ISOLATION AND CHARACTERIZATION OF HESPERIDIN FROM ORANGE PEEL

Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876 Journal home page: http:///index.php/en/ INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH ISOLATION AND CHARACTERIZATION OF HESPERIDIN FROM ORANGE PEEL Prabhakar Sharma* 1, Prakash Pandey 1, Ramchandra Gupta 1, Sunil Roshan 1, Ashish Garg 2, Ajay Shulka 3, Anil Pasi 4 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 Article history Received 13/05/2013 Available online 30/05/2013 ABSTRACT India is sitting on a gold mine of well-recorded and traditionally well practiced knowledge of herbal medicine. General phytochemical screening of Citrus sinensis (Orange) belonging Rutaceae family revealed the presence of flavones glycoside. The aim of this study is to 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). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page3892

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 antiinflammatory, 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 60 C) is filled in a 250 ml round bottom flask with 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 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, Page3893

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 (4-6 C) 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 Page3894

Yield 2.35 gm 1.8 gm conventional method modern method 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 257 C. 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 257 C 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 chromatography [n-butanol: Acetic Acid: Water (3:1:1)] Two spots observed Rf value 0.62, 0.20 Page3895

Fig. 6 TLC of Hesperidin Page3896

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), 640 642. 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), 203 210. 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). 54878478451001222 Submit your next manuscript to IAJPR and take advantage of: Access Online first Double blind peer review policy No space constraints Rapid publication International recognition Submit your manuscript at: editorinchief@iajpr.com Page3897