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Free Radical Biology & M

Original Contribution

Therapeutic use of quercetin in the control of infection and anemia

associated with visceral leishmaniasis

Gargi Sena, Suparna Mandalb, Sudipa Saha Roya, Sibabrata Mukhopadhyayb, Tuli Biswasa,TaDepartment of Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India

bMedicinal Chemistry Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India

Received 24 June 2004; revised 29 October 2004; accepted 19 January 2005

Abstract

Flavonoids are a broad class of plant phenolics that are known to possess a well-established protective effect against membrane

lipoperoxidative damages. Oxidative damage of erythrocytes has been implicated in the reduced survival of erythrocytes during leishmanial

infection. This study reveals the efficacy of five naturally occurring flavonoids in arresting the development of anemia during the

postinfection period. Among the compounds studied, quercetin was most successful in inhibiting the oxidation of proteins and lipids on the

red cell membranes of infected animals. Apart from its antianemic property, quercetin also seemed to be highly potent in lowering the parasite

load in the spleen. Combination therapy of quercetin with the antileishmanial drug stibanate produced a better decay ofSOH in the

erythrocytes of the infected animals compared to that induced by quercetin or drug treatment alone. Similar results were obtained in

successful prevention of proteolytic degradation resulting in an aversion to early lysis of red cells after simultaneous treatment with quercetin

and stibanate. Subsequent studies demonstrated the therapeutic efficacy of the combination treatment in the abatement of both anemia and

parasitemia under the diseased condition.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Flavonoids; Visceral leishmaniasis; Erythrocyte; Oxidative damage; Anemia; Free radicals

Introduction

The prevalence of visceral leishmaniasis (VL) in the

Indian subcontinent has been reported to be increasing in

recent years. The clinical spectrum of this disease is so

extensive that it ranges from asymptomatic infection to

mortality. Leishmania donovani, the causative organism for

this disease, resides and proliferates within the hostile

environment of its host macrophages [1]. VL is associated

with severe anemia, which accounts for a formidable

volume of suffering in the diseased condition [2]. The

anemia is multifactorial, early hemolysis being one of the

0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.freeradbiomed.2005.01.014

T Corresponding author. Fax: +91 33 2473 0284.

E-mail addresses: tulibiswas@iicb.res.in, tulibiswas@hotmail.com

(T. Biswas).

important factors leading to the shortened life span of

erythrocytes. Red cells become prone to oxidative assault

during the infection period, which in turn perturbs the

cellular environment, inducing degradation of membrane

proteins [3]. In recent years, flavonoids have generated

considerable interest as potential agents against a variety of

diseases [4–6]. Enhanced production of oxygen radicals by

the leukocytes was observed in patients suffering from

Fanconi anemia (FA) and rheumatoid arthritis (RA). The

natural bioflavonoid rutin (vitamin P), which seems to act

both as a free radical scavenger and a chelator, inhibited

oxygen radical overproduction in both FA and RA in an

equally efficient manner [7,8]. Progression of diabetic

complications includes parameters like glycation and

oxidative stress. Coadjuvant therapy with a combination

of flavonoids like diosmin (90%) and hesperidin (10%)

induced a decrease in glycation, which was associated with

edicine 38 (2005) 1257–1264

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–12641258

an increase in the antioxidant component, dependent on the

levels and activities of thiol-containing proteins like

glutathione peroxidase [9]. The therapeutic efficacy of

the flavonoids indicates their usefulness as pharmaceutical

agents for the treatment of free radical pathologies. They are

polyphenolic compounds and are widely distributed in the

plant kingdom, the major sources being fruits, vegetables,

olive oil, red wine, tea, and the propolis of the beehive [4].

Flavonoids display a spectrum of clinical properties such as

anti-inflammatory, antiallergenic, antiviral, antibacterial,

and antihumoral activities [5,6]. Considering the antioxidant

potential, we investigated the ability of five naturally

occurring flavonoids, quercetin, rutin, 5-hydroxy 3,6,7,3V,4V-pentamethoxy flavone (flavone A), hesperidin, and diosmin

(Fig. 1), in the protection against the degradative process in

red cells, which in turn may serve in promoting the life span

of erythrocytes during VL. Pentavalent antimonials like

sodium stibogluconate and N-methyl glucamine antimonate

are the recommended first-line drugs against VL, but seem

not to be consistently effective [10,11]. Moreover, various

side effects have also been reported after prolonged use of

these antileishmanial drugs [10]. The recommended second-

line drugs like pentamidine, mepacrine, and amphotericin B

are even less acceptable due to their more pronounced

toxicities [12,13]. All these conventional drugs are incapa-

ble of enhancing the life span of the erythrocyte to a

considerable extent and as a result culminate in the

prolongation of anemic condition associated with the

disease [11,13]. In this paper we report the role of

flavonoids in combating both the infection and the anemia

associated with VL.

