Bioflavonoid Hesperidin[1]

��

Chemistry and Pharmacology of The CitrusBioflavonoid Hesperidin

A. Garg,1 S. Garg,2 L. J. D. Zaneveld3 and A. K. Singla1*1University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India2National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S Nagar, Punjab 160062, India3Rush Presbyterian St. Luke’s Medical Centre, 1653, West Congress Parkway, Chicago, IL 60612, USA

Hesperidin, a bioflavonoid, is an abundant and inexpensive by-product of Citrus cultivation. A deficiencyof this substance in the diet has been linked with abnormal capillary leakiness as well as pain in theextremities causing aches, weakness and night leg cramps. No signs of toxicity have been observed withthe normal intake of hesperidin or related compounds.

Both hesperidin and its aglycone hesperetin have been reported to possess a wide range of pharmaco-logical properties. This paper reviews various aspects of hesperidin and its related compounds, includingtheir occurrence, physical and chemical properties, analysis, pharmacokinetics, safety and toxicity and themarketed products available. A special emphasis has been laid on the pharmacological properties andmedicinal uses of these compounds. Copyright � 2001 John Wiley & Sons, Ltd.

Keywords: hesperidin; hesperetin; bioflavonoid; pharmacology; analysis.

INTRODUCTION

Flavonoids comprise a large group of naturally occurring,low molecular weight, polyphenolic compounds widelydistributed in the plant kingdom as secondary metabo-lites. They represent one of the most important andinteresting classes of biologically active compounds andoccur both in the free state and as glycosides. They arebased on the parent compound, flavone (2-phenylchromone or 2-phenyl benzopyrone), characterized by aC6-C3-C6 carbon skeleton where the C6 components arearomatic rings (Fig. 1). Flavonoids occur in practically allparts of plants including fruit, vegetables, nuts, seeds,leaves, flowers and bark (Middleton, 1984).

Dr Albert Szent-Gyorgi, a famed Hungarian research-er, found that Citrus peel flavonoids were effective inpreventing capillary bleeding and was the first to reportthe biological activity of flavonoids on capillary fragilityassociated with scurvy (Armentano et al., 1936; Szent-Gyorgi, 1938). The broad spectrum of biologicalactivities within the group and the multiplicity of actionsdisplayed by certain individual members make theflavonoids one of the most intriguing classes ofbiologically active compounds, and thus these are oftentermed ‘bioflavonoids’.

Hesperidin is an abundant and inexpensive by-productof Citrus cultivation and is the major flavonoid in sweetorange and lemon. In young immature oranges it canaccount for up to 14% of the fresh weight of the fruit(Barthe et al., 1988). It is usually found in associationwith vitamin C. Some symptoms originally thought to be

due to vitamin C deficiency such as bruising due tocapillary fragility were found in early studies to berelieved by crude vitamin C extract but not by purifiedvitamin C. The bioflavonoids, formerly called ‘vitaminP’, were found to be the essential components incorrecting this bruising tendency and improving thepermeability and integrity of the capillary lining. Thesebioflavonoids include hesperidin, citrin, rutin, flavones,flavonols, catechin and quercetin.

Of historical importance is the observation that ‘citrin’,a mixture of two flavonoids, eriodictyol and hesperidin,was considered to possess a vitamin-like activity, as earlyas in 1949 (Scarborough and Bacharach, 1949). Thismaterial was later termed ‘vitamin P’ to indicate that itcould decrease capillary permeability and fragility,prolong the life of marginally scorbutic guinea-pigs andreduce the signs of hypovitaminosis C. Although the termvitamin P was subsequently abandoned, there was astrong indication that the material had potent antioxidant-dependent vitamin C sparing activity (Clemetson, 1989).Hesperidin deficiency has since been linked withabnormal capillary leakiness as well as pain in theextremities causing aches, weakness and night legcramps. Supplemental hesperidin also helps in reducingoedema or excess swelling in the legs due to fluidaccumulation. As with other bioflavonoids, hesperidinworks best when administered concomitantly with

��

PHYTOTHERAPY RESEARCHPhytother. Res. 15, 655–669 (2001)DOI: 10.1002/ptr.1074

Copyright � 2001 John Wiley & Sons, Ltd.

* Correspondence to: A. K. Singla, University Institute of PharmaceuticalSciences, Panjab University, Chandigarh 160014, India.E-mail: [email protected]/grant sponsor: TOPCAD, Chicago, USA.

Received 9 February 2001Revised July 2001

Accepted 20 August 2001

vitamin C. No signs of toxicity have been observed withnormal intake of hesperidin.

Hesperidin was first discovered in 1827, by Lebreton,but not in a pure state and has been under continuousinvestigation since then (Fluckiger and Hanbury, 1986).Though it has been found to possess a wide range ofpharmacological properties, no comprehensive report isavailable on this compound. The following provides acomprehensive review of this particular bioflavonoid.

OCCURRENCE

Hesperidin is isolated in large amounts from thediscarded rinds of the ordinary orange Citrus aurantiumL. (Kanes et al., 1993; Emim et al., 1994), C. sinensis(Horowitz and Gentili, 1963), C. unshiu (Kawaguchi etal., 1997) and other species of the genus Citrus (familyRutaceae). It has been reported to occur in many plantsother than Citrus, such as in genera Fabaceae (Bhalla andDakwake, 1978), Betulaceae (Pawlowska, 1980), Lamia-ceae (Kokkalou and Kapetanidis, 1988) and Papiliona-ceae. Its presence in the bark of Zanthoxylum avicennaeand Z. cuspidatum (family Rutaceae) has been reported(Arthur et al., 1956). These plants are indigenous to HongKong. The presence of hesperetin- 7-�-neohesperidoside(neohesperidin) was recently reported in C. humilis andC. cornigera, two Cynara species growing in Greece(Chinou and Harvala, 1997). Hesperidin has also beenisolated from the roots of Acanthopanax setchuenensis(family Araliaceae), collected in Sichuan Province,China (Zhao et al., 1999).

Hesperidin occurs in greatest concentration in greenfruit and its concentration in the fruit increases duringstorage (Higby, 1941). Its distribution in the epicarp,mesocarp, endocarp and juice of Citrus fruits has beenreported (Kawaguchi et al., 1997). The distribution andconcentration of hesperidin within the different tissues ofmature fruit of C. sinensis has been measured using aradioimmunoassay method and it was found to present inhigh levels in the albedo, membranes and the pithwhereas the concentration was much lower in the juicevesicles and seeds. In seeds, the hesperidin contentincreases after germination suggesting that there is a netproduction of this compound in the developing seedling,which is partly stimulated by light (Barthe et al., 1988).

Hesperidin occurs in crystalline, feather-like aggre-gates or sphaerocrystalline masses in the cells (Evans,1996). Di Mauro et al. (1999) have recently described anovel procedure for obtaining hesperidin from wasteorange peel of the Citrus industry based on the adsorptionof dilute extracts of hesperidin on a styrene-divinylben-zene resin.

PROPERTIES

Pure hesperidin occurs as long hair-like needles, tan orpale yellow in colour. Its melting point ranges from 258°to 262°C (softens at 250°C). It has a molecular formulaC18H34O15 and a molecular weight of 610.57 daltons. It iseasily soluble in dilute alkali and in pyridine giving aclear yellow solution, slightly soluble in methanol, hotglacial acetic acid and almost insoluble in acetone,

benzene and chloroform. The solubility in water is 1 in 50(Budavari, 1996). It has a property of forming complexcrystals with other similar glucosides, which greatlyaffects its solubility and other physical properties,making it difficult to obtain in a pure state (Higby,1941). It can, however, be purified by washing with hotwater and extraction with 95% methyl alcohol, followedby crystallization (King and Robertson, 1931). It istasteless and odourless (Kometani et al., 1996).

CHEMISTRY

Hesperidin (Fig. 2) is a flavanone glycoside, comprisingof an aglycone, hesperetin (Fig. 3) or methyl eriodictyol(Evans, 1996) and an attached disaccharide, rutinose.Hesperidin is, therefore, a �-7-rutinoside of hesperetin(Preston et al., 1953). The disaccharide unit (C12H22O10)is composed of one molecule of rhamnose and one ofglucose and may assume one of the two isomeric forms,rutinose or neohesperidose. Rutinose (Fig. 4) is chemi-cally 6-O-(6-deoxy-�-L-mannopyranosyl)-D-glucose orO-�-L-rhamnosyl-(1 → 6)glucose. Neohesperidose ischemically O-�-L-rhamnosyl-(1 → 2)glucose, differingonly in the configuration of the two sugar units (Har-borne, 1994). The position of the sugars in hesperidin wasdetermined by partial hydrolysis with dilute acid leadingto the formation of L-rhamnose and hesperetin-7-�-D-glucoside which could be cleaved by the enzyme �-D-glucosidase (Fox et al., 1953). Hence, in hesperidin,glucose is attached to hesperetin and rhamnose isattached to the glucose. Hesperetin (C16H14O6) chemi-cally is 3�, 5, 7-trihydroxy-4�-methoxy flavanone. Hes-peridin is thus 3�, 5, 7-trihydroxy-4�methoxyflavanone-7-(6-�-L-rhamnopyranosyl-�-D-glucopyranoside or -7-ruti-noside (Calomme et al., 1996).

Hesperidin, upon alkaline hydrolysis yields phloroglu-cinol and hesperetenic acid (Higby, 1941). Upon acid

��

�� !��

�� "�� #$��%�� & → '��(

656 A. GARG ET AL.

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 655–669 (2001)

hydrolysis, hesperidin produces one mole each of theaglycone hesperetin, D-glucose and L-rhamnose (Asahinaet al., 1930). Hydrolysis with either dilute sulphuric acidor sulphuric acid in ethylene glycol yields an opticallyactive mixture of (�)- and (�)-hesperetin, which can beseparated by fractional recrystallization. Both have beencharacterized by conversion into derivatives (Arthur etal., 1956). Under carefully controlled conditions of acidhydrolysis, hesperidin yields the optically active laevo-rotatory aglycone, (�)-hesperetin, and this on ozoniza-tion, gives L- malic acid, indicating that (�)-hesperetinhas the 2S-configuration (Arakawa and Nakazaki, 1960).

There is a known relation between the structure of thedisaccharide and the presence or absence of bitterness inthe compound (Horowitz and Gentili, 1963). Rutinosidesare tasteless, whereas neohesperidosides are intenselybitter. Hesperidin itself is a flavonoid rutinoside and isthereby non-bitter (Kometani et al., 1996). The bitterneohesperidosides mainly accumulate in grapefruitwhereas the non-bitter rutinosides predominate in orangeand lemon (Horowitz, 1961).

IDENTIFICATION AND ANALYSIS

Both hesperidin and hesperetin show a characteristicflavanone absorption spectrum with UV maxima at 286and 289 nm, respectively and an inflection of lowintensity at 330 nm (Jurd, 1962).

