A COMPARATIVE ANTIMICROBIAL EVALUATION OF EMBELIARIBES BURM. F. AND EMBELIAROBUSTA AUCT. NONROXB. FRUITS

A COMPARATIVE ANTIMICROBIAL EVALUATION OF EMBELIA RIBES BURM. F. AND EMBELIARO BUSTA AUCT. NONROXB. FRUITS

Malvika Saini^*, Gaurav Sharma and Mita Kotecha

Department of Dravyagunavigyan, National Institute of Ayurveda, Jaipur, Rajasthan, India

ABSTRACT: Background: Vidang (Embelia ribes Burm. f.) is a very famous plant for its anthelmintic activity. In Ayurveda, it is a first choice of drug for its krimighna action or to kill foreign pathogens. Embeliaro busta auct. Nonroxb., the other species of vidang is adulterated in Embelia ribes Burm. f. Therefore shastrokta or original vidang and its adulterant species were selected for the comparative study. Methods: Both aqueous and alcoholic extracts of fruits of both species of Embelia at 10, 20, 30% concentrations were tested for antimicrobial activity by agar well diffusion method against a range of gram-positive and gram-negative bacteria. Zone of inhibition of extracts were determined, then activity index and IC₅₀ value was calculated. Results: Both ethanol and Aqueous extracts of Embelia ribes Burm. f. and Embeliaro busta auct. Nonroxb. inhibited the growth of all the tested strains of bacteria. Aqueous extract Embelia ribes Burm. f. has more potent action against microorganism Staphylococcus aureus, Escherichia coli, Klebsiellaaerogenes, ZOI (16mm,14mm,19mm respectively), AI(0.64, 0.78, 0.70 resp.), IC₅₀ (1.751, 1.166, 1.641 respectively). Conclusion:  Embelia ribes Burm. f. has more potent action in comparison to Embeliaro busta auct. Nonroxb.. It concludes shastrokta vidang species (Embelia ribes Burm. f.) is better to its adulterant species (Embeliaro busta auct. Nonroxb.).

Embelia ribes burm. f.,

 Embeliaro busta auct. Nonroxb., Bacteria, Zone of inhibition,

Activity index, IC₅₀ value

INTRODUCTION: Embelia ribes Burm. f. is a threatened woody shrub belongs to the family Myrsinaceae, which is  found throughout India up to an altitude of 1600 m, from Central Himalaya to Konkan, Deccan, Western Ghats and South India. In ‘Ayurveda’, the plant is popularly known as Vidanga or Bashmak or Krimigna (Sanskrit); Baberangor Wawrung (Hindi); Vayuvilanga (Kannada) and it is used as one of the adjuvant in most of the drug preparations. The fruits contain embelin, quercitol, tannin, christembin, embolic acid, fatty ingredients, resinoid, volatile oil, vilangin.

The plant possess nematicidal, estrogenic, hypoglycaemic, anthelmintic, antibiotic, anti-tubercular, anti-implantation, anti-ovulatory, antifertility, anti-inflammatory, hypotensive, antipyretic, diuretic, hepato protective, anti-leishmanial, resorptive, anti-spermatogenic, anti-androgenic, anticancer, immunostimulant property.

Fruits are astringent, bitter useful in helminthiasis, skin diseases, leprosy, pruritis, nervous debility, amentia, dyspepsia, jaundice, flatulence, colic, constipation, strangury, tumour, asthma, bronchitis, dental caries, odontalgia, hemicrania, dyspnoea, cardiac diseases, psychological disorder, ringworm, fever, emaciation, general debility ¹.

Fruits of Embeliaro busta auct. Nonroxb. are used as adulterant to Embelia ribes Burm. f.. At some places it is used as substitute of E. ribes. These seeds are found in abundance and E. ribes now a day is considered to be in the red listed plants. E.robusta has similar properties to E. ribes. and chemical constituents like embelin etc. In Market, in the name of Vidang, both species are sold mostly in mixed form. At some places only Embeliaro busta auct. Nonroxb. is available in the name of Vidang. It is a rambling shrub or small tree, distributed from Ceylon to Malabar coast through Sylhet and Assam to Singapore ². Both the two species are different in colour and size, E. ribes is black in colour, small and rounded whereas Embeliaro busta auct. Nonroxb. auct. is reddish brown in colour, big in size as compared to E. ribes and rounded. Both possess same actions according to modern literature. But researches on both species have not been done so far. So both species were selected for the comparative study. Moreover fruit of vidang is one of the ingredient of a paste formulation mentioned for local application in wound infections like fistula in ano, piles etc. ³, So to prove this activity of Vidang experimentally, in vitro study was carried out with both species of Vidang and following wound infection causative organisms.