Fig. 1. Chemical structure of the flavonoids.

Materials and methods

Materials

Sodium stibogluconate (stibanate) was procured from

Gluconate Private Ltd. Radioactive sodium chromate (sp act

94.2 Ci/g) was purchased from Bhaba Atomic Research

Centre (Mumbai, India). Unless indicated all the chemicals

were of analytical grade and purchased from Sigma–Aldrich

Chemical Co.

Animals

Syrian golden hamsters (3–4 weeks of age), weighing

about 25–30 g, were used in this study. The hamsters were

infected with L. donovani (amastigotes), strain MHOM/IN/

1983/AG83, obtained from hamsters in which the strain

was maintained by intracardial passage every 6 weeks.

Extraction and administration of flavonoids to hamsters

infected with L. donovani

Flavone A was isolated from the leaves of Vitex

negundo [14]. Diosmin and Hesperidin were isolated

Citrus sinensis [15]. Rutin was isolated from Fagopyrum

esculentum [14,15]. The structures of the compounds

were ascertained by spectroscopic analysis, superimpos-

able infrared spectra, and undepressed mixed melting

point in comparison with authentic samples. Quercetin

was procured from Sigma–Aldrich Chemical Co.

The drugs were administered to 1-month-infected

animals at the dose of 5–40 mg/kg body wt after being

dissolved in a minimum quantity of ethanol (0.1% v/v) and

then diluted with double-distilled water. Drugs were

administered orally biweekly and animals were sacrificed

after 1 month of drug treatment. Sodium stibogluconate

was injected intraperitoneally at the dose of 20 mg/kg body

wt for 5 consecutive days followed by an interval of 15

days and then the treatment was repeated again for 5 more

days.

Parasite load of spleen

Parasite burden of spleen was assessed microscopically

from Giemsa-stained impression smears on slides after

fixing in methanol. At least 800 nucleated spleen cells

were examined for each set. Results are expressed as the

total parasite load per organ, using the following formula

[16]:

Organ weight ðmgÞ� the number of amastigotes per cell nucleus� 2�105:

Analytical determinations

Preparation of erythrocyte membrane

Blood was collected from control and infected animals

using heparin as anticoagulant. Plasma and the buffy coats

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–1264 1259

were separated from erythrocytes by aspiration. Packed red

blood cells were then washed thrice with isotonic PBS (pH

7.4). Washed erythrocytes were hemolyzed in hypotonic

lysing buffer and centrifuged at 27,000g for 30 min at 0–

48C. The process was repeated until the red cell

membranes thus obtained were almost free from hemoglo-

bin (Hb) [17].

Measurement of lipid peroxidation and protein carbonyl

content

Lipid peroxidation in the erythrocyte membrane was

measured by thiobarbituric acid (TBA) test [18]. Malon-

dialdehyde (MDA) formed from the breakdown of poly-

unsaturated fatty acid was considered an index of

peroxidation reaction. The absorbance of the reaction

product of MDA with thiobarbituric acid was measured at

532 nm. Quantitation was based upon the molar extinction

coefficient of 1.56 � 105 M�1 cm�1. Protein carbonyl was

measured as a marker of protein oxidation using 2,4-

dinitrophenylhydrazine according to the method of Levine

et al. [19]. The carbonyl content was calculated from

absorbance at 365 nm, using a molar extinction coefficient

of 22,000 M�1 cm�1.

Measurement of hydroxyl radical (SOH)

SOH radicals in erythrocytes were estimated by

salicylate trapping of the radical using an HPLC system

with electrochemical detection. The generation ofSOH

radicals was monitored by measuring hydroxyl adducts

of sodium salicylate such as 2,3- and 2,5-dihydroxyben-

zoic acid [20]. The biogenic amines were separated on

an Ultrasphere ion pair, C18 reverse-phase analytical

column (5 Am, 4.6 � 250 mm; Beckman Instruments,

Fullerton, CA, USA). The electrodetection was per-

formed at +0.74 V, employing an electrochemical

detector (Merck–Hitachi, Germany). The mobile phase

contained 8.65 mM heptane sulfonic acid, 0.27 mM

EDTA, 13% acetonitrile, 0.45% triethylamine, and 0.20%

phosphoric acid (v/v).

Total reactive antioxidant potential (TRAP)

Antioxidant potential of plasma was estimated from

TRAP [21]. Lipid peroxidation was first induced with ferric

Table 1

Effects of flavonoids on the development of anemia in hamsters infected with L.

Dose of drug (mg/kg body wt) Hemoglobin level (g/dl)

Control Infected Inf. + hesperidin

— 16.4 + 1.2 9.0 + 0.95 —

5 — — 10.8 F 0.72

10 — — 11.0 F 0.64

20 — — 11.4 F 0.78

30 — — 11.8 F 0.82

40 — — 11.8 F 0.90

Details of the course of drug treatment are given under Materials and methods. Re

values obtained after 2 months of infection. Nine animals were taken in each grou

chloride in the presence and absence of plasma. TRAP was

then calculated by using the formula

TRAP ð%Þ ¼ 100 � 1 � Ap tð Þ � Ap t0ð Þ=A tð Þ � A t0ð Þ� �

;

where A is the mean absorbance at 532 nm in the absence of

plasma, Ap is the mean absorbance at 532 nm in the presence

of plasma, t is the incubation time, and t0 is the initial time.

Analysis of physiochemical properties of red cells

Hemoglobin content and the total erythrocyte count were

taken at various time intervals during the progress of

infection using standard methods [3].

Red cell survival was estimated by enumerating the red

cell half-life over time. Labeled sodium chromate 51Cr was

injected intracardially into hamsters at a dose of 11 ACi/kgbody wt. The radioactive count of the red cells was taken at 7-

day intervals until half of the radioactivity injected had

disappeared from the circulation. The count on day 0 was

taken as 100% radioactivity. The day at which 50% radio-

activity disappeared was termed t1/2. Final results were

expressed as t1/2 in days [22].

The osmotic fragility of erythrocytes was determined by

measuring their hemolysis spectrophotometrically at 640 nm

in hypotonic saline. The extent of hemolysis of the cells in

saline was assessed in comparison to the lysis of the same

volume of red cells in distilled water [23].

Analysis of membrane proteins

The protein profile of the erythrocyte membrane was

determined by sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS–PAGE) using 4 and 10% acrylamide

as the stacking and running gels, respectively, according to

the method of Laemmli [24]. The gels were stained with

Coomassie Brilliant Blue R-250 and scanned in a laser

densitometer.

Statistical analysis

Conventional methods were used for calculation of

means and standard deviations. Comparisons between the

groups were performed using an unpaired Student t test.

donovani

Inf. + diosmin Inf. + flavone A Inf. + rutin Inf. + quercetin

— — — —

9.8 F 0.64 11.0 F 0.70 9.2 F 0.84 12.5 F 0.88

10.2 F 0.72 11.8 F 0.78 9.2 F 0.82 14.4 F 0.70

10.8 F 0.85 12.5 F 0.74 9.8 F 0.90 14.4 F 0.80

11.0 F 0.90 12.5 F 0.80 9.8 F 0.98 14.5 F 0.90

11.0 F 0.88 12.6 F 0.78 9.9 F 1.0 14.5 F 1.00

sults in the infected groups (with and without drug treatment) represent the

p and the data shown are the means F SD of four independent experiments.

Fig. 3. Protective effects of flavonoids on the decrease of TRAP in hamsters

infected with L. donovani. (Column A) Control (noninfected), (B) infected

(without drug treatment). Infected groups were treated with (column C)

hesperidin and (D) diosmin at the dose 30 mg/kg body wt each. Other

infected groups were given (E) flavone A (20 mg/kg body wt), (F) rutin (40

mg/kg body wt), and (G) quercetin (10 mg/kg body wt). Results shown are

means F SD of four separate determinations. Eight animals were taken in

each group under observation.

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–12641260

Results

Effects of flavonoids on the development of anemia

To determine the effects of flavonoids on the develop-

ment of anemia in hamsters infected with L. donovani,

animals were treated with different doses of the flavo-

noids as described under Materials and methods. The

flavonoids were effective toward the restoration of

decreased Hb level in the infected animals, although to

different extents (Table 1). Significant recovery was noted

after treatment with 10, 20, 30, 30, and 40 mg/kg body

wt of quercetin, flavone A, hesperidin, diosmin, and

rutin, respectively, which were used subsequently for in

vivo treatments.

Therapeutic effects of flavonoids on the oxidative damage

and antioxidant potential of erythrocytes

The extent of lipid peroxidation in the cells of control

and infected hamsters after treatment with flavonoids was

determined through estimation of TBA-reactive substances.

Among the compounds studied, quercetin was most

Fig. 2. Inhibitory effects of flavonoids on the oxidation of (a) membrane

lipids and (b) membrane proteins in the erythrocytes of hamsters infected

with L. donovani. Columns A and B represent oxidation in the control and

infected group, respectively, without drug treatment. Infected animals were

treated with (column C) hesperidin (30 mg/kg body wt), (D) diosmin (30

mg/kg body wt), (E) flavone A (20 mg/kg body wt), (F) rutin (40 mg/kg

body wt), and (G) quercetin (10 mg/kg body wt). Details of the course of

treatment are given under Materials and methods. Nine animals were taken

in each group and the results shown are the means F SD of four separate

determinations.

successful in rectifying the altered parameter, almost to

the normal level (Fig. 2a). The ability of the flavonoids to

inhibit protein oxidation in erythrocytes correlated closely

with their action on lipid peroxidation. Quercetin was

equally potent in suppressing the increased protein carbonyl

content in the infected animals, as evident from Fig. 2b. The

antioxidant capacity of erythrocytes was assessed by

measuring the TRAP of the cells in the presence and

absence of plasma. Fig. 3 reveals the reversal of the

decreased level of TRAP in the red cells of the infected

animals after flavonoid treatment, quercetin being slightly

more efficient in comparison to the others in this process.

Antianemic and antileishmanial properties of flavonoids

As a consequence of the oxidative stress during

leishmanial infection, Hb level and the half-life of eryth-

rocytes drop down significantly from control levels after 3

months of infection, emphasizing the anemic status of the

animals under the diseased condition. Anemia could be

checked by treating the hamsters with flavonoids as shown

in Table 2. Here also, quercetin excelled as the most

effective drug compared to others in the rectification of

anemia during VL. However, treatment with flavonoids

other than quercetin and flavone A did not have much

impact on the parasite load of the spleen. Among the two

drugs showing antileishmanial activities (Table 2), quercetin

seemed to be more potent in reducing the degree of

parasitemia in the spleens of the infected group.

Effects of combination treatment with quercetin and

stibanate on the L. donovani-infected hamsters

Sodium stibogluconate (SAG) is recommended as a

first-line antileishmanial drug, but is often associated with

inefficacy to some extent. Considering the antileishmanial

Table 2

Antianemic and antileishmanial properties of flavonoids

Group Parasite load

(No. of

parasites �108/spleen)

Hb (g/dl) Erythrocyte

life span

(51Cr t1/2in days)

Control — 16.4 F 1.2 22 F 1.60

Infected 8.6 F 0.89 9.0 F 0.95 8 F 0.55

Infected + quercetin

(10 mg/kg body wt)

2.0 F 0.11* 14.4 F 0.72* 18 F 1.02*

Infected + hesperidin

(30 mg/kg body wt)

8.5 F 0.85 11.8 F 0.82** 14 F 0.93**

Infected + diosmin

(30 mg/kg body wt)

8.3 F 0.93 11.0 F 0.90** 13 F 1.12**

Infected + flavone A

(20 mg/kg body wt)

5.6 F 0.92** 12.5 F 0.74** 16 F 1.37*

Infected + rutin

(40 mg/kg body wt)

8.6 F 0.90 9.5 F 0.62 9 F 0.78

Eight animals were taken in each group under observation and values are

means F SD of four separate determinations. Drug treatment schedule was

as described under Materials and methods. Results are from one time course

after 2 months of infection (with and without drug treatment).

*p b 0.02 and **p b 0.05 compared to infected group (without drug

treatment).

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–1264 1261

property of quercetin, we attempted to use the combination

of SAG with quercetin in our subsequent experiments.

Production ofSOH in erythrocytes

Our previous studies indicatedSOH to be the most potent

oxidant in the erythrocytes during leishmanial infection. It is

evident from Fig. 4 that SAG treatment alone led to about

23.7% ( p b 0.01) reduction ofSOH formation in the red

cells of infected hamsters compared to the untreated infected

group. Treatment of the animals with quercetin, which is a

potent antioxidant, resulted in about a 47.4% ( p b 0.01)

reduction in reactive oxygen species (ROS) formation.