The 1H NMR spectra of TMS ether of both hesperidinand hesperetin in CCl4 have been published (Mabry et al.,1970). The 1H NMR spectra of hesperidin in DMSO-d6solution has also been studied and the values for chemicalshifts and coupling constants presented (Nieto andGutierrez, 1986). Aged samples of hesperidin in DMSOshow NMR lines from most protons split into two setsof signals, the second being assigned to an isomer ofhesperidin with a change of the natural configuration atthe C2 atom. A 13C NMR spectrum of hesperidin has beenpublished (Agarwal, 1989). Markham and Ternai (1976)have described the 13C NMR of hesperidin and comparedit with those of other major flavonoid groups includingglycosides, relating to the one basic solvent system,DMSO-d6.

A method for the fluorimetric determination ofhesperidin has been developed, based on the reactionbetween rutin and dimethylformamide to form afluorescent chelate. This chelate shows two excitationmaxima at 295 and 370 nm and one emission maximumat 460 nm, indicating a 1:1 molar ratio of ligand to metalion (Kaito et al., 1979). Manual and flow injectionmethods are also used along with the spectrofluorimetry(Perez-Ruiz et al., 1999).

Other methods have been developed for the spectro-photometric determination of hesperidin by forming itscomplexes with certain metals. Copper (II)–hesperidincomplex (Kuntic et al., 1999), uranil (II)–hesperidincomplex (Kuntic et al., 1998), zirconium (IV)–hesperidincomplex (Radovic et al., 1996) and aluminium (III)–hesperidin complexes (Malesev et al., 1997) have beenformed in water–methanol systems and the determinationof the compound is found to be precise (Radovic et al.,1996).

Radioimmunoassay (RIA) methods, which combinesensitivity, specificity, high sample capacity, speed and

relative simplicity, are finding wide application for thequantification of plant secondary compounds. A RIAmethod has been developed for hesperidin utilizingantibodies raised against a hesperidin-4-O-carboxy-methyl-oxime hapten and a tritiated radiotracer preparedby direct reduction of hesperidin with NaB[3H]4. Themethod possesses a detection limit and measuring rangeat nanogram levels, a low coefficient of variation and agood correlation with HPLC (Barthe et al., 1988).

Capillary electrophoresis is another method that hasbeen used to simultaneously analyse hesperidin andneohesperidin, along with a range of other compounds.Separation could be easily achieved and the compoundscould be subsequently quantified. The method allowsrapid monitoring with great specificity (Cancalon, 1999).

A large number of scientists have worked on thedevelopment of chromatographic methods for theestimation of hesperidin and its analogues. Hoerhammerand Wagner (1962), separated and purified Citrusflavonoids by preparative TLC (silica gel) using butanol,acetic acid and water (4:1:5) as the solvent system. Theseparation of hesperidin, hesperetin, eriodictin, naringinand naringenin was successfully accomplished. Variousscientists have worked on the separation and quantifica-tion of hesperidin and/or hesperetin from a mixture of anumber of flavonoids, using HPLC. Most of these utilizea reversed phase C-18 column and an aqueous mobilephase consisting of water, acetonitrile, methanol andtetrahydrofuran coupled with minor amounts of acids(Fisher, 1978; Casteele et al., 1982; Rouseff, 1988;Rouseff et al., 1992; Mouly et al., 1993; Wang et al.,1994; Mouly et al., 1998; Saija et al., 1998; Ross et al.,2000). The detection is accomplished using a UVdetector set at 280 nm. Most of these methods are preciseand accurate and can be used for quality control ofindustrial concentrates and juices. Sheu and Lu (1995)developed a HPLC method for the simultaneousdetermination of the constituents of a Chinese herbalformula, Hsiao-cheng-chi-tang, containing hesperidin asone of the ingredients. This method utilizes a Cosmosil-5C-18 column with acetate buffer as the mobile phase andthe publication considers the effects of pH, bufferconcentration and column selectivity.

A reversed phase HPLC system has been used toseparate 141 flavonoids including hesperidin, hesperetinand neohesperidin (Casteele et al., 1982). The columnused was Lichrosorb RP-18, the mobile phase acombination of an isocratic and gradient elutions (5%aqueous formic acid and methanol) and a UV detector setat 280 nm. They also discussed the correlations betweenthe structure and retention time values of differentflavonoids. Thirty four selected flavonoids includinghesperidin and hesperetin were analysed by HPLC, usinga �Bondapak C-18 column and UV detector at 254 nm(Daigle and Conkerton, 1982). This method employstwo pumps, one eluting water–acetic acid (495:5) andthe other eluting methanol. The retention timeswere measured to calculate two chromatographic par-ameters—the capacity factors and the relative retentions.They emphasized that ‘adsorption’ and not the size of themolecule is operative in its separation. Another simpleand precise method has been established for thesimultaneous determination of five selected markercomponents, including hesperidin, in an oriental phar-maceutical decoction—‘Heii-San’, by means of HPLCusing tetra-N-amylammonium bromide (TAA) as an ion-

HESPERIDIN – CHEMISTRY AND PHARMACOLOGY 657


pair reagent. An ODS column has been used with multi-step gradient elution with 10 mM TAA (H3PO4, pH 4.0)-acetonitrile. The acetonitrile increased linearly from 24%to 90%. This method has also been compared with threeother methods, i.e. a water–acetonitrile method, aphosphate buffer method and an ion-suppression method,and was found to give comparable and satisfactory results(Yamauchi et al., 1996). In an attempt to screen theflavonoids from Larix species, needles of different larchspecies were extracted and analysed on HPLC usingDupont 830 chromatograph with a Zorbax ODS columnusing a gradient of 45% methanol in water to 100%methanol, both with 0.1% acetic acid at 50°C, withdouble detection at 254 and 360 nm. Hesperidin andhesperetin were eluted at a retention time of 6.5 and9.9 min, respectively (Niemann and Koerselman-Kooy,1977).

Hesperidin and neohesperidin have been analysed byliquid chromatography/mass spectrometry (LC-MS) witha turbo-ionspray (TBS) interface. Characterization ofisomers differing in the glycosylation was found to bepossible on the basis of different mass spectra (Careri etal., 1999). Another LC-MS method using a capillaryscale particle beam interface has been developed for thesensitive detection of flavonoid compounds in complexmatrices (Capiello et al., 1999).

Other methods used in the analysis of hesperidininclude spectrodensitometry (El-Bayoumi, 1999), gas–liquid chromatography of the trimethylsilyl ether (Dra-wert et al., 1980) and micellar electrokinetic capillarychromatography, MECK (Ferreres et al., 1994). Voltam-metry has also been utilized (Obendorf and Reichart,1995; Zoulis and Efstathiou, 1996; Volikakis andEfstathiou, 2000).

COMBINATION PRODUCTS

Hesperidin is commonly used in traditional medicines asa combination product. The most widely used combina-tion product of hesperidin is Daflon- 500 mg� (Servier;Switzerland), which contains hesperidin (50 mg) anddiosmin (450 mg). Diosmin (Fig. 5) is another bioflavo-noid with a molecular weight of 608.6 daltons and ischemically diosmetin-7-rhamnoglucoside. The aglyconediosmetin can be described by the systematic name 3�,5,7-trihydroxy-4�-methoxyflavone or 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one.It differs from hesperetin only in having a double bondbetween C2 and C3, thereby being a flavone, whereashesperetin is a flavanone.

Treatment with Daflon-500 mg� has demonstrated awide variety of pharmacological activities includingbeneficial effects in human subjects with chronic venousinsufficiency. Two hundred and fifteen human subjects

were administered one tablet of the drug, twice daily forone year. Subjects recorded an overall assessment ofefficacy and relief from venous symptoms such as crampsand evening oedema. Clinical laboratory values remainedwithin normal range throughout the study year (Guillot etal., 1989).

SAFETY AND TOXICITY

In general, Citrus bioflavonoids including hesperidinappear to be extremely safe and without side effects evenduring pregnancy (Pizzorno Jr and Murray, 1999). Basedon an experiment in male and female mice (Sieve, 1952),it was established that phosphorylated hesperidin (PH)was nontoxic both to the organism and to tissues, easilyassimilated, nonaccumulative and caused no allergicreactions. Based on these results Sieve performed a studyon humans and reported that phosphorylated hesperidincould be given to humans clinically as an anti fertilityagent, along with other substitution factors such asvitamins, endocrines, amphetamine derivatives anddecholic acid derivatives. Besides this, trauma, infectiousdiseases or systemic diseases did not inhibit itsantifertility effect.

In a study in rats, when Daflon-500 mg� wasadministered by gastric intubation for 26 weeks, nodeaths, changes in weight or abnormalities of standardfunctional tests were observed. A mixture of purecompounds in a ratio of 1:9 was used to obtain thereported effect (Damon et al., 1987). Methyl hesperidin,when administered orally to mice at a level as high as 5%in the diet, exerted no mutagenic or carcinogenic effects.No obvious toxic effects in mice of either sex wereobserved (Kawabe et al., 1993). Moreover, the ingestionof hesperidin did not affect the daily food intake, bodyweight gain or food efficiency (Kawaguchi et al., 1997).

In a study in humans hesperidin administration wasfound to result in minor side effects in only 10% of thesubjects compared with 13.9% of those treated withplacebo (Meyer, 1994). However, there have been somereports of interactions between the aglycone hesperetin(Mitsunaga et al., 2000), hesperidin (Melzig et al., 1997)and conventional drugs.

DRUG–FOOD INTERACTIONS

Hesperidin, as such has not been widely reported tointeract with drugs or food substances (Kawaguchi et al.,1997). In an early study conducted on human volunteers,PH was found to have no interaction when givenconcomitantly with vitamins, endocrines, amphetaminederivatives and decholic acid derivatives (Sieve, 1952).

In a recent study conducted to see whether thegrapefruit bioflavonoids alter the permeation of vincris-tine (an agent used in cancer therapy), across the blood–brain barrier, it was observed that hesperetin increasedthe [3H] vincristine uptake in the 10–50 �M range, but theglycoside did not have any effect (Mitsunaga et al.,2000). This effect was thought to be brought about by thestimulation of P-glycoprotein-mediated drug effluxthrough the cells. This indicated that the patients takingdrugs that are P-glycoprotein substrates, may need to�� )��

658 A. GARG ET AL.


restrict their intake of bioflavonoid containing foods andbeverages. Hesperidin along with other flavonoids wasalso found to interact with daunomycin (Melzig et al.,1997).

PHARMACOKINETICS

The pharmacokinetics of hesperidin has not beendiscussed in great detail in the literature but a fewreferences are available.

