*Corresponding author: Rajni singh, Kalka Institute for research and advance studies, Meerut (U.P.), India. 86

Indian J. Pharm. Biol. Res. 2014; 2(1):86-94

Original Research Article

Antibacterial and Antioxidant activity of Benincasa hispida using Hydrogen

peroxide scavenging model Rajesh kumar sharma

1, Rajni singh

2*, K.K.Jha

1, Abhishek B

1

1Teerthankar Mahaveer college of Pharmacy, Teerthankar Mahaveer University, Moradabad (U.P.), India.

2Kalka Institute for research and advance studies, Meerut (U.P.), India.

ARTICLE INFO:

Article history:

Received: 24February 2014

Received in revised form:

4 March 2014

Accepted: 15 March 2014

Available online: 20 March 2014

Keywords:

High scavenging activity

Hydrogen peroxide-scavenging model

Acetone extract

ABSTRACT

The seeds of Benincasa hispida commonly known as ash gourd belonging to Cucurbitaceae family is

employed as a main ingredient in kusmanda lehyam in Ayurvedic system of medicine. The seeds of

Benincasa hispida mashed with milk or the various preparations from the pulp of fruit in the form of

sweetmeats, like Kusmanda paka and petha are commonly used as a general tonic, aphrodisiac,

rejuvenative and also a brain tonic. The plant of Benincasa hispida was collected from (Central

Council for Research in Ayurveda & Siddha, Dept. of ayush, Ministry of Health & F.W, Govt. of

India New Delhi) Govt. Central Pharmacy Annex, Ashoka Pillar, Jayanagar Bangalore. The extracts

were subjected to Antioxidant activity by In hydrogen peroxide-scavenging model, model acetone

extract showed scavenging (80.1%) of hydrogen peroxide and chloroform extract showed ( 79%) in

comparison with ascorbic acid ( 94.5% ). Antioxidant activity was carried out for extract (acetone,

chloroform, and aqueous extracts) with Hydrogen peroxide-scavenging model. Acetonic extract

showed more scavenging (80.1%) of Benincasa hispida and least scavenging was done by aqueous

extract of (43%). After comparison with the standard drug (Ascorbic acid) showed (94.5%). From

this it was conclused that acetone extract of Benincasa hispida showed high scavenging activity.

Conclusively, the result revealed that Benincasa hispida seeds has antimicrobial activity &

antioxidant activity which may be due to the presence of alkaloids, phenolic compounds, flavonoids

constituents present in the sample.

1.Introduction

Benincasa hispida (Thunb.) of cucurbitacae family commonly

known as kushmanda, winter melon, wax gourd is used in

Ayurvedic system of medicine. It is cultivated throughout the plains

of India & on the hills up to 1200 meter altitude as a vegetable.

White gourd, the best vegetable fruit, & the medicine for pitta

personality has remarkable therapeutic value in pitta ailments,

epilepsy, bleeding & insanity [1]. The continuous formation of free

radicals in humans’ body can be controlled naturally by different

beneficial compounds known as antioxidants[2]. Oxidative stress

can be caused in result of free radicals formation [3]. Aging and

different chronic diseases including diabetes, cancer and

cardiovascular diseases could be caused by oxidative stress[4]. Free

radicals are stabilized or deactivated by antioxidants before they

attack cells. Antioxidants are important factor to maintain optimal

cellular and human body health. Plants are reported to contain

flavonoids, triterpenes, vitamin-c which are responsible for the

antioxidant activity. Highly reactive free radicals and oxygen

species those are present in human body that causes degenerative

processes by oxidising nucleic acid in the cells. In addition,

involvement of oxygen derive free radicals such as superoxide

anions, hydrogen peroxide and hydroxyl radicals are well

established in the injury of gastric mucosa and in the other models

of gastric mucosal damage induced by nonsteroidal anti-

inflammatory drugs and H.pyroli ethanol and feeding restriction

stress[5]. On the other hand, the expectorant effect of the Benincasa

hispida seeds extract due to facility of mucus secretion which

prevents gastric ulcer was pointed out by Kim and Shin and Grover

et al., [6-7]. In addition, Benincasa hispida seeds extract also could

enhance immunoreactions result in histamine secretion inhibition

[8-9].