FIG. 1: SHOWING FRUITS OF EMBELIA RIBES BURM. F.

FIG. 2: SHOWING FRUITS OF EMBELIAROBUSTA AUCT. NONROXB.

MATERIAL AND METHODS:

Plant material: Fruits of Embelia ribes Burm. f. were collected from Ayurveda Regional Research Institute, Itanagar and were authentified by the same i.e. Ayurveda Regional Research Institute, Itanagar with Authentication no. – 2655. Fruits of Embeliaro busta auct. Nonroxb. were collected from Joginder Nagar, Himachal Pradesh and authentication was done at herbarium section, Department of Botany, Rajasthan University, Jaipur with authentication no. RUBL211596.

Preparation of extracts: Cold maceration method was used for preparing extracts. Macerated 10 g of the air dried drug, coarsely powdered, with 200 ml of solvent i.e. for preparing ethanol extract, ethanol solvent was used and for preparing aqueous extract, distilled water was used. Procedure was done for the specified strength in a closed flask for twenty-four hours, shaking frequently during six hours and allowed to stand for eighteen hours. Filtered rapidly, taking precautions against loss of solvent, evaporated the filtrate to dryness in a tarred flat bottomed shallow dish, and dried at 105 ⁰C, to constant weight and weighed ⁴. The yield was 6.3% for ethanol extract of Embelia ribes Burm. f., 1.31% for aqueous extract of Embelia ribes Burm. f., 11.45% for ethanol extract of Embeliaro busta auct. Non roxb., 2.94% for aqueous extract of Embeliaro busta auct. Nonroxb.. Repeated the procedure for 5 times so as to collect the enough amount of extract required for antimicrobial activity. For carrying out antimicrobial activity, 10%, 20%, 30% concentrations of both ethanol and aqueous extracts were prepared by dissolving them in ethanol/water.

Antimicrobial activity:

Micro-organisms: Bacterial strains selected for the study were Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiellaaerogenes. Out of which Pseudomonas aeruginosa, Escherichia coli, Klebsiellaaerogenes are gram negative bacteria, whereas Staphylococcus aureus is gram positive bacteria. Reason for selecting these strains was, they are causative organism for surgical wound infections and mostly causes infections in piles, fistula in ano etc. The pathogenic strains of above bacteria were procured from ‘Institute of Microbial Technology’ (IMTECH), Chandigarh and the stock cultures maintainance and antibacterial study was done at ‘microbiology lab, Dravyaguna Vigyan Deptt, NIA’, Jaipur.

MTCC No.  39-Klebsiella aerogenes

MTCC No.  10239- Escherichia coli

MTCC No.   1034- Pseudomonas aeruginosa

MTCC No.    6908- Staphylococcus aureus

Revival of microbial cultures: Microbes collected from Institute of Microbial Technology were in dried form. It needed to be revived. Like all other living forms, micro-organisms need suitable nutrients and favorable environments for growth. A simple way to obtain bacteria is to grow them in a flask in broth medium.

100 ml Nutrient broth medium were transferred in conical flasks (of quantity 100ml) 20ml each. The flasks were capped with cotton plug and autoclaved at 121 ºC for 20 minutes at 15 lb pressure per square inch. Dried and freezed bacteria were transferred to conical flasks with nutrient broth media, kept at 37 ⁰C to get cultures.

Preparation of media and media plates: Muller-hington agar medium was taken for all pathogens. 38 gram of agar was dissolved in 1 litre of distilled water. Heated the agar with water at 100 ⁰C till it becomes transparent, then kept it in hot air oven for 15 minutes. The sterilized media were poured in sterile petri dishes aseptically. The Agar (solidifying agent), which was added in a broth medium, hardens at it cools. After solidifying of agar plates (nearly about 15 to 20 minutes), they were kept inverted in incubator at 37 ºC for overnight for checking any contamination. The agar plates were ready.

