC by preparing suspensions in 10% glycerol.

Antimicrobial screeningThe 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.[18] 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[19,20] was performed for screening. The NA and SDA base plates were seeded with the bacterial and fungal inoculum, respectively (inoculum size 1×108 CFU / ml for bacteria and 1×107 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.[21] The activity index for each extract was calculated Table 2. Zone of inhibition (ZOI) of the sampleActivity index (AI)

= _________________________________

Zone of inhibition (ZOI) of the standard

Determination of minimum inhibitory concentration by the broth dilution methodMinimum 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

Table 1: Name of the tested pathogens (bacteria and fungi)Pathogens Name of pathogens G+ve/

G-veSpecimen

noBacteria Escherichia coli G-ve MTCC46

Staphylococcus aureus G+ve MTCC3160Raoultella planticola G-ve MTCC530Pseudomonas aeruginosa

G-ve MTCC1934

Enterobactor aerogens G-ve MTCC111Yeast Candida albicans - MTCC183Fungi Aspergillus flavus - MTCC277



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.[16] 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 concentrationThe 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. [22] MBC was calculated for some of the extracts that showed high antimicrobial activity against highly sensitive organisms.

Total activity determinationTotal 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. [23]

Extract per gram of the dried plant partTotal Activity = ____________________________

MIC of extract

Statistical analysisMean 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.[24]

RESULTS AND DISCUSSION

Quantitative estimationThe preliminary phyto profiling for the different parts of W. somnifera were carried out according to Bokhari.[25] 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.

Antimicrobial activityAntimicrobial 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-

Table 2: Zone of Inhibition (mm) and activity index of different extracts of Withania somnifera in different polar solvents against tested pathogensName of Solvents

Parts of plant

ZOI / AI

Bio-activity of different extracts against pathogensE. c. S. a. R. p. P. a. E. a. C. a. A. f.

Hexane (0.1) Ripened fruit ZOI - - - - - 7.5 ± 0.64 -AI - - - - - 1.071 -

Fruit coat (Calyx)

ZOI - - - - - - -AI - - - - - - -

Toluene (2.4) Flower ZOI - - - 8.5 ± 0.64 - - -AI - - - 0.708 - - -

Fruit coat (Calyx)

ZOI - - - 7.67 ± 0.25 - 7.50 ± 0.64 -AI - - - 0.639 - 1.071 -

Iso propylAlcohol (3.4)

Ripened fruit ZOI - - - 8.67 ± 0.27 - 8.17 ± 0.24 -AI - - - 0.867 - 1.671 -

Fruit coat (Calyx)

ZOI - - - - - - -AI - - - - - - -

Acetone (5.1) Fruit coat (Calyx)

ZOI - - - - - - -AI - - - - - - -

Ethanol (5.2) Fruit coat (Calyx)

ZOI - - - 7.17 ± 0.22 - 7.67 ± 0.23 -AI - - - 0.896 - 1.096 -

Abbreviations: All values are mean ± SD, n=3 (P>0.005), ZOI=Zone of inhibition in mm ± S.D. (Including 6 mm of disc diameter), AI=Activity index, E. c. -Escherichia coli, S. a. -Staphylococcus aureus, R. p. - Raoultella planticola, P. a. -Pseudomonas aeruginosa, E. a. -Entrobactor aerogens, A. f. -Aspergillus flavus, C. a. -Candida albicans, mm: Millimeter

Journal of Pharmaceutical Negative Results | Jan-Dec 2012 | Vol 3 | Issue 144


Table 3: Minimum inhibitory concentration and (MBC / MFC) of different extracts of Withania somnifera in different polar solvents against tested pathogensName of Solvents

Parts of plant MICMBC / MFC

Bioactivity of different extracts against pathogens

E. c. S. a. R. p. P. a. E. a. C. a. A. f.H Ripened Fruit MIC - - - - - 3.75 -

Fruit coat (Calyx)

MBC / MFC - - - - - 7.5 -MIC - - - - - - -

T Flower MBC / MFC - - - - - - -MIC - - - 3.75 - - -

Fruit coat (Calyx)

MBC / MFC - - - 7.5 - - -MIC - - - 7.5 - 3.75 -

I Ripened Fruit MBC/MFC - - - 15 - 7.5 -MIC - - - 3.75 - 3.75 -

Fruit coat (Calyx)

MBC/MFC - - - 7.5 - 7.5 -MIC - - - - - - -

A Fruit coat (Calyx)

MBC / MFC - - - - - - -MIC - - - - - - -

E Fruit coat (Calyx)

MBC / MFC - - - - - - -MIC - - - 7.5 - 3.75 -MBC / MFC - - - 15 - 3.75 -

MIC-Minimum inhibitory concentration (mg / ml), MBC-Minimum bactericidal concentration (mg / ml), MFC-Minimum fungicidal concentration (mg / ml), H-Hexane, T-Toluene, I-Iso propyl alcohol, A-Acetone, E-Ethanol, E. c. -Escherichia coli, S. a. Staphylococcus aureus; R. p. -Raoultella planticola, P. a. -Pseudomonas aeruginosa, E. a. -Entrobactor aerogens, A. f. -Aspergillus flavus, C. a. -Candida albicans

Table 4: Total activity and % Yield of different extracts of Withania somnifera in different polar solvents against tested pathogensSolvents Parts of plant % Yield Total activity of different extracts against pathogens

E. c S. a. R. p P. a. E. a. C. a. A. f.H Ripened Fruit 9.03 - - - - - 24.08 -

Calyx 1.68 - - - - - - -T Flower 4.02 - - - 10.71 - - -

Calyx 3.30 - - - 4.40 - 8.81 -I Ripened Fruit 12.99 - - - 34.65 - 34.65 -

Calyx 6.60 - - - - - - -A Calyx 5.30 - - - - - - -E Calyx 15.71 - - - 20.95 - 41.90 -

H-Hexane, T-Toluene, I-Iso propyl alcohol, A-Acetone, E-Ethanol, E. c. -Escherichia coli, S. a. Staphylococcus aureus; R. p. -Raoultella planticola, P. a. -Pseudomonas aeruginosa, E. a. -Entrobactor aerogens, A. f. -Aspergillus flavus, C. a. -Candida albicans

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 concentrationMIC 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 activityTotal 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.

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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.

Source of Support: Nil. Conflict of Interest: None declared.

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