Absorption

To evaluate the oral absorption of hesperidin from Citrusproducts, healthy white males (human volunteers), 25years of age, were given 500 mg of the drug in water andequivalent amounts of grapefruit and orange juice. It wasabsorbed from the gastrointestinal tract after oral ad-ministration in any form, but cumulative urinary recoveryindicated low bioavailability (�25%). The aglycone,hesperetin, was detected in both urine and plasma.Absorbed Citrus flavanones were thought to undergoglucuronidation before urinary excretion (Ameer et al.,1996). Intestinal permeability to hesperidin glycosideswas investigated by using a cultured monolayer ofCaco-2 cells as a model for the small intestinal epi-thelium. Whereas hesperidin did not permeate across theCaco-2 monolayer probably owing to its low solubility,its glycosides did permeate, in a time- and dose-dependent manner. This permeation was thought to occurvia a paracellular pathway (Kim et al., 1999). Theabsorption of orally administered hesperidin in rabbitshas been found to depend on the diet. It was absorbedwhen given with a synthetic ration but not with acommercial pelleted ration (Williams, 1964).

Metabolism

The metabolic fate of six flavonoids including hesperidinand hesperetin was studied following oral ingestion inrats. The major metabolic product in the urine was m-hydroxyphenylpropionic acid along with lesser amountsof m-coumaric acid and the aglycones. The aglyconeswere free as well as conjugated with glucuronic acid.This indicated that absorption had occurred from theintestinal tract followed by dehydroxylation, demethoxy-lation or demethylation followed by dehydroxylation toyield m- hydroxyphenylpropionic acid. This study alsoindicated that hesperetin was more readily absorbed thanhesperidin in rats, rabbits, as well as in humans (Fig. 6)(Booth et al., 1958). When human volunteers ingestedhesperidin, a marked difference was observed in themetabolism. 3-Hydroxy-4-methoxyphenylhydracrylicacid was obtained as the major urinary metabolite,indicating that the pyran ring of hesperetin splits to yieldthis hydracrylic acid. A small amount of the glucuronideof hesperetin was also detected (Booth et al., 1958;Williams, 1964). Hesperidin was found to be transformedto its aglycone, hesperetin, in the intestine by the bacteriaproducing alpha-rhamnosidase and beta-glucosidase orendo-beta-glucosidase. It was also found that the anti-platelet activity and cytotoxicity of the metabolite formed

in the human intestine was more pronounced than that ofthe parent compound (Kim et al., 1998).

Excretion

Recently a study was carried out to study the plasmakinetics and urinary excretion of hesperetin and nar-ingenin from grapefruit and orange juice. Healthyvolunteers ingested the juices; blood and urine sampleswere collected and analysed by HPLC with electro-chemical detection. Hesperetin was found to be bioavail-able from the study juices with marked inter-individualvariations. The urine levels were variable enough toconclude that urinary excretion levels could not be usedas biomarkers for dietary intake (Erlund et al., 2001).Animal studies showed virtually complete elimination ofDaflon-500 mg�, 96 h after administration, without anyuntoward accumulation in any particular organ (Meyer,1994).

A resorption and excretion study was performed on14C-hesperidin methylchalcone (a hydrosoluble, semi-synthetic derivative of natural hesperidin) in rats and itwas observed that, at a dose of 10 mg/kg body weight, itwas absorbed 1–2 h after oral administration. The bloodkinetic patterns suggested an entero–hepatic cycle,demonstrated by the intravenous administration of thecompound at the same dose. The blood profiles demon-strated good bioavailability of the drug. Urinary excre-tion was lower than faecal excretion after oral ingestionbut both were comparable after administration byintravenous route. Moreover, excretion mainly occurredin the first 24 h following administration, by either route(Chanal et al., 1981).

PHARMACOLOGICAL EFFECTS

Hesperidin is effectively used as a supplemental agent intreatment programmes and protocols of broadly com-plementary settings.

Effects on the vascular system

Hesperidin supplementation has been used in patientssuffering from blood vessel disorders including fragilityand permeability complaints resulting in easy bruisingand varicosities. This effect of hesperidin has been of

�� *�� +�� + � ��



considerable interest in the last decade and a largenumber of publications have addressed this therapeuticapplication.

Increased permeability of blood capillaries is a featureof several disease states and is manifested in suchsymptoms as oedema, bleeding and hypertension. Dis-eases which are usually associated with increasedcapillary permeability include diabetes, chronic venousinsufficiency, haemorrhoids, scurvy, various ulcers andbruising. Early research on flavonoids from plant extractsestablished that they reduced the permeability andfragility of capillary walls (Bisset et al., 1991). Hes-peridin, along with other flavonoid compounds has beenwidely reported to inhibit an increase of capillarypermeability. As early as in 1939, Morii found that dailyadministration of 30 mg of hesperidin decreased thecapillary permeability and increased capillary resistancein various clinical cases suffering from pleurisy,tuberculosis, Grave’s disease and beriberi (Morii,1939). Since then, hesperidin has increasingly been usedas a therapeutic agent for increasing capillary resistance.

Scarborough (1940) used hesperidin in the treatment ofpurpurea, resulting from the use of arsenicals and in thetreatment of syphilis. He reported that hesperidin, as wellas hesperetin, were active for increasing capillaryresistance Higby (1941) reported the successful use ofhesperidin in the clinical treatment of haemorrhagicpurpurea and disorders arising from abnormal capillaryfragility. This role of hesperidin in increasing capillaryresistance has been attributed to its inhibitory effect onthe action of hyaluronidase enzyme, which in turn hasbeen known to accentuate capillary permeability andfragility (Beiler and Martin, 1947).

Struckmann and Nicolaides (1994) used Daflon-500 mg� to treat chronic venous insufficiency. Thiseffect has been ascribed to an inhibition of inflammatoryprocesses in the ischaemia-induced hyperpermeabilitythat characterizes venous stasis (Jean and Bodinier,1994). The micronized purified flavonoid fraction con-taining diosmin and hesperidin (9:1) was found to preventischaemia/reperfusion induced leukocyte adhesion inskeletal muscles localized in the ischaemic region(Korthuis and Gute, 1999). An open pilot study, on 24human patients with third stage chronic venous insuffi-ciency, indicated a beneficial haemorheological effect onoral administration, resolving the stasis with an increasein the blood cell velocity. Furthermore a concomitantincrease in relative packed cell volume and red blood cellvelocity after therapy suggests an improvement in theflexibility of red blood cells (Allegra et al., 1995). Daflonis also found to possess venotonic and vasculo-protectivepharmacological properties and reinforces venous toneby prolonging the activity of parietal norepinephrine(Amiel and Barbe, 1998).

An antihypercholesterolaemic activity of hesperidin inCCl4-induced hypercholesterolaemic rats has been re-ported (Son et al., 1991). Intragastric hesperidin has beenfound to significantly lower cholesterol, low densitylipoproteins (LDL), total lipids and triglyceride levels innormo-lipidaemic rats and in diet- or Triton-induceddyslipidaemic rats but increased high density lipoprotein(HDL) levels (Monforte et al., 1995). Tangerine peelextract and a mixture of hesperidin and naringin werealso found to significantly lower levels of plasma andhepatic cholesterol, and hepatic triglycerides comparedwith those of the control. They also reduced the excretion

of faecal neutral sterols compared with the control (Boket al., 1999). These effects are accompanied by a reduc-tion in the plasma and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA (HMGCoA) reductase and acylCoA: cholesterol transferase (Bok et al., 1999). Thehypocholesterolaemic effect of hesperetin in rats mayalso be mediated via these enzymes (Lee et al., 1999a).

Hesperidin has also been reported to exert antihyperli-pidaemic effects and the methanolic extracts of the stemsof Prunus davidiana Fr. (Rosaceae), containing hesper-etin-5-glucoside and other flavonoids, was found to beuseful for the treatment of hyperlipidaemia. Intraperito-neal administration of hesperetin-5-glucoside to rats fedon a high fat diet was found to significantly reduce thetotal cholesterol level compared with the control groupbut had no effect on the serum triglyceride level (Choi etal., 1991). In another study, the antilipaemic activity ofhesperidin in normolipaemic and induced hypercholes-terolaemic rats has been demonstrated (Monforte et al.,1995). Hesperidin exerted significant activity, which maybe due to increased hepatic cholesterol catabolism. In yetanother study, the flavonoids from C. unshiu werescreened for lipase inhibitory effect and hesperidin wasfound to inhibit lipase activity from porcine pancreas andthat from Pseudomonas. Moreover, hesperidin (10%,p.o), decreased plasma triglyceride concentration andincreased the amount of faecal lipids excreted in rats(Kawaguchi et al., 1997).

The calcium channel blocker activity of hesperidin hasbeen patented (Morita et al., 1992). It is found to inhibitnitrendipine binding to skeletal muscle membraneprotein in rabbits. The capillary antihaemorrhagicactivity of hesperidin in mice has also been reported(Jurisson, 1973). Kubo et al. (1992a,b) have reported andfiled patents relating to the myocardial depressant activityof hesperidin in human adults.

Galati et al. (1996) have demonstrated significantantihypertensive and diuretic effects of hesperidin in ratsfollowing oral administration of the drug at a dose of200 mg/kg body weight and ascribed this hypotensiveeffect to increased diuresis. However, other mechanismsare probably involved. It is well established that variousflavanones, including hesperidin, influence various en-zymes such as protein kinase, lipooxygenase and cyclo-oxygenase. This effect on enzymes is responsible for theflavonoids’ activity on some haematic parameters, low-ering of erythrocytic adhesion, platelet aggregation andblood viscosity. The antihypertensive activity of hesper-idin might therefore be due to its activity on enzymaticsystems that influence blood rheology. In addition,various flavonoids are potent inhibitors of cyclic-AMPphosphodiesterase, and probably this activity is the basisfor the observed diuretic effect (Emim et al., 1994; Galatiet al., 1994).

Antiinflammatory effects

Hesperidin has been reported to possess significant anti-inflammatory and analgesic effects (Galati et al., 1994).Emim et al. (1994) presented pharmacological datafavouring the use of hesperidin as an inexpensive anti-inflammatory agent or as a lead compound, especially forpatients with hypersensitivity to the ordinarily used non-steroidal antiinflammatory agents. Hesperidin, thoughineffective after oral administration, was found to be

660 A. GARG ET AL.


active on subcutaneous injection without remarkablechanges in rat behaviour or apparent tissue damage at thesite of injection even after repeated administration. Athigh doses, it also reduced dextran induced paw oedemain rats. This effect could be attributed to the inhibition ofthe release of histamine from basophils by hesperidin orits metabolic products.