Applied a microbial culture to the surface in a petri plate and spread them with cotton swab sticks. The prepared plates were then incubated in inverted position at 37 ºC for 24 hours. After incubation, we got the pure cultures. This procedure is termed as ‘Sub culturing’. In this way, frequent sub-culturing was done whenever required during antibacterial study.

Well diffusion method: For bactericidal assay in vitro, well diffusion method was adopted ⁵, because of reproducibility and precision. Wells (of about 4 mm diameter) were made on the plates with the help of sterile stainless steel borer. The different test organisms were proceeded separately using a sterile swab over previously sterilized culture medium plates. About 20-30µl different concentrations of plant solvent extracts were added using sterile syringe into the wells and allowed to diffuse at room temperature for 2hrs.Control experiments comprising inoculums without plant extract were set up. The plates were incubated at 37 °C for 48 hours for bacterial pathogens. The zone of inhibition were measured around sterilized wells (4 mm in diameter). The 4 readings were taken in different planes, and then the mean was calculated.

Group design:

Test group:

10%, 20%, 30% concentrations of ethanol extracts of fruits of Embelia ribes Burm. f..

10%, 20%, 30% concentrations of aqueous extracts of fruits of Embelia ribes Burm. f..

10%, 20%, 30% concentrations of ethanol extracts of fruits of Embeliaro busta auct. Non roxb..

10%, 20%, 30% concentrations of aqueous extracts of fruits of Embeliaro busta auct. Non roxb..

Standard group- 5% w/v Vancomycin

Negative control group- Distilled water and Ethanol

Determination of activity index: The activity index of the crude plant extract was calculated as ⁶: Activity index (A.I.) = Mean of zone of inhibition of the extract/ Zone of inhibition obtained for standard antibiotic drug.

IC₅₀: The half maximal inhibitory concentration (IC₅₀) is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. The IC₅₀ of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist on reversing agonist activity. IC₅₀ values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist. IC₅₀ values can be used to compare the potency of two antagonists ⁷. In general, the higher the concentration of inhibitor, the more agonist activity will be lowered. IC₅₀ value increases as agonist concentration increases.

Determination of IC₅₀⁸                                

y-axis=% inhibition and x-axis= log conc.

linear equation y=mx + c.

Y= 50, x=log conc.

IC₅₀- Antilog of X

RESULTS:

Zone of Inhibition in mm:

TABLE 1: SHOWING ZONE OF INHIBITION OF TEST DRUGS AND POSITIVE, NEGATIVE CONTROL GROUPS

Where AEErib- aqueous extract of Embelia ribes Burm.f., EEErib- ethanol extract of Embelia ribes Burm.f., AEErob- aqueous extract of Embeliaro busta auct. Non roxb., EEErob- ethanol extract of Embeliaro busta auct. Non roxb.at 10%, 20%, 30% concentrations

GRAPH 1: SHOWING ZONE OF INHIBITION OF TEST SAMPLES WITH POSITIVE AND NEGATIVE CONTROLS WHERE C” STANDS FOR CONTROL GROUP

FIG. 3: ZONE OF INHIBITION OF + VE AND -VE CONTROL AGAINST FOUR STRAINS OF BACTERIA

FIG. 4: ZONE OF INHIBITION OF TEST GROUPS AGAINST FOUR DIFFERENT STRAINS OF BACTERIA

Activity index:

TABLE 2: SHOWING ACTIVITY INDEX OF TEST DRUGS

GRAPH 2: SHOWING ACTIVITY INDEXES OF TEST DRUGS

IC₅₀ value:

TABLE 3: SHOWING IC₅₀ VALUE OF TEST DRUGS

    
GRAPH 3: SHOWING IC₅₀ VALUE OF TEST DRUG

DISCUSSION: The bioassay results for antimicrobial activity of the Aqueous and Ethanol extracts of Embelia ribes Burm. f. and Embeliaro busta auct. Nonroxb. are presented in Table 1. From the results it is very clear that both ethanol and Aqueous extracts of Embelia ribes Burm. f. and Embeliaro busta auct. Nonroxb. inhibited the growth of all the tested strains of bacteria.