However, a combined treatment of SAG with quercetin

produced better reduction, 57.9% ( p b 0.005), ofSOH

production in the erythrocytes of the infected animals.

Fig. 4. Inhibition of ROS production in the erythrocytes of L. donovani-

infected hamsters after treatment with quercetin and SAG. (Column A)

Control (noninfected), (B) infected (without drug treatment). Infected

groups were treated with (column C) quercetin, (D) SAG, and (E) a

combination of quercetin and SAG. Treatment schedule is given under

Materials and methods. Percentage reductions in O2S� production in the

drug-treated groups with respect to column B are shown in parentheses.

Six animals were taken in each group and the results are means F SD of

four separate experiments.

Protein profile of erythrocyte membrane

Oxidative attack on the cells modifies the proteins and

enhances their degradation by intracellular proteolytic

systems. Table 3 elucidates the accentuated degradation of

both band 3 and band 4.1 in the erythrocyte membrane of

infected animals, which was partially rectified in the SAG-

or quercetin-treated groups. Simultaneous treatment with

both drugs induced significantly higher reduction in the

proteolytic degradation compared to that observed with

either of the drug treatment groups alone.

Osmotic fragility and life span of red cells

Experiments were also carried out to determine the

effects of altered structural integrity on the mechanical

stability and survival of erythrocytes during leishmanial

infection. Fig. 5a shows marked decay in the life span of red

cells after 3 months of infection. Combination therapy with

SAG and quercetin was highly effective in prolonging their

survival and eventually prevented the early hemolysis

observed in the infected group of animals.

Destabilization of the cell membrane induced ionic

imbalance, making the erythrocytes of the infected animals

highly fragile (Fig. 5b). The figure further depicts the

therapeutic efficacy of the combined treatment in decreasing

the osmotic fragility of these cells and their susceptibility to

early destruction during leishmanial infection.

Parasite load of spleen and Hb level in the red cells

Infected hamsters were treated with the drugs and their

splenic parasite load was estimated subsequently. Whereas

administration of quercetin resulted in about 77% ( p b 0.01)

reduction, treatment with SAG resulted in an 82% ( p b

0.01) reduction in the parasite burden of the infected

animals compared to the untreated controls. Fig. 6 shows

93% ( p b 0.001) reduction of parasitemia after simulta-

neous treatment with both drugs (Fig. 6a). Significant

recovery from the anemic status was observed in the

infected group after treatment with quercetin and SAG.

Combination treatment with both induced higher enhance-

ment of Hb level than either of the drugs alone (Fig. 6b).

Table 3

Degradation of bands 3 and 4.1 in the erythrocyte membrane of

L. donovani-infected hamsters after treatment with quercetin and SAG

Group Intensity (%) of band protein remaining

after degradation with respect to control

Band 3 protein Band 4.1 protein

Infected 35.0 F 4.2 40.0 F 5.28

Infected + SAG 50.9 F 6.06** 59.0 F 5.43**

Infected + quercetin 70.2 F 5.8* 72.6 F 7.7*

Infected + SAG + quercetin 88.9 F 6.2* 94.0 F 5.6*

The intensity of band proteins on the SDS–PAGE was determined from

densitometric scan, and their intensities (%) remaining after degradation

were calculated taking the control level as 100%. Values shown are

representative results of four independent experiments.

*p b 0.01 and **p b 0.05 in comparison to the infected group (without drug

treatment).

Fig. 5. Effects of treatment with quercetin and SAG on the (a) survival and

(b) osmotic fragility of erythrocytes in hamsters infected with L. donovani.

Results are means F SD of four independent experiments and six animals

were taken in each group. (Column A) Control (noninfected), (B) infected

(without drug treatment), (C) infected + quercetin, (D) infected + SAG, and

(E) infected + quercetin + SAG. Life span was measured using 51Cr and

osmotic fragility was assessed from the lysis in 0.45% (w/v) NaCl as

denoted under Materials and methods. *p b 0.01, **p b 0.02, and ***p b

0.05 compared to (B).Fig. 6. Therapeutic effects of treatment with quercetin, SAG, and a

combination of quercetin + SAG on the (a) splenic parasite load and (b) Hb

level in erythrocytes of hamsters infected with L. donovani. Values obtained

in the control and experimental groups of animals (six in each group) are

presented as means F SD of four independent experiments. Percentage

reductions in parasite load in the drug-treated groups with respect to

infected group (without drug treatment) are given in parentheses. *p b 0.01

and **p b 0.02 compared to infected group (without drug treatment).

