Phytochemical Screening of Ficus benjamina (Linn.) Fruit Extracts

Collection and Authentication

Fruits and other parts of Ficus benjamina were collected from Forest Research Institute, Dehradun (Uttarakhand) India. Fruits were identified and authenticated at Botanical Survey of India (BSI), Dehradun, Uttarakhand, India vide reference number- 118762. The voucher specimen is available at BSI laboratory for the future reference.

Preparation of Extracts

Successive Soxhlet extraction method using petroleum ether (60-80°C), chloroform, ethyl acetate, ethanol and aqueous was employed for the preparation of fruit extracts. Prior to each extraction, plant material was dried at about 40°C in the oven. Final marc obtained was digested at 50°C with distilled water in order to obtain aqueous fruits extract. All prepared extracts of the fruits of the plant were concentrated in rotary vacuum evaporator. All prepared extracts were stored at 4°C. Finally, the yield of each extract was calculated on the basis of air dried weight.

Phytochemical Screening

Different extracts viz., petroleum ether (60-80°C), chloroform, ethyl acetate, ethanol and aqueous prepared after successive extraction method were phytochemically screened following standard methods. All extracts were screened for different types of phytochemical groups such as carbohydrates, alkaloids, flavonoids, phenols, tannins, proteins, coumarins, lipids, fats, oils etc [7, 8, 9].

Alkaloids Test: For alkaloids test, extracts were treated with dilute hydrochloric acid and mixed with following reagents-

• Mayer’s reagent test: The filtrate after treating the extract with dilute hydrochloric acid was mixed with Mayer’s reagent. Cream colored precipitate formation shows that alkaloids are present in the test sample.
• Dragendorff’s reagent test: Dragendorff’s reagent was added to filtrate of sample. Reddish brown colored precipitate formation indicates that alkaloids are present in the test sample.
• Wagner’s reagent test: Few drops of Wagner’s reagent was added to the filtrate. Formation of reddish-brown precipitate formation indicates that alkaloids are present in the test sample.
• Hager’s reagent test: Hager’s reagent was added to filtrate. Formation of yellow colored precipitate shows that alkaloids are present in the test sample.

Flavanoid Test

• Shinoda test: In the test solution of drug few fragments of magnesium ribbon were added after that drop wise 2 ml concentrated HCl was added. Appearance of pink scarlet crimson red shows that flavonoids are present in the sample.
• Zinc hydrochloride test: Test sample was treated with zinc dust and 2 ml conc. Hydrochloric acid. Red colored formation shows that flavonoids are present.
• Tollen’s reagent test: Test sample was treated with 2 ml of Tollen’s reagent. Appearance of silver mirror shows flavonoids presence in the sample.
• Ferric chloride test: 2-3 drops of FeCl3 solution was added to the test solution, appearance of dark green color shows that flavonoids are present in the test drug.

Tannins and phenolics Test

• Lead acetate test: 10% lead acetate solution was added to the test sample. Formation of white precipitate shows that phenolic compounds are present in the sample.
• Ferric chloride test: 5% FeCl3 solution was added to the test solution. Formation of intense green color shows that phenolics are present in the test drug.
• Gelatin test: 10% Gelatin solution was added to the test solution, formation of white precipitates shows that phenolic compounds are present in the test sample.

Glycosides Test Test for cardiac glycoside

• Keller-killani test: Test solution was treated with 2 ml CH3COOH containing in FeCl3 in H2SO4, change of reddish brown color to blue color shows that cardiac glycoside are present in the test sample.
• Legal’s test: In this test pyridine (2 ml) and alkaline sodium nitroprusside solution (2 ml) was added to the solution of test drug. Formation of blood red color indicates those cardiac glycosides are present in the test drug.
• Baljet’s test: Test solution was treated with 2 ml picric acid solution. Orange colored formation shows those cardiac glycosides are present in the test sample.

Anthraquinone glycosides Test

• Borntrager’s test: Test drug was boiled with H2SO4 solution (1 ml) for 5 min in a test tube. Filter and filtrate was mixed with organic solvent like ether or chloroform. Organic layer was separated and shaken with dil. NH3 solution. Formation of pink to red color indicates that anthraquinone glycosides are present in the test drug.
• Modified Borntrager’s test: Test solution was boiled with H2SO4 solution (1 ml) for 5 min in a test tube and treated with the solution of 5% FeCl3 followed by shaking with same volume of organic solvent like ether or chloroform. Organic layer was separated and shaken with dil. NH3 solution. Formation of pink to red color indicates that anthraquinone glycosides are present in the test drug.

Saponin/steroidal glycosides Test

• Foam test: About 2 ml test sample was shaken persistent foam formation shows that saponins are present in the test drug sample.
• Libermann’s-Burchard test: Test sample was treated with 2 ml acetic anhydride and 2 ml conc. H2SO4, bluish- green color formation shows the presence of steroids in the sample.
• Salkowski’s test: Test sample was treated with 2 ml chloroform and 2 ml conc. H2SO4 after shaking well, greenish color formation in acidic layer while red color formation in chloroform layer shows that steroidal compounds are present in the test sample.

Test for amino acids/proteins

• Ninhydrin test: Test sample was boiled with 5% ninhydrin solution. Formation of violet color shows that amino acids are present in the test sample drug.
• Million’s test: Million’s reagent was added to the test solution. White precipitate formation which turns to red after gentle heating shows that proteins are present in the test drug sample.
• Biuret test: Test drug sample was treated with 4% NaOH and 1% CuSO4 solution. Development of violet color shows the presence of proteins in the sample.

Carbohydrate Test

• Molish’s test (General test): 1 ml of test drug sample was treated with α-napthol (alcoholic) then from the side of test tube, few drops of conc. H2SO4 was added slowly. Momentary purple to violet colored ring formation at the junction shows that carbohydrates are present in the sample.
• Fehling’s test: The test solution was boiled with few drops of equally mixed Fehling’s A and Fehling’s B. Brick red colored precipitate formation indicates that reducing sugars are present in the drug sample.
• Barfoed’s reagent test: 2 ml Barfoed’s reagent was added to the test sample. Red cupric oxide formation indicates that carbohydrates are present in the test drug sample.
• Bendict’s reagent test: 2 ml Bendict’s reagent was mixed with test sample and heated for 5 min appearance of yellow, green or red color indicates presence of various types of carbohydrates in sample.

Test for oil and fats

Sample extract was pressed between two filter papers. If oil is present, it will stain filter paper which indicates presence of oil and fat in the test sample.

Phytochemical Screening

Various phytochemical groups like carbohydrates, alkaloids, coumarins, cardiac glycosides, phenols, flavonoids, tannins, steroids, amino acids, proteins, fats and oils were screened following standard methods. The findings of phytochemical screening of various extracts are presented in Table 2.