2. Materials & methods

2.1 Plant authentication

Plant materials were authenticated at National Ayurveda

Dietetics Research Institute, (Central Council for Research in

Ayureda & Siddha, DEPT. OF AYUSH, Ministry of Health &

F.W, Govt. of India New Delhi) Govt. Central Pharmacy Annex,

Ashoka Pillar, Jayanagar, Bangaluru. Drug

Authentication/SMPU/NADRI/BNG/2011-12/767.

CODEN (USA): IJPB07 ISSN: 2320-9267

Indian Journal of Pharmaceutical and Biological Research (IJPBR)

Journal homepage: www.ijpbr.in

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 87

2.2 Extraction

The extraction was done by successive solvent extraction

method using the various solvents like Acetone, chloroform,

water. The seeds of Benincasa hispida were dried under shade &

than powdered with a mechanical grinder. The powder was

passed through sieve no. 40 & store in an airtight container for

further use. The dried powdered seed of Benincasa hispida was

defatted with petroleum ether (60-80˚C) in a soxhlet apparatus.

The defatted powder material these obtained was further extract

with chloroform, acetone, water. The solvent were removed by

distillation under reduced pressure & the resulting semisolid

mass was flask evaporated[10].

2.3 Antimicrobial Activity

Preliminary screening for Antibacterial activity

The preliminary screening for antibacterial activity was carried

out on the basis of procedure described by Hegazi. The method

is based upon the diffusion of antibacterial principle in extract

from a vertical hole into solidified seed nutrient agar layer of a

petridish to such an extent that growth of" the added

microorganism is prevented entirely in a circular zone.

The 20 extracts of various propolis samples were subjected to

antibacterial activity by using disk plate diffusion method. For

the present study, two gram positive and two gram negative

organisms were selected viz. Staphhylococcus aureus, and

‘Escherichia coli.

Preparation of Standard

50 mg of the Cefotasime sodium was dissolved in the 100 ml of

sterile water.

Preparation of Assay Medial

Beef extract 4.0

Peptone 5.0

Agar 20.0

Distilled water q. s. 1000 ml

The above mentioned quantities of different ingredients were

accurately weighed and dissolved in appropriate amount of

distilled water. The prepared media was sterilized by autoclaving

at 1210C for 15 minutes.

Procedure

The petridishes were thoroughly washed and sterilized

in hot air oven at 1600C for one hr. Inoculum was added to 30 ml

of sterile nutrient agar medium and was poured into sterile

petridishes for solidifying. Bores were made on the medium

using sterile borer. 0.l ml of test solution was added to the

respective bores, 0.1ml of the Ampicillin at a concentration of

100 ug/ml 0.1 ml was taken as standard reference. A control

having only DMSO in the cup was maintained in each plate.

The petridishes were kept in the refrigerator at 400C for

45 minutes for diffusion to take place. After diffusion, the

petridishes were incubated at 37oC for 24 hr and zones of

inhibition were observed and measured using a scale.

Antibacterial activity of all the Extracts was carried out

against all two microorganisms. The same media was used both

for subculturing and for estimating antibacterial activity. All the

reading was taken in triplicate and is reported in Standard Error

Mean (i SEM). [11].

2.4 Antioxidant Activity

Scavenging of Hydrogen Peroxide

The antioxidant activity of the different extracts of Benincasa

hispida was assessed on the basis of their hydrogen peroxide

scavenging ability. The standard ascorbic acid and the extracts

were prepared in phosphate buffer, pH 7.4. Sample and standard

(0.5) were taken in the different test tubes and to each test tube;

0.6 ml hydrogen peroxide solution (2mM hydrogen peroxide in

phosphate buffer, pH 7.4) was added. A control was prepared by

replacing the sample/standard with phosphate buffer. These

solution were kept at room temperature for 10 min. The

absorbance was measured at 230nm against the blank solution

containing phosphate buffer without hydrogen peroxide. All the

samples were prepared and assayed in triplicate and averaged.

