Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

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ISOLATION AND CHARACTERIZATION OF HESPERIDIN FROM ORANGE PEEL

Prabhakar Sharma*1, Prakash Pandey1, Ramchandra Gupta1, Sunil Roshan1, Ashish Garg2, Ajay
Shulka3, Anil Pasi4
1
Department of Pharmacognosy
2
Department of pharmaceutics
3
Department of Pharmaceutical Chemistry, Guru Ramdas Khalsa Institute of Science and Technology (Pharmacy) Jabalpur, M.P.
4
College of pharmacy, IPS Academy, Indore, M.P.

ARTICLE INFO ABSTRACT

Article history India is sitting on a gold mine of well-recorded and traditionally well practiced knowledge
Received 13/05/2013 of herbal medicine. General phytochemical screening of Citrus sinensis (Orange) belonging
Available online Rutaceae family revealed the presence of flavones glycoside. The aim of this study is to
30/05/2013 identify and characterize the bioactive principle compound (Hesperidin) from the peel of C.
sinensis fruit. It has wide folk medicinal uses. The isolation and characterization of
phytoconstituents was done from the alkaline (KOH) extract of peels.

Keywords
Citrus sinensis,
Hesperidin, Orange
Peel, Orange

Corresponding author:
Prabhakar Sharma,
Email ID: mr.pks19@gmail.com
Contact No.: +91-8269426820

Please cite this article in press as Sharma et.al. Isolation and Characterization of Hesperidin from Orange Peel. Indo American
Journal of Pharm Research.2013:3(4).
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Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which
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permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Vol 3, Issue 5, 2013. Sharma et al. ISSN NO: 2231-6876

INTRODUCTION
Throughout the ages, plants have been used by humans as a source of food, cosmetics, medicine,
clothing and even shelter. Plant products also play an important role in the health care systems of the remaining
20% of the population who mainly reside in developed countries [1]. The plants of the genus citrus (Rutaceae)
are widely distributed in most tropical and subtropical countries. Hesperidin, a flavanone-type polyphenolic
bioflavonoid, is found abundant in the peel and membraneous parts of orange peel and other citrus fruits.
Hesperidin is an antioxidant that enhances the action of vitamin C (ascorbic acid) to lower cholesterol levels.
Hesperidin is comprised of the flavanone hesperitin and the disaccharide rutinose and has been reported to have
many biologically important properties, including anti-inflammatory, antimicrobial, anticarcinogenic,
antioxidant and capillary strengthening effects it is also known to have pharmacological action as an anti-
inflammatory, antihistaminic, and antiviral agent [2-5] as well as flavoring agent.

MATERIAL AND METHOD

1) Collection of Plant Material: - The fruits of Citrus sinensis (Orange) were purchased from local market of
Jabalpur, M.P and they were peeled off and peels were dried under shade.
2) Isolation of Hesperidin: - Hesperidin was isolated by the method given in literature and reference book
with little modification.
2.1) Conventional Method: - The dried orange peels (250gm) were macerated with 800ml of aq. alkaline
solution (10% KOH) pH 8-9 for overnight (fig. 1), After complete maceration the mixture was filtered by large
Buchner funnel and filtrate was evaporated to make it syrupy mass [6].

Fig. 1 Maceration of orange peel

2.2) Modern Method: - 800 mL petroleum ether (40 60C) is filled in a 250 mL round bottom flask with
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magnetic stir bar. 250g 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 (fig. 2). The petroleum ether extract is
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discarded. In order to remove the adherent petroleum ether, the content of the extraction sleeve is laid out in an
extensive crystallisation dish. Afterwards the substance is placed again in an extraction sleeve and, like before,

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Vol 3, Issue 5, 2013. Sharma et al. ISSN NO: 2231-6876

but with 800 mL methanol, extracted unless the solvent leaving the extraction sleeve is color less (1 to 2 hours)
[7]
.

Fig. 1 Soxhlation of orange peel

After complete Soxhlet extraction and maceration the filtrate was then acidifying (pH 3-4) with 6%
acetic acid, Keep the concentrated residual liquid in refrigerator (4-6C) over night when a solid crystalline
substance appears (fig. 3 and 4). It was again filtered and the crude hesperidin was separated out on Buchner
funnel as amorphous powder. The hesperidin was further characterized and identified according to various
physicochemical parameters [8]. The compound was characterized according to various factors. The yield of
hesperidin was analyzed by graphical representation (fig. 5)

Fig. 3 Hesperidin by conventional method fig. 4 Hesperidin by modern method

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Vol 3, Issue 5, 2013. Sharma et al. ISSN NO: 2231-6876

Yield

1.8 gm
conventional method
2.35 modern method
gm

Fig. 5 Graphical representation of yield of hesperidin

RESULT AND DISCUSSION

The compound was isolated from maceration and Soxhlation shows a positive ferric chloride and
Shinoda test for flavonoids, indicating that the compound may be a flavonoid [9-10]. It is yellow brown in color,
characteristic aromatic in odor, with melting point 257C. There are two spots were observed in thin layer
chromatography of crystal using n-Butanol: Acetic Acid: Water (3:1:1) as mobile phase at 0.20 and 0.62 (fig.
6), physical and analytical characters are listed in table 1 and 2 respectively. The yield of hesperidin from two
different methods is 1.80 gm and 2.35 gm respectively.

Table 1:- Physical Characters

Sr. No Parameter Observation
1 Color Yellowish brown
2 Odor Aromatic and characteristic
3 Melting point 257C
4 Yield 1.80 gm and 2.35 gm

Table 2:- Analytical Characters

Sr. No. Analytical Test Observation Result
1 Ferric chloride test Wine red color Present
2 Shinoda test Bright pinkish violet Present
color
3 Thin layer Two spots observed Rf value 0.62, 0.20
chromatography
[n-Butanol: Acetic
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Acid: Water (3:1:1)]

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Vol 3, Issue 5, 2013. Sharma et al. ISSN NO: 2231-6876

Fig. 6 TLC of Hesperidin

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REFERENCES
1. Kumarasamy, Y. et al, 2002. Screening seeds of Scottish plants for antibacterial activity. Journal of
Ethnopharmacol, 83: 73-77.
2. Zhou, W. et al, 2011, Determination of Hesperidin from Pericarpium Citri Reticulatae by Microwave
Assisted Extraction- High Performance Liquid Chromatography, Herald of Medicine, 30 (5), 640642.
3. Seo, C.S. et al, 2011, Simultaneous Determination of Liquiritin, Hesperidin, and Glycyrrhizin by HPLC-
Photodiode Array Detection and the Anti-Inflammatory Effect of Pyungwi-san. Arch. Pharmacal Res., 34 (2),
203210.
4. Kakadiya J. et al, 2010, Effect of Hesperidin on Renal complication in Diabetes an Experimentally Study in
Rats, Journal of Current Pharmaceutical Research; 3(1): 35-40
5. Garg A et al, 2001, Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res.; 15:
655 -669.
6. Jarald, E. Edvin and Jarald S. E., Textbook of Pharmacognosy For Phytochemistry, 2009, CBS Publishers
And Distributers, 142

7. http://kriemhild.uft.uni-bremen.de/nop/en/instructions/pdf/1023_en.pdf
8. Aghel N. et al, 2008, hesperidin from citrus sinnesis cultivated in dejful, iran, Pakistan journal of biological
science, 11(20), 2451-2453
9. Geissman, A. (Ed.,) (1962) The Chemistry of Flavonoid Compounds pp. 72 (New York: The MacMillan
Company).
10. Markham, K. R. (ED.,) (1982) Techniques of Flavonoid Identification (London: Academic Press).

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