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EVALUATION OF ANTIOXIDANT ACTIVITY OF FICUS GLOMERATA FRUIT

 

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About Authors:
Gurvinder Pal Singh
Senior Lecturer, Department of Pharmacognosy,
Adesh Polytechnic College, Sri Muktsar Sahib, Punjab
gp_singh352000@yahoo.com

Introduction
An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells (Sies H., 1997).

Reference Id: PHARMATUTOR-ART-1966

Materials and methods
Plant material
The plant materials were collected from the Botanical garden of National Botanical Research Institute (NBRI), Lucknow, India during the month of August. The materials were authenticated at National Botanical Research Institute (NBRI), Lucknow, India. Sample specimens were made and deposited (Voucher no. 97836) to the National Herbarium of NBRI, Lucknow, India.


Chemical and reagent
The chemicals used in present study were tannic acid, saturated sodium carbonate, folin ceocalteus reagent, 2% aluminum chloride, quercetin, ethanol, methanol, ascorbic acid, rutin, DPPH.


Preparation of sample
Sample preparation for total phenolics content
Extracted 2 gm  powdered plant material with 100 ml distilled water by boiling on water bath for 6-8 hrs., filtered and made up the volume to 100 ml volumetric flask.

Preparation of methanolic extracts for total flavanoid and antioxidant
Methanolic extract of Ficus glomerata fruits was prepared through cold percolation by using 10 gm of powdered fruits and 100 ml of methanol.


Sample preparation for total flavanoids
A stock solution of methanolic extract of concentration 1 mg/ml was prepared for each Ficus glomerata.

Sample preparation for free radical scavenging method
A stock solution of methanolic extract of concentration 1 mg/10ml was prepared for each Ficus glomerata

Preparation of standards test solution

Standard preparation for total phenolic content
Sodium carbonate saturated solution: It was prepared by adding 35g anhydrous sodium carbonate in 100 ml distilled water. Dissolved it at 70-80oc and get cool overnight. Filter through glass wool.

Tannic acid standard solution: (0.1 mg/ml) dissolved 10 mg of tannic acid in 100 ml of distilled water.

Folin – Ceocalteus reagent

Standard preparation for total flavanoids
10% AlCl3 solution was dissolved in methanol.
10 mg quercetin was dissolved 100 ml of methanol.

Standard preparation for free radical scavenging method
A solution of 0.52 mg/ml DPPH was dissolved in 100 ml methanol solvent, Ascorbic acid was dissolved in 1mg/ml and makeup its volume upto 10 ml in methanol solvent, Rutin 1mg/ml was dissolved in 10 ml in methanol solution.

Evaluation of antioxidant activity in Ficus glomerata fruits

1. Total phenols Content in Ficus glomerata fruits (Bray H.C. and Thorpe W.V., 1954)
Total phenols estimation can be carried out with Folin-Ciocalteu reagent (FCR).

Principle
Phenols react with an oxidizing agent phosphomolybdate in Folin-Ciocalteu reagent under alkaline conditions and result in the formation of a blue colored complex, the molybdenum blue which is measured at 650nm colorimetrically.

Reagent
A.
Sodium carbonate saturated solution: It was prepared by adding 35 g anhydrous sodium carbonate to 100 ml distilled water. Dissolved it at 70-80oc and get cool overnight. Filter through glass wool.

B.Tannic acid standard solution: (0.1 mg/ml) dissolved 10 mg of tannic acid in 100 ml of distilled water.

C.Folin – Ceocalteus reagent

Methodology
Extracted 2 g powdered plant material with 100 ml distilled water by boiling on water bath for 6-8 hrs., filtered and made up the volume to 100 ml volumetric flask. Took 1 ml aliquot of it, added 5 ml folin phenol reagent, 10 ml saturated sodium carbonate and made up the volume to 100 ml in volumetric flask. Set the instrument to zero through blank and took the corresponding absorbance of different samples at 760 nm by Double beam UV-Visible spectrophotometer. The results were tabulated at Table no.1 and Fig. no. 1.