The percentage inhibition was calculated using the below

formula

Where:

Control – Hydrogen peroxide in phosphate buffer

Sample – Standard or extract solution in methanol

The antioxidant activity of the plant extracts was

expressed as IC50. The IC50 value is defined as the concentration

(in pg/ml) of extracts that inhibits the formation of hydrogen

peroxide radicals by 50 %. All the tests were performed in

triplicate and the graph was plotted with average of three

observations [12].

Percentage inhibition= Absorbancecontrol – Absorbancesample× 100

AbsorbanceControl

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 88

3. Result

Antibacterial Activity

Table 3.17 Evaluation of antibacterial activity

Plant

Treatment Dose (mg/ml) Zone of inhibition (mm) (Mean+SEM)

Gram negative bacteria Gram positive bacteria

E.coli S. aureus

Benincasa hispida Acetone extract

300 5.33±0.76** 5.33+0.90**

400 6.66±0.76** 6.43 ±0.90**

500 8.66±0.76** 8.76+0.90**

Benincasa hispida chloroform extract 300 3.66+0.76** 3.66+0.83**

400 4.33+0.76** 4.0+0.83**

500 5.66+0.76** 6.33+0.83**

Benincasa hispida Aqueous Extract

300 4.66±0.76** 4.33+0.73**

400 5.0±0.76** 6.0+0.73**

500 6.66+0.76** 7.5+0.73**

Benincasa hispida Standard (cefotasime sodium)

0.2ml

24.6+0.76

0

0

Benincasa hispida Blank

The value represents mean ±S.E.M, One-way ANOVA followed by Dunnet test through Instat software, compare all vs. standard

applied.

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 89

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

acetone 500chloro 500 aq. 500 std. Blank

Acetone

chloro

aqueo.

std.

blank

Column2

Concentration (mg/ml)

Graph 1 – Graphical representation of Antibacterial activity against E. Coli

Zone of inhibition of Benincasa hispida extracts against E. coli

Acetone extract

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 90

Chloroform extract

Aqueous extract

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 91

Zone of inhibition S. aureus

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

00.10.20.30.40.50.60.70.80.9

Acetone

chloro

aqueo.

std

blank

Concentration (mg/ml)

Graph 2– Graphical representation of Antibacterial activity against S. Aureus

Zone of inhibition of Benincasa hispida extracts against S. Aureus

Acetonic extract

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 92

Chloroform extract

Aqueous extract

Singh et al. / Indian J. Pharm. Biol. Res., 2014; 2(1):86-94

Original Research Article 93

Antioxidant Activity

Table 3.18 Antioxidant activity of Benincasa hispida using Hydrogen peroxide scavenging model

Concentration

(µg/ml)

% Inhibition of Antioxidant activity

Ascorbic acid Benincasa hispida of extracts

Acetone extract Chloroform extract Aqueous extract

10 10 9 7 6

20 20.5 15.4 14.6 10.9

40 40 30.9 29.5 29.4

60 54.6 57.2 55.6 25.8

80 68.5 70 69.0 30.4

100 94.5 80.1 79 43

% Antioxidant activity using Hydrogen peroxide-scavenging model

0

20

40

60

80

100

10 20 40 60 80 100

ascor…

Concentration (microgram/ml)

Graph 3 - Graphical representation of Hydrogen peroxide-

scavenging model

Conclusion

Antioxidant activity was carried out for extract (acetone,

chloroform, and aqueous extracts) with Hydrogen peroxide-

scavenging model. Acetone extract showed more scavenging

(80.1%) of Benincasa hispida and least scavenging was done by

aqueous extract of (43%). After comparison with the standard

drug (Ascorbic acid) showed (94.5%). From this it was conclused

that acetone extract of Benincasa hispida showed high scavenging

activity. Conclusively, the result revealed that Benincasa hispida

seeds has antimicrobial activity & antioxidant activity which may

be due to the presence of alkaloids, phenolic compounds,

flavonoids constituents present in the sample.

Conflict of interest statement

We declare that we have no conflict of interest.

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Cite this article as: Rajesh kumar sharma, Rajni singh, K.K.Jha, Abhishek B.Antibacterial and Antioxidant activity of

Benincasa hispida using Hydrogen peroxide scavenging model. Indian J. Pharm. Biol. Res.2014; 2(1):86-94.