Isolation of Hesperidin from Orange Peel Using

Soxhlet Extractor

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-inflammatory, antihistaminic, and
antiviral agent.

Hesperidin alone, or in combination with other citrus bioflavonoids (diosmin, for

example), is most often used for blood vessel conditions such as hemorrhoids, varicose veins,
and poor circulation (venous stasis). It is also used to treat lymphedema, a condition involving
fluid retention that can be a complication of breast cancer surgery. The molecular mechanism of
the inhibitory effect of hesperidin on bone resorption is not clear.
Hesperidin improves the health of capillaries by reducing the capillary permeability so
that it could treat leg ulcers caused by poor circulation, when used in combination with diosmin.

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 Hesperidin, in combination with diosmin and a compression dressing, seems to improve
healing of small ulcers (less than 10 cm) caused by poor blood circulation. Hesperidin is used to
reduce hay fever and other allergic conditions by inhibiting the release of histamine from mast
cells. The possible anti-cancer activity of hesperidin could be explained by the inhibition of
polyamine synthesis.

The following doses have been studied in scientific research:

- For the treatment of hemorrhoids inside the anus: 150 mg of hesperidin plus 1350 mg of
diosmin twice daily for 4 days, followed by 100 mg of hesperidin and 900 mg of diosmin
twice daily for 3 days.
- For preventing the return of hemorrhoids inside the anus: 50 mg of hesperidin plus 450
mg of diosmin twice daily for 3 months.
- For the treatment of leg ulcers caused by poor blood circulation (venous stasis ulcers): a
combination of 100 mg of hesperidin and 900 mg of diosmin daily for up to 2 months.

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.

Work Procedure:
1) 150 mL petroleum ether (40 60C) are filled in a 250 mL round bottom flask with
magnetic stir bar.
2) 50g dried and powdered orange peel are placed in the extraction sleeve of a Soxhlet
extractor and covered with a little glass wool.

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 3) 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.
4) 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.
5) 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).
6) 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.
7) It is sucked off with a Buchner funnel, washed with 6% acetic acid and dried 60 C until
it is constant in weight.
8) For recrystallization, a 5% solution of the crude product in dimethyl sulfoxide is
produced under stirring and heating to 6080 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.

Analytics
Reaction control with TLC
TLC-Conditions:
adsorbant: TLC plates GF254 normal silica
Mobile phase: n-butanol : acetic acid : water = 4 : 1 : 5
Rf (hesperidine) = 0.6

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Extraction Scheme: