Hesperidin from Citrus sinensis Cultivated in Dezful, Iran
This study described procedures for extracting and quantitation
of hesperidin in the waste orange peel of Dezful. Two extracting procedure
were used. In procedure A hesperidin was isolated from orange peel by
extracting the dry peel first with petroleum ether, removing the essential
oil and then with methanol. In procedure B alkaline extraction followed
by acidification of the extract was used. It was purified by treatment
with formamide-activated charcoal. Detailed analysis of UV, IR, 1HNMR,
13CNMR and Mass spectroscopic data confirm the structure and
extent of purity of extracted hesperidin. The spectroscopic results of
two extract showed that procedure A produced high extraction yield and
more purified hesperidin. Pure hesperidin in gram quantity (11.7% for
procedure A and 7.39% for procedure B) was obtained in one purification
Flavonoids are an important group of secondary metabolites, which are
synthesized by plants as a result of plant adaptation to biotic and a
biotic stress conditions (infection, wounding, water stress, cold stress,
high visible light). Protective phenylpropanoid metabolism in plants has
been well documented (Shetty, 2004; Harborne and Williams, 2000; Korkina,
2007). In recent years flavonoids have attracted the interest of researchers
because they show promise of being powerful antioxidants that can protect
the human body from free radicals for their hydrogen radical donating
abilities (Jeong et al., 2007; Lemanska et al., 2001). Many
epidemiological studies have shown that consumption of edible plants rich
in phenolic compounds is associated with a lowered risk of degenerative
diseases such cancers (Harris et al., 2007), cardiovascular diseases
(Naruszewicz et al., 2007) and immune dysfunctions (Kale et
al., 2008). These epidemiological results are corroborated by many
in vitro and in vivo studies demonstrating the impact of
flavonoids on mammalian biology (Turner et al., 2004) and displaying
the remarkable scope of biochemical and pharmacological actions of these
compounds, among others their antiviral (Materska and Perucka, 2005),
antiinflammatory (Wu et al., 2006) and antiallergic (Sökmen
et al., 2004) properties.
Hesperidin is a flavanone glycoside abundantly found in sweet orange
and lemon and is an inexpensive by-product of citrus cultivation (Garg
et al., 2001).
Hesperidin may be associated with potential benefits in the prevention
of diseases, such as decreasing capillary permeability, anti inflammatory,
antimicrobial and anti carcinogenic effects. Hesperidin also regulates
hepatic cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methlyglutaryl
coenzyme A (HMG-CoA) reductase (Uehara, 2006). Hesperidin is effectively
used as a supplemental agent in the treatment protocols of complementary
settings. Its deficiency has been linked to abnormal capillary leakiness
as well as pain in the extremities causing aches, weakness and night leg
cramps. Supplemental hesperidin also helps in reducing oedema or excess
swelling in the legs due to fluid accumulation. A number of researchers
have examined the antioxidant activity and radical scavenging properties
of hesperidin using a variety of assay systems (Orallo et al.,
2004; Cho 2006; Hirata et al., 2005; Fujisaw et al., 2002).
This study will explore the isolation of the natural product, hesperidin,
from orange peel and will also chemically identified and then characterized
it by several spectroscopic methods.
MATERIALS AND METHODS
Plant material: Mature orange fruit, harvested in November 2005,
was obtained from Safieabad Research Center, Dezful, Southwest of Iran
and identified as Citrus sinensis (Rutaceae). Chopped green peels
were sun dried for 36 h.
Chemicals: All chemicals were of reagent grade. Hesperidin was
obtained from Sigma (USA). Acetic acid, ferric chloride, calcium hydroxide,
isopropanol, methanol, formamide, hydrochloric acid, Mg metal, petroleum
ether, celite, activated charcoal, ethanol and dimethyl sulfoxide were
obtained from Merck (Germany). Distilled water was used through out.
Extraction of hesperidin: Sun-dried green peel of orange was grinded
into powder with two procedures (Ikan, 1991).
Procedure A: Two hundred grams of this powder was placed in a
reflux condenser. One liter of petroleum ether was added and refluxed
on a water bath for 1 h. After filtration of hot mixture through a Buchner
funnel, the powder was allowed to dry at room temperature. The dry powder
was returned to the flask and 1 L of methanol was added. The contents
were heated under reflux for 3 h again and then hot mixture was filtered
and washed with 200 mL hot methanol. The filtrate was concentrated under
reduced pressure, leaving a syrupy residue crystallized from dilute acetic
acid, yielding white needles, mp 252 °C.
Procedure B: Two hundred grams of powder was transferred in an
Erlenmeyer flask, 750 mL of calcium hydroxide was added and mixed thoroughly.
The mixture was stayed at room temperature overnight. The mixture was
filtered through Buchner funnel containing a thin layer of celie. The
pH of the filtrate was adjusted to 4. An amorph powder was separated which
was again filtered and washed with distilled water and kept for recrystallization
Purification of hesperidin: The crude hesperidin was added to
dimethylformamide (7 mL g-1 of syrup), prepared by warming
to about 60 °C and then was treated for 30 min with activated charcoal
previously boiled with dilute hydrochloric acid. The formamide solution
should be slightly acidic; a little glacial acetic acid was added. The
solution was then filtered through celite, diluted with an equal volume
of water and was allowed to stand for a few hours in order to crystallize.
The crystals of hesperidin were filtered off and washed, first with hot
water and then with isopropanol. Two such crystallizations were given
a white crystalline product melting at 260-262 °C.
Identification of hesperidin
Ferric chloride test: Addition of alcoholic ferric chloride
solution to hesperidin produce a wine red color.
Magnesium-hydrochloric acid reduction test (Shinoda test): Dropwise
addition of concentrated HCl to an ethanolic solution of hesperidin containing
magnesium developed a bright violet color (Ikan, 1991).
Spectroscopic studies of hesperidin: (a) NMR spectra were recorded
at 500 MHz for 1H and 13C by Bruker NMR spectrometer
using DMSO-d6 and chemical shifts were given on a δ (ppm)
scale with tetramethylsilane as internal standard, (b) Mass spectra were
recorded on Finnganmat TSQ 70 USA spectrometer (c) UV-Visible recording
spectrometer 7850, Jasco (Japan) was used for UV spectrum and (d) IR spectra
were recorded on IR 700, Jasco (Japan).
RESULTS AND DISCUSSION
Structure of hesperidin is shown in Fig. 1. Hesperidin
extracted from orange peel was identified by spectroscopic data. The UV
spectrum of the methanolic extract in DMSO showed maximum absorption at
290 nm. The pattern of the spectrum was the same as standard for both
The IR spectrum as KBr disk showed a strong band of OHstr at
3544 and 3470 cm-1, CH (aliphatic) at 2976, 2916 and 2848 cm-1,
C=C (aromatic) at 1606, 1519, 1467 and 1443 cm-1 and of C=Ostr
at 1648 cm-1, C-Ostr at 1298, 1276, 1240, 1203,
1182, 1154, 1131, 1094, 1050, 1033 and 1009 cm-1.
1HNMR and 13CNMR of extracted hesperidin by procedure
A showed the following chemical shifts in ppm which are as those of standard.
Those of procedure B showed a few more different chemical shifts.
1HNMR: δ 12.00 (OH-5), δ 6.91 (H-6´, 5´), δ6.12
(H6, 8), δ 5.49 (H2), δ 3.79 (OCH3).
13CNMR: δ 78.4 (C-2), δ 42.0 (C-3), δ 196.7
(C-4), δ 163.3 (C-5), δ 96.7 (C-6), δ 165.2 (C-7), δ
|| Chemical structure of Hesperidin
δ 162.5 (C-9), δ 103.5 (C-10), δ 131.0 (C-1´), δ
114.3 (C-2´), δ 146.7 (C-3´), δ 148.1 (C-4´), δ 112.7 (C-5´),
δ 117.8 (C-6´) and δ 56.0 (Ome).
Mass spectrum showed m/e of 483.0 (100%), 485.1 (45%), 349.0 (50%), 238.0
No additional peaks were seen in this spectroscopic spectrum of procedure
A. So, it can be concluded that the extracted hesperidin is almost pure.
However the percent of hesperidin in orange peel of Dezful was determined
by weighing. 11.7 g hesperidin was extracted from 100 g of Dezful orange
peel by procedure A.
These data also showed that hesperidin can easily be extracted and purified
from orange peel by procedure A.
So, it is economic to use orange peel as a source of hesperidin for pharmaceutical
Cho, J., 2006. Antioxidant and neuroprotective effects of hesperidin and its aglycone hesperetin. Arch. Pharm. Res., 29: 699-706.
Fujisaw, S., M. Ishihara and Y. Kadoma, 2002. Kinetic evaluation of the reactivity of flavonoids as radical scavengers. SAR QSAR Environ. Res., 13: 617-627.
Garg, A., S. Garg, L.J. Zaneveld and A.K. Singla, 2001. Chemistry and pharmacology of the Citrus bioflavonoid hesperidin. Phytother. Res., 15: 655-669.
Harborne, J.B. and C.A. Williams, 2000. Advances in flavonoid research since 1992. Phytochemistry, 55: 481-504.
CrossRef | PubMed | Direct Link |
Harris, C.S., F. Mo, L. Migahed, L. Chepelev, P.S. Haddad, J.S. Wright, W.G. Willmore, J.T. Arnason and S.A. Bennett, 2007. Plant phenolics regulate neoplastic cell growth and survival: A quantitative structure-activity and biochemical analysis. Can. J. Physiol. Pharmacol., 85: 1124-1138.
Hirata, A., Y. Murakami, M. Shoji, Y. Kadoma and S. Fujisawa, 2005. Kinetics of radical-scavenging activity of hesperetin and hesperidin and their inhibitory activity on COX-2 expression. Anticancer Res., 25: 3367-3374.
Ikan, R., 1991. Natural Products: A Laboratory Guide. 2nd Edn., Academic Press, London, UK., ISBN-13: 9780123705518, Pages: 360.
Jeong, J.M., C.H. Choi, S.K. Kang, I.H. Lee, J.Y. Lee and HJung, 2007. Antioxidant and chemosensitizing effects of flavonoids with hydroxy and/or methoxy groups and structure-activity relationship. J. Pharm. Pharm. Sci., 10: 537-546.
Kale, A., S. Gawande and S. Kotwal, 2008. Cancer phytotherapeutics: Role for flavonoids at the cellular level. Phytother. Res., 22: 567-577.
Korkina, L.G., 2007. Phenylpropanoids as naturally occurring antioxidants: From plant defense to human health. Cell Mol. Biol., 53: 15-25.
Lemanska, K., H. Szymusiak, B. Tyrakowsla, R. Zielinski and A.E.M. Soffers et al., 2001. The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Free Radic. Biol. Med., 31: 869-881.
Materska, M. and I. Perucka, 2005. Antioxidant activity of the main phenolic compounds isolated from hot pepper fruit (Capsicum annuum L.). J. Agric. Food Chem., 53: 1750-1756.
CrossRef | PubMed |
Naruszewicz, M., I. Laniewska, B. Millo and M. Dluzniewski, 2007. Combination therapy of statin with flavonoids rich extract from chokeberry fruits enhanced reduction in cardiovascular risk markers in patients after Myocardial Infraction (MI). Atherosclerosis, 194: 179-184.
Orallo, F., E. Alvarez, H. Basaran and C. Lugnier, 2004. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin. Naunyn Schmiedebergs Arch. Pharmacol., 370: 452-463.
Shetty, K., 2004. A model for the role of proline-linked pentose phosphate pathway in biosynthesis of plant phenolics for functional food and environmental applications: A review. Process Biochem., 39: 789-804.
Sökmen, M., J. Serkedjieva, D. Daferera, M. Gulluce, M. Polissiou, B. Tepe, H.A. Akpulat, F. Sahin and A. Sokmen, 2004. In vitro antioxidant, antimicrobial, and antiviral activities of the essential oil and various extracts from herbal parts and callus cultures of Origanum acutidens. J. Agric. Food Chem., 52: 3309-3312.
Turner, R., T. Baron, S. Wolffram, A.M. Minihane and A. Cassidy et al., 2004. Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein. Free Radic. Res., 38: 209-216.
Uehara, M., 2006. Prevention of osteoporosis by foods and dietary supplements. Hesperidin and bone metabolism. Clin. Calcium., 16: 1669-1676.
Wu, Y., C. Zhou, X. Li, L. Song and X. Wu et al., 2006. Evaluation of antiinflammatory activity of the total flavonoids of Laggera pterodonta on acute and chronic inflammation models. Phytother. Res., 20: 585-590.