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ISOI Journal of Microbiology, Biotechnology and Food Science

Volume 1 Issue 1; Page No. 06-09

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Antibacterial activity of isoflavone extracted from Curcuma longa linn. Zingeberaceae.

Gaikwad A, Bodhankar M*, Ittadwar A, Waikar S.

Gurunanak of College of Pharmacy, Nagpur-440 026, Maharashtra, (India)

ARTICLE INFO

ABSTRACT

Received 17 Nov. 2014 Accepted 24 Nov. 2014

Corresponding Author: Gurunanak of College of Pharmacy, Nagpur-440 026, Maharashtra, (India)

The dried rhizome of Curcuma longa was used for isolation of Isoflavone by

using the extraction process known as maceration in which the rhizomes were

macerated in hydro-alcoholic solvent in proportion (1:1). The extraction was

carried out for 15 days. The dark yellow colored extract obtained after

extraction was sub fractioned by chloroform and the isoflavone was obtained

which was confirmed by Shinoda test for flavonoids.

The extract (isoflavone) was subjected to carry out the antibacterial activity.

The activity was carried out for different concentrations and solvent (DMSO)

taken as blank using agar diffusion technique by cup plate method. The

extracts were added at concentration of 500 μg/ml, 250μg/ml, 125μg/ml,

62.5μg/ml, 31.25μg/ml into the five wells. The plates were kept in incubator

for 18-24hr at 37ºC, zones of inhibitions were measured and compared with

the standard drugs.

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INTRODUCTION The dried rhizome of Curcuma longa L. (Zingiberaceae) (1) nearly ovoid (about 3 cm in diameter and 4 cm long), Lateral rhizome, slightly bent (2–6cm), externally yellowish brown and internally orange yellow was taken for isolation of isoflavone and determination of activity. It contains major Chemical constituents such as volatile oil (6%) composed of a number of monoterpenes and sesquiterpenes, including zingiberene, poly phenolic compounds, isoflavones, curcumene, α- and β- turmerone among others. The coloring principles (5%) are curcuminoids, 50– 60% of which are a mixture of curcumin, mono-desmethoxycurcumin and bis-desmethoxycurcumin. (1, 6, 25) The extract of curcuma longa is used as potent anti-cancer and anti- inflammatory agent. It helps to reduce the inflammation by acting on body metabolism. It also helps to reduce the cholesterol level in kidney and liver tissues. It helps to clean wounds, remove excess secretion and helps the growth of healing tissues in the wounds.The inhibition of the microbial growth under standardized conditions may be utilized for demonstrating the antibacterial activity. Experimental: The plant material (Rhizomes) were extracted with hydro-alcohol (1:1) system, this hydro-alcoholic extraction was done by cold maceration for fifteen days. Generally these many days were needed for complete extraction by cold maceration with the

solvents. After extraction the solvent was removed by evaporation and the residue obtained was dried, weighed and percentage yield was determined. The extractive value (%yield) was found to be 12.1g% w/w The hydro-alcoholic extract of Curcuma longa was evaporated and residue was sub-fractionated with the help of solvent Chloroform by soxhlation. The soxhlation was continued for 25 numbers of cycles as these many cycles were needed for the complete extraction with the solvent. After completion of soxhlation with chloroform, chloroform was removed from the extract and the residue left was dried, weighed and % yield was determined. The % yield of sub-fraction of hydro-alcoholic extract of Curcuma longa was 29.22 %. The above extract was subjected to carry out the antibacterial activity of the compound. The following two are the methods available for screening the antimicrobial activity. 1. Turbidimetric / photometric/ tube dilution method. 2. Agar diffusion/cup-plate/cylinder plate method. The agar diffusion technique was used to assess antibacterial activity of new compound. In agar diffusion methods, following three techniques are used. Agar cup-plate method Agar streak dilution method Paper disc method

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Out of these cup-plate agars diffusion method was used to evaluate the antibacterial activity. It is one of the in-vitro bacterial susceptibility tests. Cup-Plate Agar Diffusion Method: Preparation of solutions of compounds: Stock solution of the extracted isoflavone was prepared in dimethyl sulfoxide (DMSO) in the concentration of 500μg/ml, and its dilutions were prepared in the concentrations 250μg/ml, 125μg/ml, 62.5 μg/ml, 31.25 μg/ml. The solvent DMSO was also tested for its antibacterial activity. Test cultures: For evaluation of antibacterial/antifungal activity, following cultures were used. 1) S. aureus (Gram-positive bacteria)

2) C. albicans (Fungi) PREPARATION OF INOCULUMS: The bacterial cultures were grown on nutrient agar slants for 24hr at 37˚C in incubator. They were stored at 4 0C and sub culturing was done after one week. For evaluation of antibacterial activity, 24hr fresh culture of bacteria was suspended in sterile distilled water to obtain a uniform suspension of bacterial strains. The culture of bacteria was obtained from Gurunanak College of pharmacy, Nagpur. PREPARATION OF CULTURE MEDIUM4: 1. Nutrient Agar Medium: The medium used was obtained from Hi-media having the following composition:

Composition: Table: 1

Peptone 5.0g

Sodium chloride 5.0g

Beef extract 1.5g

Yeast extracts 1.5g

Agar 15g

Water up to 1000ml

Final PH (at 25C) 7.4 +0.2

The media for antibacterial activities were prepared by dissolving 28g of above medium in 1 Litre of distilled water and was sterilized in an autoclave (temp. 121ºC at 15 lbs/inch pressure) for 30 minutes PROCEDURE FOR CUP-PLATE METHOD5: A sterilized medium was inoculated with bacterial suspension of test organism having uniform turbidity at temperature of 40-50ºC and immediately poured into the sterile petridishes to give a depth of 3-4mm of uniform thickness in different plates. After complete solidification of inoculated medium, the wells were prepared by cork borer (6mm). In one plate six cups were made in which the compounds with different concentrations in DMSO and one for solvent (DMSO) taken as blank was poured. The solution was added at concentration of 500μg/ml, 250μg/ml, 125μg/ml, 62.5

μg/ml, and 31.25μg/ml into the five wells. Then it was kept in refrigerator for half an hour to ensure the proper diffusion of the extract in the medium and then plates were kept in incubator for 18-24hr at 37ºC.After 24hrs, zones of inhibition were measured and recorded. Table no 1. Observations and Result The antimicrobial activity of the isoflavone was determined from the zone of inhibition against the standard antibiotics (Ampicilin as antibacterial and Miconazole as antifungal) similarly MIC (Minimum inhibitory concentration) was determined by observing the zone of inhibition of different concentrations. The Minimum concentration at which the zone of inhibition was observed was considered as MIC. It was found to be 62.5μg/ml conc.

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Ampiciline (10 μg/ml) Miconazole (10 μg/ml)

Table 2: Results of antibacterial activity of all compounds are presented in:

Compound Concentration (μg/ml)

Zone of inhibition

S.aureus C.albicans (DMSO)

Isoflavone 500 μg/ml 14 13 Nil

250 μg/ml 13 11 Nil

125 μg/ml 11 09 Nil

62.5 μg/ml 09 07 Nil

31.2 μg/ml 00 00 Nil

Ampicillin 10 μg/ml 13 ------- Nil

Miconazole 10 μg/ml -------- 11 Nil

CONCLUSION The isolated compound shows antibacterial as well as antifungal activity which when compared with the standard drugs shows same inhibition but at different concentration of the isoflavone. REFERENCES:

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