In vitro Antioxidant activity and phytochemical screening of Aegle marmelos extracts


Nirmala Gupta1, R.C.Agrawal1, Vinoy Shrivastava2 Amit Roy3 and Pushpa Prasad3

1Jawaharlal Nehru Cancer Hospital and Research Centre, Idgah hills, Bhopal (MP), India

2Dept. of Biosciences, Barkatullah University, Bhopal (MP), India

3Columbia Institute of Pharmacy, Raipur



Antioxidant activity and phytochemical screening of hydromethanolic extracts of Aegle marmelos fruit, leaves and bark were investigated in this work. Phytochemical screening of the crude extracts revealed the presence of Alkaloids, glycosides, Terpenoids, Saponins, Tannis, Flavonoids, and Steroids. Antioxidant activity of the extracts was evaluated using Fenton reaction: an in vitro assay and the extracts showed high hydroxyl radical scavenging property when compared with Ascorbic acid, which was used as reference standard. These findings demonstrated that Aegle marmelos extracts possess free radical and hydroxyl radical scavenging activity as well as antioxidant activity in vitro.


KEYWORDS: Aegle marmelos, Antioxidant, Phytochemical constituents, hydromethanolic.




Medicinal plants form the backbone of traditional system of medicine in India and the  traditional medicine is increasingly solicited through the tradipractitioners and herbalists in the treatment of various infectious diseases because throughout the history of mankind, many infectious diseases have been treated with herbals. Among the remedies used, plant drugs constitute an important part. A number of scientific investigations have highlighted the importance and the contribution of many plant families i.e. Asteraceae, Liliaceae, Apocynaceae, Solanaceae, Caesalpinaceae, Rutaceae, Piperaceae, Sapotaceae used as medicinal plants. Medicinal plants play a vital role for the development of new drugs (Samy et. al., 2007; Rajasekaran et. al., 2008). All parts of the A. marmelos tree have been recorded as having medical use in traditional medicine (Kamalakkanan et. al., 2003) lists a number of uses for A. marmelos in the Ayurvedic, Siddha and Unani traditional systems of herbal medicine. Jagetia et. al. (2003) quoted the Ayurvedic author Charaka (1500 B.C.)  as saying:” No drug has been longer or better appreciated by the inhabitants of India than the Bael.”


Various phytochemicals and their effect on health, especially the suppression of free radicals by natural antioxidants have been studied and they are potentially useful for decreasing the risks of several chronic diseases (Salawu et. al., 2006). The medicinal importance of a plant is due to the presence of these special substances like alkaloids, glycosides, resins, volatile oils, gums and Tannins etc. and these active principles usually remain concentrated in the storage organs of the plants viz., roots, seeds, bark, leaves etc. Considering all these facts present investigation was designed to find out phytochemical analysis and antioxidant activity of Aegle marmelos extracts.



Selection and Authentification of Plants

Plant was selected on the basis of literature and indigenous traditional knowledge. The selected plant was Aegle marmelos, Identification of the plant was done by competent botanist Dr. Shaukat S. Khan and the voucher no. given was AM\003, Department of Botany Saifia Science College, Bhopal, MP.


Preparation of plant extracts

The plant material (fruit, leaves and stem bark) was carefully cleaned, shade dried and powdered. About 100 gm of plant material was kept in petroleum ether to defat the extract for 1 hour. The crude extract was defatted to remove the lipid present in plant material and it was then subjected to separating funnel extraction using 50% methanolic solvent by refluxing for 36 hrs. at 50-600c and the powder of the drug were obtained.


(I) Preliminary Phytochemical Screening

Standard screening tests for the three Aegle marmelos hydromethanolic extracts were carried out for various plant constituents. The crude extracts were screened for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, flavonoids, saponins, tannins, using standard procedures (Trease and Evans, 1989; Harborne, 1993; Debela, 2002)


Test for carbohydrate

Molisch test:  A small quantity of the extracts was dissolved separately in 4 ml of distilled water and filtered. The filtrate was subjected to Molisch’s reagent and formation of brick red colour confirmed the presence of reducing sugar.