2. Estimation of total Flavanoidsin Ficus glomerata fruits(Marinova D.,et al,2005)
Flavanoids, with various biological activities, are considered as key compounds. In this study, quantitative determination of flavanoids in were conducted by two complementary colorimetric methods, aluminum chloride method and 2,4-dinitrophenylhydrazine method. Results suggested that the sum of flavanoids contents determined by the above two individual methods may represent the real content of total flavanoid.

Methodology

Aluminum chloride colorimetric method
Quercetin was used to make the calibration curve. Ten milligrams of quercetin was dissolved in 100 ml methanol and then diluted to 0.2, 0.4, 0.6, 0.8 and 1 ml of the diluted standard solution was separately mixed with 0.1 ml of 10% aluminum chloride, make up volume 5 ml with methanol. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm.

Blank
The amount of .1 ml of 10% aluminum chloride was makeup 5 ml with methanol. The results were tabulated in Table no.1 and Fig. 2.

3. Free radical scavenging method (Navarro M.C., et al, 1992; Thabrew M.I., 1998; Namiki M., 1990)

Preparation of Reagent
A solution of 0.52 mg/ml DPPH was dissolved in 100 ml methanol solvent, Ascorbic acid was dissolved in 1mg/ml and makeup its volume upto 10 ml in methanol solvent, Rutin 1mg/ml was dissolved in 10 ml in methanol solution, dried extract of Ficus glomerata was dissolved in 10mg/ 10 ml methanol solvent. The blank sample was having only methanol solvent and control sample having equal amount of DPPH solution and methanol solvent.

Methodology
This method was used for the determination of scavenging activity of DPPH free radical in the extract solution. A solution of 0.52mg/ml DPPH in methanol was prepared and 1 ml of this solution is mixed with 1 ml of extract prepared in methanol. The reaction mixture was vortexed thoroughly and was left in dark at room temperature for 30 min. The absorbance was measured at 517 nm. The ability of the plant extract to scavenge DPPH radical was calculated by the following equation:

DPPH radical scavenging activity=

(Absorbance control - Absorbance sample) / (Absorbance control) × 100

Where;
Absorbance control = absorbance of DPPH radical + methanol

Absorbance sample = absorbance of DPPH radical + sample extract or standard

The results were showed in Table no. 3, 4 and Fig. no. 3, 4

Table 1:  Total phenolic content present in methanolic extract of Ficus glomeratafruits

S.No.

Drug

Absorbance

(nm)

Concentration

(mg/ml)

% of Total phenolics content

1

Tannic acid (Standard)

0.154

0.001

0.0035

0.260

0.002

0.353

0.003

0.446

0.004

0.530

0.005

2

Ficus glomerataextract

0.123

0.0007

Fig1: Standard curve of tannic acid for determined the % of total phenolicscontent in concentration mg/ ml

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Table 2: Total flavonoids content present in methanolic extract of Ficus glomeratafruits

S.No.

Drug

Absorbance

(nm)

Concentration

(µg/ml)

% of Total Flavonoids content

1

Quercetin (Standard)

0.438

0.02

0.0917

0.553

0.04

0.653

0.06

0.769

0.08

0.867

0.10

2

Ficus glomerataextract

0.507

0.0417

Fig.:2 Standard curve of quercetin for determined the % of total flavonoidscontents in concentration mg/ml

Table: 3 Absorbance of antioxidant present in methanolic extract of Ficus glomerata fruits in concentration mg/ml

Concentration mg/ml

0.02

0.04

0.06

0.08

0.10

Absorbance of ascorbic

0.372

0.340

0.332

0.304

0.294

Absorbance of rutin

0.358

0.325

0.310

0.290

0.264

Absorbance of sample

0.437

0.425

0.417

O.409

0.378

Fig.:3 The curve of ascorbic, rutin and Ficus glomerata fruits for determined the absorbance of antioxidant by DPPH assayin concentration mg/ml

Table.4: The antioxidant potential of methanolic extract of Ficus glomeratafruits standard by DPPH assay (scavenging activity in %)