Hesperidin in combination with diosmin, shows amarked protective effect against inflammatory disorders,both in vivo and in vitro, possibly through a mechanisminvolving an inhibition of eicosanoid synthesis and/orantioxidant free radical scavenger activity (Jean andBodinier, 1994). Lonchampt et al. (1989) studied thescavenging properties of Daflon 500 mg� on activeoxygen radicals in vitro and in vivo, implying itspharmacological action on capillary hyperpermeabilityas well as antiinflammatory and antioedematous actions.

S-5682 (Daflon) was demonstrated to improve multi-ple histological aspects of the acute inflammatoryreaction (diapedesis of polymorphs, lymphocytes, histo-cytes and macrophages) and features of the chronicinflammatory reactions (newly formed micro vascular-ization of the granuloma tissues, perivascular oedema,presence of collagen fibres). Its effects in the chronictreatment of inflammatory granuloma in the rat werestudied. It possessed potent antioedematous activity andreduced the synthesis of PGE2 and PGF2� in inflamma-tory granuloma (Damon et al., 1987).

The antiinflammatory effect of hesperidin in the acutestage of trinitrobenzene sulphonic acid (TNBS) model ofrat colitis was evaluated. Pretreatment with hesperidinreduced colonic damage compared with TNBS controlrats. In addition, hesperidin after oral administrationreduced the areas of colonic necrosis and hyperaemia,scored according to the severity and extension ofinvolved tissue compared with the control group. Asignificant reduction in the colonic weight/length ratiowas also observed. This intestinal antiinflammatoryeffect was also shown biochemically since hesperidinwas able to reduce colonic myeloperoxidase activity, anenzyme that is considered as a biochemical marker forneutrophilic infiltration in damaged tissue (Crespo et al.,1999). Another possible mechanism that contributes tothis effect is its antioxidant property (Jean and Bodinier,1994). Hesperidin has been reported to amelioratecolonic oxidative stress that occurs in an experimentalmodel of inflammatory bowel disease (Loguercio et al.,1996). When given intragastrically to mice at a dose of25 mg/kg, it was found to reduce adjuvant-inducedarthritis (Kim et al., 1990).

Pretreatment with Daflon-500� prior to the inductionof tourniquet ischaemia significantly lowered the numberof adherent leukocytes thereby controlling oedema inclinical situations (Friesenecker et al., 1995). This pro-tective effect is associated with the decreased platelet andcomplement system activation, leading to a loweredrelease of histamine and decreased leukocyte-dependentendothelial damage.

Action on enzymes

Hyaluronidase is an enzyme which depolymerizes themucopolysaccharide hyaluronic acid. It has been knownto play a part in the typical changes in the morphology ofthe connective tissue, particularly of the interfibrillator

cement substance, which is believed to contain hyaluro-nic acid. This enzyme plays a role in regulating thepermeability of capillary walls and supporting tissues.Hyaluronidase causes a breakdown of hyaluronic acid,thereby increasing tissue permeability. Also, variousbacteria produce hyaluronidase, thereby increasing thepermeability of the tissue and favouring the invasion oftissue by these and other microorganisms (Hahn, 1952).Various scientists and medical researchers have beenworking on the hyaluronidase inhibitory effects ofdifferent flavonoids. Beiler and Martin, as early as in1948, prepared various derivatives of hesperidin ashyaluronidase inhibitors. The sulphonated and phos-phorylated hesperidins proved to be extremely potentinhibitors, and acetylated hesperidin caused a potentia-tion in the inhibitory action of ascorbic acid onhyaluronidase. They demonstrated that, although purehesperidin and hesperetin were active as hyaluronidaseinhibitors, this action could be greatly potentiated by theformation of the above mentioned derivatives of the purecompound (Beiler and Martin, 1948). Based on aninvestigation on the isolated connective membranes frommice, it has been reported that the permeability increas-ing action of hyaluronidase could be abolished by contactwith phosphorylated hesperidin for 15 min or more. It notonly abolishes the hyaluronidase action but also restoresthe original degree of permeability of the membranes. Iteven reduces the permeability of normal (untreated)membranes (Steincke, 1956).

Besides hyaluronidase, hesperidin has also beenreported to inhibit human acrosin, a sperm enzyme, invitro (Jackson, 1959). It also shows an inhibitory effecton aldol reductase (lens) in vitro (Varma and Kinoshita,1976), alkaline phosphatase in rat serum (Son et al.,1991) and a weak activity against alpha-glucosidase invitro (Iio et al., 1984). Inhibition of alkaline phosphatasewas, however, found to be absent in a study on rat liver(Son et al., 1991) and another in vitro study where theenzyme was derived from calf intestinal mucosa (Iio etal., 1980). Hesperidin, however, did not inhibit xanthineoxidase in vitro (Iio et al., 1985). It was also found to beinactive as an inhibitor of the enzyme reverse transcrip-tase, when tested on virus-raucher murine leukaemia invitro (Ono et al., 1990).

In a study carried out on some flavonoids, onnonenzymatic lipid oxidation and enzymatic oxidationof arachidonic acid in vitro, hesperidin was found tostimulate the enzyme cyclooxygenase (Iio et al., 1984).However, inhibition of 1,5-lipoxygenase by Citrus peelflavonoids has been reported (Malterud and Rydland,2000).

When a range of flavonoids were screened forinhibitory effects against partially purified aromataseprepared from human placenta, hesperetin was found topossess a significant inhibitory effect on the same (Jeonget al., 1999). A series of flavonoids were tested for theireffects on low Km phosphodiesterase with cyclic AMP asthe substrate and for their lipolytic activity, employing ratadipocytes. Hesperetin showed inhibition of epinephrineinduced lipolysis, in a dose dependent manner but did notinhibit phosphodiesterase significantly (Kuppusamy andDas, 1992).

Hesperidin and diosmin, alone and in combination(1:9), were found to inhibit prostaglandins, when usedagainst sponge-induced granuloma in rats, significantlylowering the prostaglandin levels of the treated animals



compared with those of untreated controls, through 16days after intragastric sponge implantation (Damon et al.,1987).

Antimicrobial activity

Hesperidin and hesperetin, among other flavonoids, haveshown antiinfective and antireplicative activities in vitroagainst several plant and animal microbes.

Antibacterial. In a study involving the investigation ofanti-Helicobacter pylori (HP) activity, in vitro, of someflavonoids and their metabolites, hesperetin and otherflavonoids were found to inhibit the growth of HP (Bae etal., 1999). In patients with chronic gastritis, HP promotesthe alteration from gastritis to gastric cancer (Correa,1988). However, Islam and Ahsan (1997), recentlyshowed hesperidin to be inactive in vitro on agar plates,against Bacillus subtilis, Staphylococcus aureus, Strepto-coccus hemolyticus, Escherichia coli, Klebsiella species,Pseudomonas aeruginosa, Salmonella typhi, Shigelladysenteriae, Shigella flexneri and Vibrio cholera.

Antifungal. The antifungal activity of hesperidin hasbeen reported at a dose ranging from 1 to 10 �g, againstBotrytis cinerea, Trichoderma glaucum and Aspergillusfumigatus (Krolicki and Lamer-Zarawska, 1984). It was,however, found to be inactive against Aspergillusfumigatus, Aspergillus niger and Trichoderma species(Islam and Ahsan, 1997).

Antiviral. Wacker and Eilmes, in their two studies,reported hesperidin to be active as an antiviral agent, invitro, at different concentrations on cell culture, againstvirus-vesicular stomatitis. Hyaluronidase was found toreverse its antiviral activity suggesting that this activityof hesperidin was mediated by its antihyaluronidaseeffect. They also reported hesperidin to be active againstinfluenza virus (Wacker and Eilmes, 1975; Wacker andEilmes, 1978). In a recent study, hesperidin was found topossess a weak activity against herpes simplex virus (Leeet al., 1999b). It was found that certain flavonoids,including hesperidin, possessed antiviral activity againstherpes type-I, para influenza-3, poliovirus type-I andrespiratory syncytial virus (RSV) in tissue cell mono-layers (Middleton, 1984). Mucsi and Pragai (1985)demonstrated the inhibitory effect of hesperidin amongother flavonoids on human herpes simplex virus type-I(HSV-I) and suid (alpha) herpes virus type-I (Pseudo-rabies virus). A direct relationship between the antiviralactivity of the flavonoid and its ability to stimulate cyclicAMP synthesis in the cells was exhibited. Quercetin andhesperetin are also known to possess antireplicativeactivity (inhibition of virus replication after establishedinfection) if introduced at the time of cell activation.Hesperetin was, however, found to be inactive againstHIV-virus (Hu et al., 1994), pseudorabies virus, herpessimplex virus (Mucsi and Pragai, 1985) and rhinovirus(Tsuchiya et al., 1985). In a recent study, the inhibitoryeffects of some flavonoids was tested on infectivity ofrotavirus, which predominantly causes sporadic diar-rhoea in infants and young children (Bae et al., 2000) andhesperidin was found to have a potent inhibitory effect.Hesperetin, however, did not possess this activity,indicating that the rutinose moiety is essential in

protecting against the invasion of rotavirus into the cells.The fruit of C. aurantium, having hesperidin andneohesperidin as main constituents, was also found topossess a potent inhibitory effect on rotavirus infectivity(Kim et al., 2000).

When tested for an effect on the infectivity andreplication of HSV-1, polio-virus type-1 and para-influenza virus type-3, hesperetin was found to have noeffect on the infectivity but reduced intracellularreplication of each of the viruses (Kaul et al., 1985).Hesperidin was also found to have a significant effectagainst Staphylococcus aureus on infected mice (Pana-siak et al., 1989).

Anti yeast. Hesperidin was found to have no inhibitoryeffect on Candida albicans and Saccharomyces cerevi-siae, when tested in vitro on agar plates (Islam andAhsan, 1997).

Anti fertility activity

Hesperidin and its derivatives have been under investiga-tion as anti fertility factors for a very long time. As earlyas 1948, phosphorylated hesperidin (PH) was reported toact as an effective anti fertility agent mediating this effectby inhibiting the sperm enzyme hyaluronidase (Beilerand Martin, 1948; Martin and Beiler, 1952). In a study onrats, PH was found to have an antifertility effect whengiven orally or intraperitoneally (Martin and Beiler,1952). In another attempt to assess the antifertility effect,PH (20 mg/kg) was administered to both male and femalemice, intraperitoneally for 8 days. On mating, the percentpregnancies were significantly reduced. The oestrus cycleof the females remained unchanged. Microscopic exam-ination of the seminal fluid from males showed a normalsperm count and sperm motility. No permanent sterilityin either sex was observed. Based on these results, a studywas further carried out on 300 married human couples.PH tablets (100 mg) were given orally to both thepartners, over varying periods, up to 30 months. Contra-ception occurred in all the cases except for two, where thecouples were non cooperative regarding the studyparameters. Merely omitting the drug for a period of48 h could restore fertility. It thereby offers promise as anoral contraceptive without any toxic effects or permanentinhibition of sterility. Trauma, infectious diseases orsystemic diseases did not inhibit the antifertility effect(Sieve, 1952).

Later it was reported that the fertilizing capacity ofrabbit sperm was inhibited to a certain extent when thesperm was suspended in a 1% solution of PH and in-seminated into female rabbits (Chang and Pincus, 1953).PH also exhibited significant contraceptive efficacy onvaginal application in rabbits, even at nonspermicidalconcentrations (Joyce and Zaneveld, 1985). In yetanother study, PH was proven to inhibit bovine fertiliza-tion by mouse spermatozoa by specifically inhibiting thesperm enzyme hyaluronidase. Hyaluronidase is reportedto be required for sperm penetration through the folliclecell layer of the egg. PH, by inhibiting sperm hyaluroni-dase can thereby successfully be used as an antifertilityagent (Joyce et al., 1986).

662 A. GARG ET AL.


Anticarcinogenic activity

During the past decade, a considerable amount ofresearch has been carried out on the anticancer activitiesof hesperidin and its aglycone hesperetin. Encouragingresults of carcinogenesis inhibition were observed byusing a hesperidin/diosmin combination on male mouseurinary bladder. The combination inhibited N-butyl-N-(4-hydroxybutyl)nitrosamine-induced carcinogenesis whentreated with the combination for 8 weeks during thetumour initiation phase (Yang et al., 1997). An inhibitionof carcinogenesis in rats by hesperidin, at a concentrationof 500 ppm/kg body weight has also been reported. Thecompound was also found to inhibit 4-nitroquinoline-1-oxide-induced oral carcinogenesis and to decrease thenumber of lesions, polyamine levels in tongue tissue andcell proliferation activities (Tanaka et al., 1994). Thesame group later reported a similar activity of hesperidinalone, and in combination with diosmin, where it not onlyinhibited 4-nitroquinoline- 1-oxide initiated tumorigen-esis but also showed chemoprevention of azoxymethane-induced rat colon carcinogenesis. These results arethought to be due to increased suppression of cellproliferation (Tanaka et al., 1997a,c).

Hesperidin, when administered subcutaneously to CD-1 mice, did not inhibit 7,12-dimethylbenz(�)anthracene-induced tumour initiation but inhibited 12-O-tetra-decanoyl-13-phorbol acetate (TPA)- induced tumourpromotion, thus indicating its potential use as a chemo-preventive agent (Berkarda et al., 1998). Later theyestablished the protective effect of hesperidin againstTPA-stimulated infiltration of neutrophils. It alsoafforded significant protection against TPA inducedhyperplasia in the dorsal skin of CD-1 mouse, throughmultiple applications prior to TPA (Koyunku et al.,1999).

The mutagenic activity of hesperetin was assessedalong with several other flavonoids and their derivativesusing Salmonella typhimurium mutants that reveal basepair substitutions and frameshift mutagens. Neitherhesperetin nor neohesperetin were found to be mutagenic(Bjeldanes and Chang, 1977). Much later when theantimutagenicity of various Citrus flavonoids includingnaringin, hesperidin, nobiletin and tangeritin was eval-uated, hesperidin was found to possess a weak antimuta-genic effect on Salmonella typhimurium against benz (�)pyrene-induced mutations (Choi et al., 1994; Calomme etal., 1996). An antimutagenic effect has also been reportedagainst N-methyl-N-amylnitrosamine-induced tumori-genesis, in rats (Tanaka et al., 1997b). When a numberof plant flavonoids were tested for mutagenicity in theSalmonella/microsomal activation system, both hesperi-din and hesperetin were found to require metabolicactivation for any mutagenicity (Hardigree and Epler,1978). Hesperetin has shown an antimutagenic effectagainst aflatoxin B1 with more than a 70% inhibition ratein S. typhimurium, in the presence of a mammalianmetabolic activation system (Choi et al., 1994).

When the effects of predominant dietary flavonoidswere tested for inhibiting neoplastic transformationinduced by 3-methylcolanthrene in C3H 10T1/2 murinefibroblasts, hesperidin and hesperetin were found to bethe most potent agents inhibiting this transformationcompletely (Franke et al., 1998). Structure–activitycomparisons were also made and favourable structuresdiscussed.

Platelet and cell aggregation inhibition

Hesperidin was found to increase the survival time of ratsby 16–71 days when they were placed on a thrombogenicdiet but produced variable results when the rats were onan atherogenic diet (Robbins, 1967). In studies carriedout on human adult platelets (Zaragoza et al., 1985;1986), hesperidin was reported to inhibit epinephrine-and ADP-induced platelet aggregation at a concentrationof 0.08 mg/ml. In yet another study carried out on horses,hesperidin was found to effectively reduce aggregation oferythrocytes. This decrease in the blood cell aggregationmay explain the beneficial effects of hesperidin onabnormal capillary permeability and fragility, the reduc-tion of disease symptoms and their protection againstvarious traumas and stresses (Robbins, 1971). Hesperidinand diosmin, in combination, were found to decreaseleukocyte adhesion to the endothelium in post capillaryvenules, after ischaemia/reperfusion, as assayed throughskin fold window chamber in guinea- pigs followingintragastric administration of the compounds (Friese-necker et al., 1994). This antiischaemic effect ofhesperidin was again reported in the hamster, at a doseof 20 mg/kg body weight after intragastric administration(Bouskela and Donyo, 1995). In addition a micronizedpurified flavonoid fraction containing hesperidin anddiosmin was found to significantly decrease ADP-induced platelet aggregation and increase plateletdisaggregation, in rats. Fibrinogen binding to ADP-induced platelets was also reduced significantly (Mc-Gregor et al., 1999).

Ultraviolet protecting activity

Recently much research has been focused on the potentialuse of flavonoids as free radical scavengers to preventoxidative skin damage (Schoemaker et al., 1995;Mortimer, 1997). The oxidative stress caused by ultra-violet irradiation might be an initiator in the pathogenesisof skin cancer and photoaging (Dalle Carbonare andPathak, 1992; Darr and Fridovich, 1994). Bonina et al.(1996, 1998) investigated the ability of topically appliedhesperetin, alone and in a crude extract of C. sinensisalong with other flavonoids, to reduce UV-B-inducedskin erythema. Employing phosphatidylcholine (PC)vesicles as model membranes they studied the effect ofhesperetin on UV irradiation induced peroxidation. Invitro human skin permeation of these compounds wasalso measured. Hesperetin was effectively found toprotect PC liposomes from UV-irradiation inducedperoxidation probably by scavenging oxygen freeradicals generated by UV irradiation. It was also able topermeate through the stratum corneum. It was laterconcluded from in vitro and in vivo data, obtained afterapplying a gel formulation uniformly on the skin sites(human volunteers) and exposing them to UV-B irradia-tion and monitoring the extent of erythema by means ofreflectance spectrophotometry, that hesperetin might besuccessfully employed as a topical photo-protectiveagent (Saija et al., 1998).

In another study, transglycosylation of hesperidin bycyclodextrin glucanotransferase was conducted and itsmono- and di-glucosides were prepared, both having thesame absorption spectra as that of hesperidin. When thesewere exposed to ultraviolet light, they seemed to stabilize



the colour of the pigments by absorbing the UV rays. Inaddition they did not have strong spectra in visible light.The authors propose its use as a colour stabilizer in foodproducts (Kometani et al., 1994).

Miscellaneous effects

Analgesic and antipyretic activity. Hesperidin hasexhibited analgesic activity in mice on subcutaneousadministration. This effect has been explained as beingexerted through a peripheral and not a central mechanism(Galati et al., 1994). It also showed analgesic activity inmice following intraperitoneal administration. Also,hesperidin decreased the fever induced by yeast in rats.This effect may be related to the inhibition of yeast-induced prostaglandin biosynthesis. Hesperidin is knownto inhibit both histamine and prostaglandin release,thereby acting as a defensive gastric factor and prevent-ing acid secretions and lesions of the gastric mucosa(cited in Emim et al., 1994).

Antioxidant effect. Hesperidin has been reported topossess antioxidant properties. For example, it has beenfound to reduce superoxide in electron transfer plusconcerted proton transfer reaction in vitro (Jovanovic etal., 1994). This activity was also exhibited in liverhomogenates for hydroperoxide-induced chemilumines-cence (Fraga et al., 1987). It has been suggested that thehesperidin/diosmin combination could function as anantioxidant, which may explain its beneficial therapeuticeffect in chronic venous insufficiency where oxidativestress is involved in the pathological mechanism(Bouskela et al., 1997). A number of researchers haveexamined the antioxidant activity and radical scavengingproperties of hesperidin using a variety of assay systems(Kroyer, 1986; Brasseur et al., 1986; Ratty and Das,1988; Yuting et al., 1990; Wang and Zheng, 1992;Miyake et al., 1997; Deng et al., 1997; Miller and Rice-Evans, 1997; Suarez et al., 1998; Malterud and Rydland.2000). Results from different assays varied considerably,but in most, hesperidin was found to be inactive or onlymoderately active in comparison with other flavonoidsand nonflavonoidal antioxidants. Hesperidin was alsofound not to inhibit the liberation of reactive oxygenspecies from stimulated neutrophils (Limasset et al.,1993). Thus hesperidin appears not to be a particularlyactive antioxidant in comparison with most otherflavonoids.

Immuno-modulatory activity. It has been reported thathesperidin possesses an immuno-suppressant activity(Kim and Cho, 1991). It suppresses the bacterial alpha-amylase antibody production in mice on intragastricadministration at a dose of 50 mg/kg. In another study,intragastric administration of 50 mg/kg of hesperidin tomale mice, significantly increased the development ofimmunological memory in cellular immune response(Kim and Cho, 1991).

Recently, the colony stimulating factor (CSF) inducingactivity of various bioflavones was tested, in order toevaluate their immuno-modulating activities. Samples ofdifferent bioflavonoids, suspended in saline were injectedintraperitoneally into mice at a dose of 1 mg/kg, 6 hbefore bleeding. Hesperidin exhibited the strongest CSF

inducing activity and the response was dose dependent(Kawaguchi et al., 1999).

Antiallergic effects. Hesperidin’s antiallergic and anti-anaphylactic activities have been reported and patented(Kubo and Matsuda, 1992). It was shown to possessantianaphylactic activity where it delayed hypersensi-tivity against sheep red blood cells, prevented passivecutaneous anaphylaxis, induced mast cell degranulationand also prevented histamine-induced anaphylaxis afterintragastric administration to mice at different doses(Kim and Chung, 1990). Later, it was found to inhibitvascular permeability in the homologous passive cuta-neous anaphylaxis test conducted on rats after intragastricadministration, at a dose of 0.1 mmol/kg (Kubo andMatsuda, 1992). An inhibition of 48 h homologouspassive cutaneous anaphylaxis in rats has also beenreported with the use of hesperidin (Matsuda et al., 1991).

Activity on haemorrhoids and IUCD-induced bleed-ing. Oral tablets of a hesperidin/diosmin combinationwere clinically tested in situations involving intrauterinecontraceptive device (IUCD)-induced bleeding. Thirtyone women were given the combination over a period ofthree consecutive cycles. Eighty three percent of thepatients showed improvement in the signs and symptomsassociated with the bleeding. The acceptability wasexcellent with minimal side effects (Daftary et al., 1995).Daflon-500 mg� has also been found to reduce inflam-mation by inhibition of prostaglandin and thromboxaneB2 release from macrophages and also to reduce the freeradicals. The investigators postulated that inhibition ofthe inflammatory response in the haemorrhoid surgicalwound site reduced defaecation stress and bacterialfibrinolysis, decreasing the risk of secondary bleeding(Ho et al., 1995). The effectiveness and tolerability of asimilar hesperidin/diosmin combination was evaluatedfor the treatment of haemorrhoids. Overall improvementof signs and symptoms was quicker and greater with goodacceptability and minimal side effects (Godeberge,1995). Recently, a similar combination was provided ina micronized form, for a median of 8 weeks beforedelivery and 4 weeks after delivery, to 50 pregnantwomen with acute haemorrhoids and was found to bevery successful with 66% of the patients reporting reliefof symptoms by day 4 of treatment and fewer sufferingrelapses during the antenatal period. The treatment waswell accepted and did not affect pregnancy, fetaldevelopment, birth weight, infant growth or feeding(Buckshee et al., 1997).

Hormonal disorders. Hesperidin was reported (Smith,1964) to help in regulating oestrogen levels anddecreasing related pain, inflammation and swelling. In aclinical study, 94 women suffering from hot flashes andother menopausal symptoms were given a formulacontaining 900 mg of hesperidin, 300 mg of hesperidinmethylchalcone and 1200 mg of vitamin C daily. At theend of 1 month, symptoms of hot flashes were relieved in53% of the patients and reduced in 34%. Improvementswere also noted in nocturnal leg cramps, nose bleeds andeasy bruising. The only side effect noted was a slightlyoffensive body odour with a tendency for the perspirationto discolour the clothing.

Antiulcer activity. Hesperidin demonstrated no ulcero-

664 A. GARG ET AL.


genic effect in male rats at a dose of 100 mg/kg bysubcutaneous administration (Emim et al., 1994). How-ever, when given to rats orally at a dose of 100 mg/kg, thecompound exhibited a positive antiulcer effect on coldstress-induced ulcers but had no effect on ethanol-induced ulcers. No increase in gastric mucus wasobserved. It was found to stimulate hexosamine secretionin cold stress-induced ulcers but inhibited the same inethanol-induced ulcers (Suarez et al., 1996).

Effect on wound healing. Recently a micronizedflavonoid fraction, comprising of 90% diosmin and10% hesperidin, was tested for its effect on clean woundsand those infected with S. aureus on oral and topicaladministration. The study showed that while there was nosignificant effect on clean wounds, the combination had abeneficial effect on the infected wounds, when adminis-tered both orally and topically (Hasanoglu et al., 2001).

MARKETED THERAPEUTIC SYSTEMS

Hesperidin, though not yet very widely used therapeuti-cally, has been listed by the Martindale Extra Pharma-copoeia to be an ingredient of various formulationsinternationally used for vascular disorders (Angiopan—Gentili, Italy; Circovenil—Wyeth, Spain; Daflon—ther-apia, Germany; Varico Sanol Forte—Sanol, Germany),capillary fragility (Cepevit-K—Darcy, France), haemor-rhoids (Daflon 500—Servier, Switzerland; Hamamelis

complex—Blackmores, Australia), rheumatic and jointdisorders (Guaiacum complex—Blackmores, Australia;Ostochort—Adenylchemic, Germany), vitamin C defi-ciency and dietary supplement (HY-C—Solgar, USA;Min-Detox-C—Eagle, Australia), skin trauma (Prove-no—Madaus, Germany; Ondascora—Servier, France),obstetric disorders (Rubus complex—Blackmores, Aus-tralia), gingival inflammation (Peridin-C—Hamilton,Australia), fluid retention and gastrointestinal disorders(Hepanephrol—Rosa Phytopharma, France) (Reynolds,1996).

CONCLUSION

From the above review, it can be concluded thathesperidin, its aglycone hesperetin, and their variousderivatives possess significant potential as therapeuticagents for a wide range of diseases and disorders. Thispromise needs to be effectively studied at the clinicallevel, so as to firmly establish the usefulness of thesecompounds in the treatment or prevention of disease inhumans.

Acknowledgements

The authors wish to thank TOPCAD, Chicago, USA, for financing thisproject and for helping to further research in this area.

REFERENCES

,��-�� ./� &0102 3� �� 2 4��5,��2

,�� 6� 7�� *� �� 6�� 7� 6�� )2 &00�2 ,�� ++�� + )��88 �� +9��2 �� :�� &5�8;�!2

,�� 7� <�� ",� =�� =>� ?�� ",� "��++ "%2&00'2 @�� +�� 2 �� !�"� � �!#� �5 �!;!82

,�� *� 7�� "2 &0012 :�� + �� + )��88 ��2 �� $� � % ��& ��5&1�;&112

,��A�-� �� B�A��A� * &0'82 ,�� 9�� + �� C��2 �!#" �� %�#�&5 ��2

,�� %� 7�� ,� 7�� "��A :� :��D�E��,2 &0�'2 F�� 4�� + �� A��2 >�� .2'��! �#� � �!#��!� 5 &��;&��12

,�� "� <� <�� *� 6B2 &0�'2 ,� � �� + ��"�� + �� /��2 .��32 @�� +��(��! �)�*" �� + �� 2 � �!#" + � %�&5 '��;'��2

,�� ?� B�A�� D� 3�� *2 &0�82 @�� >�5 "�� + �� 2 � !�" + �� 5 �&�;��2

7�� 4,� �� *=� /�� )�2 &0002 �� + �� 2 ��#� �5 !!�;!!�2

7�� 4,� �� *=� %�� *� /�� )�2 �8882 �� ++�� + �� +��2 ,� � !�" ,*�� 5 &&��;&&�!2

7�� D,� =�� .:� *3�� 6�� *�� "%2 &0112"�� +�� C�� +�� + �� *��#��2 !)� �!#"��) �5 �!0;��!2

7�� =*� *�� D=2 &0!�2 3�� + ��.�� 2 � ,� � �!#" ��5 �8�;�&&2

7�� =*� *�� D=2 &0!12 3�� + �� + ��2 � ,� � �!#" ��5 �&;��2

7��A�� 7� /�� :�� D� 7��A� @2 &0012 3��++�� + �� A��2 �#� �� #� %,#��& ��5 0�;002

7�� B.� )�A-�A� "B2 &0�12 6�� + ��$ �#�%��2 � �� , � + � ��5 &18;&1�2

7�� BD� �� .=� �� .=� &00&2 3� �!# "#�� *��)- �#�� *��)2 6"6 .��5 7��"�� @%2

7G�� %@� 6�� D<2 &0��2 *�� +C�� 2 +��#��# ��5 ��;��12

7�� ,�� %� .�� =� )�� 62 &01'2 .�� 2 ,*�� .� *�# �)�!#� �� 5 ��;��02

7�A :�� %�� :�� .��A ?7 #� �- &0002 .�� + �� 6�, �� 6�,H 6�� +�� -�� +�� 6�� +6�� 2 � �*�� 5 &&1�;&&1�2

7�� @� %�� *� *�� % #� �- &00'2 @�� A��2 �� !�" ��5 1�;0!2

7�� @� :��G� ,� �� ,� %� 6�� "� )�� =62 &0012�� ++�� + � �� 2 �� " �� 5 ��&;�!�2

7�� ,B� =�� @�� )�4�� @2 &0�12 *�� +�� +�� 2 �,� � �!#" �5 ''&;''12

7��A�� 4� 6�� @I� %�� %2 &00�2 4++�� + �� + ��++�� + ��9�� +�� +��J��+�� A ��2 ,� � !�"� � �5 &'&&;&'&'2



7��A�� 4� )�� /,2 &00�2 4++�� + �� +��9�� +�� J��+�� 2 �� 5 �0�;�882

7A�� /� ��AA�� )� ,��-�� B2 &00�2 *�� 2�� .)�#� � /��#� ��5 &!�;&�&2

7�� :2 &00'2 3� �!#�#��0 ��#�2 *��A �� 6�2� 3�25 B=26�� *� .�� %� >��A ,� 7�� )>2 &00'2

3�� + �� 6�� 2 �� #� 5 ��;��'2

6�� .@2 &0002 ,�� + �� G�� 2 � �/�� 5 &0�;�8'2

6�� ,� @�� D� *�� @� 6�� *� %�� .�*�� 62 &0002 %�C�� J�� + �� +��2 � �!� "� $� ��5 �&�;��2

6�� *� 4�� %� *�� ,2 &0002 >�� + � ��C�� +�� + �� 2�� ""*� �� +�#�� " �5 ��00;�!8�2

6�� />� D�� >�� :�� 6@2 &01�2 :�� +�� +�� C��2 � �!� "� $� ��5 1&;0!2

6�� =%� 6�� :�� *�� 7�� 7� *�� "2&01&2 ,�� + &!6� �� 2 �*� � '�*$ �#�� !�"� �0��#� 5 &�&;&��2

6�� *6� .�� D2 &0��2 )�� ++�� +��K +��#��# ��5 ��!;��'2

6�� 3� �� 62 &00�2 .�� +�� + �)�� -�� D��2 �� #� 5 !'0;!�82

6�� =:� .��A /?� *�� :�� "�� :�� ?�� :2 &00!2,�� ++�� + �� :�� 2 ��! !�" �#� ��5 �&;��2

6�� =:� ?�A��-� �� $�� 2 &00&2 ,�� ++��+ �� +�� *�*� ��2 � �� 5 �&1;��!2

6�� 6,72 &0102 ��"�� - 6"6 .��5 7�� "��@�� &8&;&�12

6�� .2 &0112 , �� + �� 2��#� �#� ��5 ��!;��'82

6�� *4� D�� =� 6�� $�� *,� I�� ,2 &0002,�� + �� B7:2 �� #� �5 '�&;'��2

)�+�� :B� 3�� =:� �� ?2 &00�2 �� + �� +�� )��88�� 3F6)�� 2 '�*$� � �) �5 !&;!�2

)�� )=� 6��A�� 4=2 &01�2 ��+�� C�� + �! �� 2 � �!� "� $��5 �8�;�8�2

)�� 6�� *� .��A *,2 &00�2 :A�� + �� 2 � ! � �!#" ! � �� , ��5 &8�;&�!2

)�� *� @�� $� *�� @� %�� 6� 6�� .�� ,2&01�2 4++�� + �� -�� 9�� +�� 2 :�� +�� 4� �� @� �� 7�� 2 ��1�#�"��#�� ! �5&&!0;&&��2