Fehling’s test: Equal volume of Fehling A (copper sulphate in distilled water) and Fehling B (potassium tartrate and sodium hydroxide in distilled water) reagents were mixed with few drops of crude extract is added and boiled, a brick red precipitate of cuprous oxide forms, if reducing sugar are present.


Test for glycosides

Borntrager’s test: 200 mg crude extract was mixed with 2 ml of dilute sulphuric acid and 2 ml of 5 % aqueous ferric chloride solution, boiled for 5 minutes which lead to oxidation to anthroquinones, indicating the presence of glycosides.


Kedde’s test:  Crude extract was mixed with chloroform, one drop of 90 % alcohol and 2 drops of 2 % 3, 5 dinitrobenzoic acid in 90 % alcohol and made alkaline with 20 % sodium hydroxide. A purple colour so produced, suggested the presence of glycosides.


Test for Alkaloids

Mayer’s test: Crude extract was mixed with Mayer’s reagent (potassium mercuric iodide solution). Cream colour precipitate was formed, indicating the presence of alkaloids.

Dragondroff’s test: Crude extract was mixed with Dragondroff’s reagent (potassium bismuth iodide solution). Reddish brown precipitate was formed which suggested the presence of alkaloids.


Test for Steroids and terpenoids

Libermann-Buchard test: Crude extract was mixed with few drops of acetic anhydride, boiled and cooled, conc. H2SO4 was then added from the sides of the test tube. A brown ring at the junction of two layers was formed. The upper layer turned green which showed the presence of steroids and formation of deep red colour indicated the presence of triterpenoids.


Salkowski test: Crude extract was mixed with chloroform and a few drops of conc. H2SO4, shaked well and allowed to stand for some time. Red color appeared at the lower layer indicated the presence of steroids and formation of yellow coloured layer indicated the presence of triterpenoids.


Test for Flavonoids

Alkaline reagent test: Crude extract was mixed with few drops of sodium hydroxide solution. An intense yellow colour was formed. Yellow colour turned to colorless on addition of few drops of diluted acid, marked the presence of flavonoids.


Lead acetate test: To a solution of 0.5 g extract in water, about 1ml of 10% lead acetate solution was added. Production of yellow precipitate is considered as positive for flavonoid.


Test for Saponins

Froth test: 0.5g extracts were dissolved in 10ml of distilled water for about 30 seconds. The test tube was stoppered and shaken vigorously for about 30 seconds. The test tube was allowed to stand in a vertical position and observed over 30 minutes period of time. If a “honey comb” froth above the surface of liquid persists after 30 minutes the sample is suspected to contain saponin.


Test for Tannins

Ferric chloride test: Crude extract was mixed with ferric chloride. Blue green colour appeared, suggested the presence of tannins.


Test for Phlobatanin

Weigh about 0.5 gm of plant extract in a test tube and dissolve with distilled water and filter. The filtrate was boiled with 2% Hydrochloric acid solution. Observe for a red precipitate that shows the presence of Phlobatanin.


(II) Determination of in vitro antioxidant activity

Fenton reaction was used for determination of in vitro antioxidant activity. It was performed as per the method reported by John M.C. Gutteridge (1981). The hydroxyl radical attached deoxyribose and initiated a series of reaction that eventually resulted in the formation of thiobarbituric acid reaction substance (TBARS).  The measurement of TBARS thus gives an index of free radical scavenging activity. The reaction mixture consisted of  deoxyribose (3 mM, 100μl), ferric chloride (Fe3++, 0.2 mM, 50μl), EDTA (0.1mM, 50 μl), ascorbic acid (0.1 mM, 100 μl), stock solution of fruit, leaves and bark extracts at 10 mg/ml were prepared from which 10-100 μl were added in reaction mixture, the final volume was made up to 1ml by adding adequate quantity of phosphate buffer saline (pH, 7.4) and incubated for 1 hour at 37°C. The reaction was stopped by adding 0.5 ml of 5% TCA and 0.5 ml of 1% TBA the mixture was then incubated for 20 minutes in a boiling water bath. The absorbance was measured at 532 nm. Ascorbic acid was used as positive control. The results are expressed as the percentage inhibition of TBARS.