Conc. mg/ml

0.02

0.04

0.06

0.08

0.10

Ascorbic

29.00

35.11

37.64

41.98

43.89

Rutin

31.67

37.97

40.83

44.65

47.61

Sample

16.60

18.89

20.41

23.94

26.86

Fig.:4 The effect of methanolic extract of Ficus glomeratafruits on DPPH scavenging activity

Result and discussion
Methanol extract of Ficus glomerata fruits contained relatively higher levels of total phenolics than the other extracts. Antioxidants from figs can protect lipoproteins in plasma from oxidation and produce a significant increase in plasma antioxidant capacity like total phenolic content, total flavonoids and DPPH method. The antioxidant potential of the extracts can be assessed by employing different in vitro assays. Total phenolics content was found in Ficus glomerata fruits to be 0.0035 %w/w. Total flavanoids content was found to be 0.0917 %w/w. The results were showed in Table no.1 and 2 and Fig. no. 1 and 2.

Free radical scavenging potential of the methanolic extracts of Ficus glomerata fruits was evaluated in vitro by using diphenyl-picrylhydrazyl (DPPH) assay. In this method the antioxidants present in the plant extracts reacted with DPPH, which is a stable free radical and converted it to 1,1-diphenyl-1,2-picryl, hydrazine which is measured at 517 nm. The scavenging effect of plant extracts and standard (Ascorbic acid and Rutin) on the DPPH radical decreases in the following order: Rutin and ascorbic acid were found to be 47.61 % and 43.89 % at concentration of 0.10 mg; 44.65 % and 41.98 % at concentration of 0.08 mg; 40.83 % and 37.64 % at concentration of 0.06 mg; 37.97 % and 35.11 % at concentration of 0.04 mg and 31.67 % and 29.00 % at concentration of 0.02 mg, respectively. In the present study, methanolic extracts of Ficus glomerata fruits showed concentration dependent free radical scavenging activity.

Antioxidant activity of Rutin and Ascorbic acid were performed by using free radical scavenging on DPPH Radical and it was observed that free radicals were scavenged by extracts in a concentration dependent manner. The % inhibition on DPPH was in methanolic extract Ficus glomerata fruits 26.86 % concentration of 0.10 mg, ascorbic acid 43.89 % and rutin 47.61 % showed at a concentration of 0.10 mg.

The results showed a significant decrease in the concentration of DPPH radical due to the scavenging ability of methanolic extract Ficus glomerata fruits as compared to standard. The results were showed in Table no.3 and 4 and Fig. no.3 and 4

The Antioxidant and free radical scavenging activities of Methanolic extract of Ficus glomerata fruits might be due to the presence of high amounts of phenolic and flavonoids compounds. The fruits are used in India for the treatment of various diseases. Methanol extracts of Ficus glomerata fruits contained relatively higher levels of total phenolics than the other extract. Antioxidants from figs can protect lipoproteins in plasma from oxidation and produce a significant increase in plasma antioxidant capacity. So there are many scopes are there in Ficus glomerata fruits and more number of studies can be undertaken like oxidative stress hepatoprotective, anticancer activities and etc. In future we look forward to check the potency of the fruits by means of In-vivo antioxidant studies.

REFERNCES

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  • Marinova D., Ribaroca F.,Atanassova M.,Total phenolics and total flavonoids inBulgarin fruit and vegetables. “J. the university of chemical technology and metallurgy”, Vol. 40(3), 2004, PP.231-237
  • Rahman N. N., Khan M., and Hasan R., Bioactive components from Ficus glomerata. “Pure & App. Chem”, Vol. 66, 1994, PP. 2287-2290.
  • Rao R.A, Rehman F, Adsorption studies on fruits of Gular (Ficus glomerata): removal of Cr(VI) from “synthetic wastewater.”2010 Sep.15 Vol.181(1-3), PP. 405-412.
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  • Thabrew M.I., Hughes R.D., Farlane IG., Antioxidant activity of Osbeckia aspera. “Phytother Res.”; Vol.12, 1998, PP.288–290

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