)�� )� @�� 32 &00!2 @�� 2 ��#�� '#�"� � ��5 '�&;'��2

)�� <� @�� <3� < =2 &00�2 @�� +�� 5 �� K �� !)� �!#" ��5 ��&;��'2

)� *�� ,� @�� 7� .�� ,� "�� .� *�� 42&0002 "�� + �� +�� + � �� 2 � �$�� 2 � �!#" ��5 !�0&;!�0�2

)��-�� @� %�� D� .�� 2 &0182 L�� 7�� "�� B�� $��+�2 �!#" ��0� �� #�!� �#�#��" 5 &10;&0&2

4��7�� ,2 &0002 *��9�� +�� +�� + �� 2 �� #�� 5 �1�;!882

4�� =,):� $�� ,7� %�� ,=2 &00!2 .�� + �� + � 6�� C�� 2 � !�" !�"�� 5 &&1;&��2

4�� 3� *�� 4� ,�+�� D� ,�� ,2 �88&2 .�� A�� + �� +�� + �� G�� +�� G��2 � �*�� 5 ��;�!&2

4�� <6� &00'2 3� ��#�# �� 3 !�"� $� �)2 <2 72:��5 �� ,�� .�� $ +�� F/2

@�� @� 7��C�� *,� D�� *,� ��7�� @,� &00!2:�� + �� A�� 2 � �!� "� $� � �5 �'1;��!2

@�� =@2 &0�12 , �.%6 �� +�� C��9�� +�� G��2 � �$�� 2 � �!#" 5 &!�0;&!'82

@�A�� @,� �� )� &01'2 3� !�"� $��!�4�5�� ) � �!# ��# '�*$� � �#$#��# /��$��23�� 7��A )��5 )�� 3��H &8!;&8�2

@� )<� :�� <%� <�� :�2 &0��2 �� +�� 2 ��" �!#" + � ��5 ��8!;�!8�2

@�� 6D� *�� >:� @�� D4� D�� =)� 7�� ,2&01�2 @�� * �� 2 ,� �!#" !�"� � 5 �&�;��82

@��A� ,,� 6�� ">� 6�� %=� *�� %=� ��A� ?2&0012 3�� + �� +�� + �� 2 �� #� ,� ��5 ��;�!12

@��A�� 7� �� ,D� 3�� *2 &00�2 6�� +�� + )�� 88��2�� 5 &�;�&2

@��A�� 7� �� ,D� ,�� 6� 3�� *2 &00!2 $�� + ��9�� +�� A�� +��G��5 �� A�� +��2 �� 5 �8;��2

D�� 4*� *��+�� *�� /��G�� :� @�� ,*�� **2 &00!2 7�� ++�� + �� 6�� 2 .�� &2 ,�� 2 2�"� ��5 �80;�&�2

D�� 4*� �� ,� /��G�� :� @�� ,*� *��+��*�2 &00'2 7�� ++�� + �� 6��2 .�� 2 ,�� 2 2�"� ��5 �&0;��&2

D�� .2 &00�2 )��88 ��5 3�� + �� +�� 2'�*$� � �) �5��;'�2

D�� 7� D�� ==� )� 6�� *2 &0102 , �� -�� 5 "�� +9�� +�� + )��88 �� +9��2�� $� � �5 '�;�&2

�� %2 &0��2 3�� + ��2 ��*�# ��5 �1�;�1�2

�� =72 &00!2 3� �!# 2�� 4��#� ��#�#��! +��# �6782 6�� 5 %��H �!82

�� ,,� 4�� =%2 &0�12 6�� +�� 2�*�� #� ��5 ��&;��02

�� ,� ,�� 6� $�� :� /�� /� 4�� 42 �88&2 4+9�� +�� +�� + �� +�� -��2 �� $� � ��5 !&;!!2

�� "�2 &0!&2 �� + �� + ��.5 ,��-2 � �" !�" �� +�� 5 '�02

�� ?�� @�� 6%� :��-�6�� @2 &00�2 .�� + � �� +�� +�� 2 ,� � +*�$ �5&8�!;&8��2

��E �� %� <�� 2 &0'�2 6�� 2 '#*�� !#0#� (�$ ��5 ��0;�'�2

��-�� "*2 &0'&2 �!# /��$#- F�� + 6��+��.��5 7��A�� 6,2

��-�� "*� D�� 72 &0'�2 @�� + 6�� >32�#��!#�� 5 ��;�1�2

� 6L� 6�� /� :�� L� /��A�A�� "4� 6�� ?6� %�� /�2 &00!2,��,3): ��2 � �� 5 !�;�&2

3�� * *�� ,� *�� ?� ��A�A� B� @A�� *2

666 A. GARG ET AL.


&01�2 3�� + �� 2 �$�,� � �!#" ��5 �&��;�&�'2

3�� * F��G�� /� @G�� *� *�� *� *��A� :2 &01824++�� + �� A�� 2 �� $#� 9$0* 9��!� ��5 &�&;&��2

3�� * ?��A� ,� 3�� ?� /�� 6� *�� ,2&01!2 4++�� + �� +�� +�� 2 �$� ,� � �!#" ��5 &��0;&�'�2

3�� :/B� ,�� *2 &00�2 7�� + �� +�� (�#�� "��!� ��(��! �)�*" #�#�!�� 2 !)� �!#� �#� ��5 '!;''2

=�A�� 2 &0�02 ,��+�� 2 !�"� � �#� ��5&��;&��2

=�� 7�� *62 &00!2 *�� 5 4++�� + )��88�� 2 ��$� � $)��5 ��!;��02

=�� =� :�� ?D� /�� 3�� .�� =*2 &0002 3�� +�� 2 ��! !�" �#� 5�80;�&�2

=�� :>� :��A�� :� �� *� *��G�� 7� :�� *D2&00!2 @�� 2 � �" �!#" + � �� 5!1!';!1�&2

=�� 6%� *�A :"� ,�� ",� I�� %=)2 &01'2 4++��+ �� + �� 2 ,� � �#�� 5 ��';�!'2

=�� 6%� I�� %=)2 &01�2 >�� + �� 2 2#�� +�#�� 5 !�';!�12

=�� %2 &0'�2 :�� + �� 2 3��!# �!#"��) � 2�� "� *�� D�� , ��2 *��5 B�- ?��AH &8�;&��2

=�� :2 &0��2 @�� + ��#�� ,*�� 2 2�"��) %� �� :& 5 �!;�12

/�� ?�� :�� /2 &0�02 @�� + ��2 � !�" +�� #�!� � �� 5 �&8;�&�2

/�� /� �� 7� 7��-*� >��A 62 &00�2 .�� + �� + "�� 2 !)� �!#"��) �5 0'�;0�!2

/�� B� *�� 4=� $�� .%2 &01�2 ,�� ++�� +�� 2 � �#� �� 5 �&;�02

/�-�� *� �� :� :�� *,� �� *� @A�� :�3�� B2 &00�2 :�� + �� 2 � � #�� 5 ��;!!2

/�-�� /� /�A�� :� ��A�� /� ?��A�-� ��/��-� ?2 &0002 6�� +�� + ��2 �� #� �5 �'�;�''2

/�-�� /� *�� ,�� /� F�� /2 &00�2 �� + �� +�� #*� " ��2 ,� �� ,� �#�! ,� �!#" �5 &8�;&8!2

/�� 6=� 6�� :/2 &00&2 .�� + �� 33�M:�� + �� 2 ��! !�"� � �#� ��5 &!�;&�02

/�� 6=� 6�� =*2 &0082 .�� +�� 3�� "�� + �� +�� 2 ;0!0 5 # �!� �5 �!1;�'!2

/�� 6=� : �/� =�� =�� 6�� :/2 &0082 .�� + ��2 33� "�� + �� 2 ;0!0 5 # �!��5 !8�;!&!2

/�� )�� =�� 4,� :�� 3:� �� =,� /�� *=2 &00123�� + �� 2 ��! !�" �#��5 &�;��2

/�� )�� :�� *=� 7�� 4,� �� *=2 �8882 3�� ++�� +�� +��2 ,� � !�" ,*��5 ��';��12

/�� *� /�� $A�� :� :�� *2 &0002 .��+ �� 6�� -��2 ,� �� ,� �#�!� � ,� �!#" 5 �&1�;�&112

/�� @4� "�� ,2 &0�&2 B�� .�� 2 ��!#" � � %��&5 &�8!;&�802

/�AA�� 4� /�� 32 &0112 @�� + �� + �� *�# �#��2 !�" �� 5#�� 5 &�8;&��2

/�� B�� B�A�� A�� $A�� :2 &00'2:�� + ��

�� +�� +�� A�� ,��*� :��2 ,� �� ,� �#�! ,� �!#" �5'!�;'!02

/�� ?� B�� A�� $A�� :2 &00!2�� + �� +�� +�� A�� 7�� A�� + �� 2 ,� ��,� �#�! ,� �!#" ��5 &&08;&00!2

/�� "=� D�� )62 &0002 ,�� +��5 ,�� + �� 9�� +��2 � �� #� 5 &�;��2

/��A �� 7��A�� 7� 7��A� @ #� �- &0002 .�� ++��+ �� 6)�& �� A�� 2 ��#� �#� ��5 ��;��!&2

/��A� I� %��I��-�A� 42 &01!2 3�� + ��+�� ++�� + ��2 .�� &2 5#�� 5 ��;��2

/�� D2 &01'2 F�� ,A��E � �� I��+��2 ( ��<!�*�$�:�� 5 '�;'02

/�� *� *�� 2 &00��2 ,�� 2 �#�� 9 0� � 00) 9 ! '!�0�� !�12

/�� *� ?��-� �� :��A� �� /�� "2 &00��2 %�� +�� +�!�1 �#�#��#�*�� 2 �#�� 9 0� � 00) 9 ! 8! �81� ��&2

/�� >� 7��G�� :� *�� )� "�� I2 &0002 :�� + �� 6 33�� 2 !�"1�# ��5 �!12

/�� >� /�� *� *�� )� "�� I� *�� F2 &0012:�� + �� 33�� 8N ��2 � +#�� !#" + � 5�'�;��2

/�� F"� )�� B.2 &00�2 4++�� + �� ,*. �� 2 ,� �!#" !�"� � ��5 &�8�;&�&�2

%�� :�� =�� :� .��A ?7� /-�� ?/� 6�� *:� 7�A :�2&000�2 �� ++�� + �� + �� , �� ,5 ��+�� +�� 2 �*�� #��5 &�!�;&��12

%�� =�� /�� ?:� %�� 6/� %�� /� �� ::2 &000�2 ,�� + �� 29 �#� !�"� $ �5 �!;�02

%�� 7� %� )�� 6� )�� =6� $G�� )�� *�6�� .�� ,2 &00�2 4++�� + �� + �� 2 ,� �!#" !�"� � � 5 &��;&��&2

%�� 6� )O,�� D� )�� * #� �- &00'2 )�� + � �� + � �� 2 '�$ '�� +�� 5&�8!;&�&&2

%�� *� D�� 7� :�� B� 7�� B� .�� %�)�� =2 &0102 .�� ++�� + � ��9�� +�� 2 ��1�#�"��#�� ! �5 11�;11�2

*�� =� *��A�� /"� �� *72 &0�82 3� �!# ��+�#�� 2�� 2 :��>��5 B�- ?��A2

*�� )� "�� I� /�� /� /�� *2 &00�2 :�� + �� ,� 333�M�� -��M�� 2 ��#�� 5 0&�;0�'2

*�� /4� "�� /*2 �8882 3�� + &�� +�� 2 � �$�� 2 � �!#" ��5 ��';��182

*��A�� /"� �� 72 &0�'2 &�6 B*" �+ ��332�#��!#�� 5 �'8�;�'&�2

*�� D=� 7�� =*2 &0��2 4++�� + �� +�� 2 +��#��# ��5 !8�2

*�� ?�� *� /�� *� 3�� *� $�� *� *��*2 &00&2 .�� + 6�� +�� 33� ,�� ++�� + +�� + ��*� *��!�*2 ;0*$0* (��!��5 &0�;&012