The Aqueous extract Embelia ribes Burm. f. was found privileged Zone of Inhibition against S. aureus, E. coli, K. aerogenes in comparison of ethanolic extract Embelia ribes Burm. f., Aqueous Embeliaro busta auct. Nonroxb. extract, ethanolic Embeliaro busta auct. Nonroxb. extract. But ethanolic extract of Embeliaro busta auct. Non roxb. shows significant results on P. aeruginosa in comparison to ethanolic extract of Embelia ribes Burm. f. and aqueous Embeliaro busta auct. Non roxb. extract. The activity index of the test substance above 0.5 is considered as significant activity ⁹. Aqueous extract of Embelia ribes Burm. f. has activity index of 0.9, 0.64, 0.78,0.70 against P. aeruginosa, S. aureus, E. coli, K. aerogenes respectively which again proves its better efficacy than other tests samples i.e. ethanolic extracts of Embelia ribes Burm. f., aqueous extracts and ethanolic extracts of Embeliaro busta auct. Nonroxb.. Ethanolic extract of Embeliaro busta auct. Non roxb. Shows activity index of 0.85 against Pseudomonas aeruginosa which shows its potent action in comparison to other samples. Concentration required to inhibit 50 % of Pseudomonas aeruginosa is lowest in ethanolic extract of Embeliaro busta auct. Nonroxb. (IC₅₀=1.063) which depicts its more potent action as compare to other Extracts of Embelia ribes Burm. f. and Embeliaro busta auct. Nonroxb.. Against Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes, IC₅₀ is lowest in aqueous extract of Embelia ribes Burm. f. i.e. 1.751, 1.166, 1.641 respectively required to kill 50% microorganism.

CONCULSION: The difference in the activity may be due to the different secondary metabolites present in the ethanol and water extracts. Different solvents have various degrees of solubility for different phytoconstituents ¹⁰. This indicates that the secondary metabolites act as an antimicrobial compounds, which either inhibit or kill the bacteria by different mechanisms.

Aqueous extract of Embelia ribes Burm. f. shows potent anti-microbial action against Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes microorganism in comparison of ethanolic extract of Embelia ribes, aqueous  extract of Embeliaro busta, ethanolic extract of Embeliaro busta auct. Nonroxb.. Ehanolic extract of Embeliaro busta auct. Nonroxb. shows potent action against Pseudomonas aeruginosa, microorganism.

Out of four tested microorganisms, three organisms are affected by aqueous extract of Embelia ribes Burm. f. upto a significant level which infers that Embelia ribes Burm. f. has more potent action in comparison to Embeliaro busta auct. Non roxb.. It represents shastroktavidang species (Embelia ribes Burm. f.) is better than its adulterant species (Embeliaro busta auct. Nonroxb.).

CONFLICTS OF INTEREST STATEMENT: There are no conflicts of interest.

REFERENCES:

1. Padhi MM, Mangal A.K. et al: Database on medicinal plants. Published by CCRAS, New delhi,vol.-5, 2008: 478-482
2. Bapalal Vaidya: Some controversial drugs in Indian Medicine. Chaukhambha Orientalia publication, Varanasi, 2014 : 256
3. Brahmanand tripathi: Charak Samhita: Sutrasthan. Chaukhamba Surbharti Prakashan, Varanasi, 2008: 60.
4. Lavekar G.S. al. Laboratory guide for analysis of Ayurveda and siddha formulations. Published by CCRAS publications, 2010:29,30
5. Mounyrbaulouiri, Moulay Sadiki al.: Methods for in-vitro evaluating antimicrobial activity. Journal of pharmaceutical analysis. April 2016; 6 (2): 71-79.
6. B Singh, PM Sahu: MK Sharma. Phytomedicine. 2002; 9: 355-359.
7. IC₅₀: Wikipedia online resource. Cited on 2016/June. Available from :https://en.wikipedia.org/wiki/IC50
8. Calculation of IC₅₀: protocol online resource. Cited on 2016/June. Available from: http://www. Changbio science. com/stat/ec50.html
9. V. Hosamath: International Journal of Pharmaceutical Applications. 2011; 2(1):105-114.
10. Majorie MC: Plant products as antimicrobial agents. Microbial. Rev. 1999; 12(4): 564-582.
    

    ---

 How to cite this article:

Saini M, Sharma G and Kotecha M: A comparative antimicrobial evaluation of Embelia ribes burm. F. and Embeliaro busta auct. Nonroxb. fruits. Int J Pharm Sci Res 2017; 8(1): 342-49.doi: 10.13040/IJPSR.0975-8232.8(1).342-49.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.