Phytochemical screening of the extracts

The results confirmed the presence of alkaloids, glycosides, steroids, saponin, terpenoids, tannins and flavonoids in all the extracts of the plant. Some of the constituents were observed in one or the other extracts. These phytochemical constituents are good source of antimicrobial and antioxidant activity. The results of phytochemical screening are reported in Table.1



Table-1:  Phytochemical constituents in hydromethanolic extracts of Aegle marmelos fruit, leaves and bark extract.

Test parameters

Reagents Used

Aegle marmelos extracts





Fehling’s test




Molisch test





Borntrager’s test




Kedde’s test





Dragendroff’s test




Mayer’s test




Steroids and Terpenoids





Salkowski test





Alkaline reagent test




Lead acetate test





Froth test





Ferric chloride test









Key: + = Presence, - = Absence



In vitro antioxidant potential of Extracts:

Now-a-days, there is considerable interest in finding out the antioxidants that are consumed in the habitual diet. It is also known that polyphenols are involved in reducing the risk of diseases associated with oxidative stress (Muselik et. al., 2007) and phenolic compounds such as Flavanoids present in medicinal plants found to have a potent role in prevention of various diseases through their antioxidant activity (Rajani et. al., 2009). The antioxidant activity of Aegle marmelos (fruit, leaf and bark) extracts using Fenton reaction and it showed promising hydroxyl radical scavenging property (Table- 2, Fig. -1). Ascorbic acid was used as reference standard. In antioxidant screening, the IC 50 values of Aegle marmelos (fruit, leaf and bark) extracts and Ascorbic acid was found to be 550, 350, 300 and 200 µg/ml, respectively. In this investigation, the Aegle marmelos leaf extract showed the highest antioxidant activity with IC 50 value of 300µg/ml. Aegle marmelos bark extract showed moderate antioxidant activity with IC 50 value of 300µg/ml. These results denote the presence of antioxidant principles in the extractives. The reference standard Ascorbic acid was found to be the most potent antioxidant as compared to Aegle marmelos extracts. Ferrous salts can react with hydrogen peroxide and form hydroxyl radical via. Fenton reaction.  The iron required for this reaction is obtained either from the pool of iron or the heme-containing proteins. Deoxyribose is incubated with H2O2 and Fe2+-EDTA complex in the presence of a reducing agent such as ascorbic acid, the resulting deoxyribose degradation is inhibited by the addition Aegle marmelos extracts, which acts as scavenger of OH radical. And when this incubated with TBA at low pH, it forms thiobarbituric acid malondialdehyde (TBA-MDA) adduct, which forms a pink chromogen that was measured by its absorbance at 532 nm (Halliwell et. al., 1987).


Table.2. Showing hydroxyl radical scavenging activity of Aegle marmelos extracts (Fruit, leaves and bark) and Ascorbic acid as positive control.


% TBARS value




Ascorbic acid

100 µg/ml





200 µg/ml





300 µg/ml





400 µg/ml





500 µg/ml





600 µg/ml





700 µg/ml





800 µg/ml





900 µg/ml





1000 µg/ml






Figure.1. Showing comparison of hydroxyl radical scavenging activity of Aegle marmelos extracts (Fruit, leaves and bark) and Ascorbic acid as positive control.


In the present investigation, it is revealed that there are several phytochemical constituents present in the hydromethanolic extracts of Aegle marmelos. These phytochemical constituents are responsible for antioxidant activity of the plant. The results are in accordance with the results of in vitro antioxidant activity as revealed by Fenton’s reaction. Further studies on isolation and characterization of the specific constituent (s) are needed to validate our results. The study thus can be further utilized to formulate the natural antioxidant which can be used as a dietary supplement to fight against several diseases such as ageing, artherosclerosis etc. which caused due to Reactive Oxygen Species (ROS).



The authors are thankful to research staff of Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal (M.P), India for their contribution in the piece of study.



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Received on 24.08.2011

Modified on 20.09.2011

Accepted on 11.10.2011        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 4(2): March-April  2012, 80-83