Isolation of hesperidin from orange peel

1. Isolation of hesperidin from orange peel

2. Introduction: Hesperidin, a polyphenolic bioflavonoid, is the predominant flavonoid in orange peel and other citrus fruits. The highest concentration of hesperidin can be found in the white parts and pulps of the citrus peels. Hesperidin can also be found in green vegetables. Hesperidin is a flavanone glycoside consisting of the flavone hesperitin bound to the disaccharide rutinose. The sugar causes hesperidin to be more soluble than hesperitin.Figure below shows its structure. Hesperidin is an antioxidant that enhances the action of vitamin C to lower cholesterol levels. It is also known to have pharmacological action as an anti-inflammatoryand antiviral agent.

4. Introduction Hesperidin was first isolated by Leberton in 1828 from the albedo (the spongy inner portion of the peel) of oranges of the family Hesperides, and was given the name hesperidin , Its presence was detected in lemons by Pheffer as early as 1874. Neohesperidin. An isomer of hesperidin, has been isolated together with hesperidin from unripe sour oranges cultivated in Europe. Horowitz and Gentiliisolated it from Ponderosa lemon. Hesperidin was isolated from Citrus mitisby Sastry and Row. Neohesperidin, a bitter compound, occurs in the bitter orange, Citrus aurantium. While hesperidin, a non-bitter compound, is the predominant flavonoid in lemons and the ordinary sweet orange, Citrus sinensis. It was found that hesperidin increases the fragility of blood capillaries.

5. Uses of Hesperidin • Hesperidin is thought to have beneficial effects on the blood vessels. It used as a natural remedy for a number of health problems, including: • allergies • Hemorrhoids • Chronic Venous Insufficiency • High blood pressure • High Cholesterol • Cancer , Anti oxidant • Hesperidin Side Effects • Hesperidin may trigger a number of side effects, including abdominal pain, diarrhea and nausea.

6. Principle • Hesperidin can be isolated by two different methods: • The first method involves extracting the dried citrus peel successively with petroleum ether followed by methanol. The petroleum ether removes the essential oils in the peel and the methanol will extract the glycoside (hesperidin). • The second method uses an alkaline extraction of chopped orange peel and acidification of the extract. The hesperidin can then be crystallized from the acidified extract. Because of its highly insoluble, crystalline nature, hesperidin is one of the easiest flavonoids to isolate. • .

8. Work Procedure: • 150 mL petroleum ether (40 – 60°C) are filled in a 250 mL round bottom flask with magnetic stir bar. • 50g dried and powdered orange peel are placed in the extraction sleeve of a Soxhlet extractor and covered with a little glass wool. • A reflux condenser is put on the Soxhlet extraction unit, and then the reaction mixture is stirred and heated for 4 hours under strong reflux. • The petroleum ether extract is discarded. In order to remove the adherent petroleum ether, the content of the extraction sleeve is laid out in an extensive crystallization dish. • Afterwards the substance is placed again in an extraction sleeve and, like before, but with 150 mL methanol, extracted unless the solvent leaving the extraction sleeve is colourless (1 to 2 hours). • The extract is evaporated at the rotary evaporator until syrup consistency is reached. The residue is mixed with 50 mL of 6% acetic acid; the precipitated solid is the crude hesperidine. • It is sucked off with a Buchner funnel, washed with 6% acetic acid and dried 60 °C until it is constant in weight. • For recrystallization, a 5% solution of the crude product in dimethyl sulfoxide is produced under stirring and heating to 60–80 °C. Afterwards the same amount of water is added slowly whilst stirring. When cooling to room temperature the hesperidine precipitates. It is sucked off, first washed with little warm water and then with iso-propanol and dried in the desiccators until it is constant in weight.

10. Purification of hesperidin with formamide A 10% solution of hesperidin in formamide, prepared by warming to about 60°, is treated for 30 minutes with activated charcoal previously boiled with dilute hydrochloric acid. The formamidewhen tested in a 50 %aqueous solution should be slightly acid. If it is not acid, a little glacial acetic acid or formic acid should be added. The solution is then filtered, diluted with an equal volume of water, and allowed to stand for a few hours in order to crystallize. The crystals of hesperidin are filtered off and washed, first with hot water and then with isopropanol. Two such crystallizations give a white crystalline product melting at 261-263°.

11. Method b 200 g chopped orange peel and 750 ml 10% calcium hydroxide solution are placed in a 2-1 Erlenmeyer flask and thoroughly mixed, then left overnight at room temperature. The mixture is filtered through a large Buchner funnel containing a thin layer of Celite on the filter paper. The yellow-orange filtrate is acidified carefully to pH 4-5 with concentrated hydrochloric acid. Hesperidin separates as amorphous powder. It is collected on a Buchner funnel, washed with water, and recrystallized from aqueous formamide. If the precipitation of hesperidin on addition of hydrochloric acid is slow, it is advisable to concentrate the solution under reduced pressure.

12. Ferric chloride test Addition of ferric chloride Solution to hesperidin produces a wine red color. Magnesium-hydrochloric acid reduction test Drop wise addition of concentrated hydrochloric acid to an ethanolic solution of hesperidin containing magnesium develops a bright violet color. Acidic degradation of hesperidin