*D�� %� 7�� *� 6�� 4� %�� %� "��6� *D�� =%2 &0002 4++�� + � ��9�� +�� +�� 2 �!� "��#� ��5 ��;�!82

*�� *@� %�� "� :�� D2 &00�2 4++�� + �� 2 !�"1�# �5 �0�;�0'2



*�� $62 &00!2 :�+�� + )��88 �� +9�� 2��$� � $) ��5 ��0;�1!2

*�� 4=2 &01!2 �� 2 ��#�� !�"� � +�� 5��;��12

*�� B=� "��4�� 6,2 &00�2 �� +�� + �� +�� G�� A�� A2 2 � �!#" �5 ��&;��2

*�� ?� ��A�� *�� #� �- �8882 4++�� +�� 2 �*� � !�"� � ��5 &0�;�8&2

*��A� ?� ?�� /� *�� ?� $��-� �2 &00�23�� + �� +�� + �� +��2� �$�� 2 � �!#" ��5 !'&0;!'��2

*��+�� *�� ,� /��G�� :� @�� ,*� %�6�� "72 &00�2 7�� ++�� + �� 6��2 .�� 2 �� 2 2�"� ��5 �0�;�002

*�� :2 &0�02 "�� +�� .2 � ,� �!#"�� 0) �5!1�;�8&2

*�� $� :��A� �� :�� :2 &00�2 6�� 2 �#�� 9 0� � 00) 9 ! �8! �!�� 1��

*�� .:2 &00�2 �� +�� 2��$� � $) ��5 1�;0&2

*�� .� D�� 4*� ,++�� ,2 &0012 :�� + �� G�� C�� 2 � �!� "� $� � ��5 &�&;&�02

*�� .� D�� 4*� 4�� =2 &00�2 6�� C�� + �� 6��2 ,�� +�� G��2 � �!� "� $� �5 &�0;&�!2

*�� 3� .�� 7*2 &01�2 3�� + �� + �� ,*. �� 2 ��#��#�� 5 0�8;0�&2

B�� D=� /��/�� =<2 &0��2 .�� +��%�� 2 �� #� �5 �0�;�8&2

B�� =%� D�� ,*2 &01'2 &� B*" �� '8*�� +�� )*:$ ��2 +�#�� #��5 !��;!�!2

$��+ )� "�� 4 &00�2 )�� + �� G�� 2 ��#�� )�� 5 &8��;&81&2

$�� /� B�A�� @A�� *� 6�� =6� 7��:�� @2 &0082 )�++�� ++�� + �� + �� )B, �� "B, ��2 �*� � ,� �!#" ��5!'0;!�'2

.��A <� <��A��A *� $��-�A� ,� %��A *2 &01023�� + �� +�� -�� 4*6 �� +��!)� � ��*� *�#*� �� 2�� 5 &1�;&112

.�-��-�A� %2 &0182 @�� + ,- �#��*� "�� ,- ��*� /�� 2 �� + � , � � ��5 �1&;�0'2

.��"�� *��%�� 6� �� >� @�� =2 &0002:�� + �� - ��G�� 2 2�#�#��*� � �� !#" �5 ��0;�1�2

.�� =4� *�� *�2 &0002 3� �#�� 0 � ��*��"#��#2 6�� %��5 4��H �02

.�� "/� ,��A�� :� 7�� =*� *�� =B� *�� D=2 &0��2�� + �� 2 �� #� +*�$ ��5 &;12

"�� I� *�� )� =��A��:��A�� *2 &00'2 :�� + �� I� 3>�� 2 !�"1�# ��5 '8!2

"�� ,/� )�� B.2 &0112 4++�� + �� 5 �� 2 ,� �!#" �#� �#�� ,� � �5 '0;�02

"�� =4@2 &00'2 3��#= �!# �� !�"� � #2�� "�� .�� 5 %��2

"�� "62 &0'�2 4++�� + �� + ��+�� 2 � ��!#� ��#��#� �5 �;&82

"�� "62 &0�&2 4++�� + ��

�� 5 7��+�� + �� + ��2 �� !#" ��5 !��;!��2

"�� :,� I��A� ):� I�� /� 4�� *,2 �8882 >�� +�� + ��+�� G��2 2�� #�� 5 &�!;&'&2

"��++ "%2 &0112 %�C�� +�� + ��+��G�� G��2 � �� !#" ��5 �01;18�2

"��++ "%� )��-�� D"� :-�� "*� B�� *� I�� F2&00�2 :�� .%6 �� +!�� +�� G��2 � �!� "� $� +�� 5 �1�;�1�2

:��G� ,� �� ,� �� )� D�� *� )� .��C�� ,�7�� @2 &0012 3�� + ��++�� A�� ++�� + ��2 �� !�" ��5 1�;0!2

:�� 2 &0!82 )�9�� + ��6 �� .�� 2 ��#� ��5 '!!;'!�2

:�� 7�� %2 &0!02 >��2 3� P��"�� 5 �" �#�Q� �� ":� �� /> ��2 ,��.��5 B�- ?��AH &;��2

:��A�� =�� :��A�� *�� I�G�� >�� @,2 &00�2 �� + �� -�� 2 '#�"� � $) ��5 �';�12

:�� :=� % 6@2 &00�2 )�� + �� +�� +�� C�� 2 � �!� "� $� � ��5 �&1;��2

:�� 7=2 &0��2 , ��- ��+�� +��2 +��#��# �� 5 ��;�1�2

:�� 6=2 &0'!2 B�� + �� 2 �!�� #� �5 &0�;&0�2

:�� :� /�� :� = =:2 &00&2 4++�� + �� A�� 2 5�$*0 � �$!: 50! # �!� �5 �&1;��'2

:��A� /2 &0�'2 �� + �� 2 �� !�"� � � �� 5 &�';&�'2

:��A�� ="� B�� ,B2 &00!2 @��5 , ��- �+�� +9�� + )��88�� -�� +9�� 2 ��$� � $ � ��5 !&0;!�12

:�� =� �� *)� *�� 42 &00'2 �� ++�� + �� 2 !)� �!#� �#� ��5'&';'&12

:�� =� �� *)� *�� 42 &0012 �� + �� 2 !)� "#��# �5 !'0;!��2

:��D�E�� ,2 &0�12 .�� + ��2 !)�� !#"�5 &�';&�&2

��A� �� *�A�� /�-�� /� *�� 2 &00��2 6�� + �� 2 �� $#�#�� 5 0��;0'�2

��A� �� *�A�� /�-�� /�*�� /�A��*2 &00��2*�� + B� ��B�� +�� + �� 2 �� $#�#�� 5 �'&;�'02

��A� �� *�A�� $�� *� �� ?2 &00!2 6�� + !��C��&�� 5 6��-�� ++�� + �� 2 ��#� �#� ��5!'��;!'�02

��A� �� *�A�� $�� * #� �- &00�2 6��+ !��C��&�� 2 ��#� �#� ��5 �!';��2

�� ?� :�� *� �� ?� 3�� /2 &01�2 ,�� + �� 2 �!#" !�" ,*�� 5 �11&;�11'2

>�� :)� /�� =�2 &0�'2 3�� + �� M�� + �� 2 ,� �!#" !�"� � �5��8�2

>��A�A�� D=� 4+�� 642 �8882 )�� + �� -��G��J��

668 A. GARG ET AL.


�� G�� 2 �� 5 ��;�1�2

<�A�� >,� 4�� D2 &0��2 >�� 2��*�:��#��!��#� 5 �8&2

<�A�� >,� 4�� D2 &0�12 ,�� + ��2 ��1�#�"�� ! �5 �!�;��82

<�� .@� I�� "%2 &00�2 3�� + �� +�� 2 �!#" !)� �� 5 ��;!82

<�� =I� 6�� )?� <�� =2 &00!2 )�� +�� #��*" �� "�� .%62�!*�$ 9* �!*�$ ; �� !�! ��5 !�!;!��2

<�� "�2 &0'!2 *�� + �� 2 3�P,� �!#"��) � !#� �� "� *��Q2 �� =7 ��2�2 ,�� .��5 B�- ?��AH �!8;�!&2

?�� ?� F�� =� $��-� /2 &00'2 , �� + �� P��Q �� +�� C�� 2 ;0*$0* (��!�� 5 ��';�1�2

?�� *� ��A� �� ?� )�� *�� /�-�� ?2

&00�2 6�� ++�� + �� !�� 36" ��2 �� 5 �&0;��!2

?�� 6� "�� I� I��G�� =� ?�� =� &0082 @�� 2 2�## �� ,� ��#� �5 &0;�&2

I�� @� @��6�� .� 3�� 3� 7�� =2 &01'2 B�-�� + �� 2 .�� 326�� 2 �� #� �� 2�" �5!0�;�8!2

I�� @� 3�� 3� 7�� =� @�� 6�� .2 &01�2 4++��+ �� 2 2�� #�� 5 �!�;�!�2

I�� <�� L�� D<� R ": #� �-� &0002 6�� +�� + ��! �� #��!*#�#��2 2�� #�� 5��0;��&2

I�� B4� 4+�� 64� &00'2 .�� + �� 2 ��!�" �� 5 ��;�'&2



Documents

Bioflavonoid Hesperidin[1]