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–12641262

Serum albumin level

Development of hypoalbuminia was detected in the

infected group of hamsters (Fig. 7). Treatment with SAG

showed a tendency for the reversion of the serum albumin

level toward the normal range. Combination therapy of this

drug with quercetin showed an additive effect, resulting in

the complete retrieval of the hypoalbuminia to the control

level.

Fig. 7. Correction of hypoalbuminia after treatment with quercetin and SAG

in hamsters during leishmanial infection. Serum albumin levels in the

control (noninfected) and infected (without drug treatment) groups are

shown in columns A and B, respectively. Drug treatment groups are shown

as (column C) infection + quercetin, (D) infected + SAG, and (E) infected +

quercetin + SAG. Six animals were taken in each group and values are

means F SD of four separate experiments. *p b 0.01 and **p b 0.02 in

comparison to infected group (without drug treatment).

Discussion

Most of the beneficial health effects of flavonoids are

attributed to their antioxidant and chelating activities [25–

27]. Oxidative damage of erythrocytes has been reported to

be a possible mechanism for premature hemolysis in

experimental visceral leishmaniasis in hamsters [3,28].

Oxidation of unsaturated fatty acids leads to the formation

and propagation of lipid radicals, uptake of oxygen,

rearrangement of double bonds, and eventual destruction of

erythrocyte membrane lipids.

The antioxidant capacity of the flavonoids examined in

this study varied considerably from one kind to another.

Quercetin exhibited the highest potency in inhibiting lipid

G. Sen et al. / Free Radical Biology & Medicine 38 (2005) 1257–1264 1263

peroxidation and protein oxidation and had an edge over the

others in activating the decreased antioxidant potential of

erythrocytes during leishmanial infection (Table 1). The

antioxidant role of flavonoids contributes to the pathopre-

vention by virtue of their ability to donate electrons or

hydrogen from hydroxyl groups to free radicals [29]. Similar

behavior of quercetin in rectifying the anemic response of

VL (Table 2) can be explained by the contribution of an

oxidative threat on the reduced survival of erythrocytes

during the disease.

To be specific, Fenton-induced oxidation is strongly

inhibited by flavonoids with 3V,4V-catecol, 4-oxo, and 5-OH

arrangements. Chelating complexes with divalent cations

may form between the 5-OH and the 4-oxo group or between

3V- and 4V-OH [30–32]. Transition metal complexes of

bioflavonoid rutin, Fe(rut)Cl3 and Cu(rut)Cl2, not only

retained the antioxidant properties of rutin, but also exhibited

enhanced free radical scavenging activity. This may be

achieved by the acquisition of an additional superoxide

dismuting center in the flavonoid molecule after complex-

ation with transition metal without the formation of new

covalent bonds [33]. The superiority of quercetin in

inhibiting both oxidative damage and premature destruction

of erythrocytes can be ascribed to its 3-OH and greater

number of hydroxyl groups than was observed in the rest of

the flavonoids studied. This enables quercetin to offer

considerable benefit over the others as an inhibitor of the

Fenton reaction in vivo by virtue of both metal-chelating

properties and radical-scavenging ability [34,35]. In compar-

ison to rutin, quercetin seems to be more hydrophobic in

nature, which resulted in its greater concentration in the cell

membrane, thereby exerting greater antiperoxidative effect

over the former [36]. On the other hand, decreased

antioxidant properties of flavone A, diosmin, and hesperidin

may be related to the increase of glycosylation and O-

methylation, which substantially reduces their activity

against ROS [29].

Quercetin has been found to inhibit the growth of

leukemic cells and ascites tumor cells [6]. Quercetin was

also shown to potentiate the cytotoxicity of DNA-damaging

anti-cancer drugs, like cisplatin [6]. The efficacy of quercetin

in suppressing the parasite load is in good accordance with

the results of Mitra et al., who further described the

interaction between the flavonoid and the DNA topoiso-

merases, promoting site-specific DNA cleavage resulting in

the growth inhibition of L. donovani promastigotes and

amastigotes [36,37]. Thus, in addition to being an antiox-

idant agent, quercetin seems to possess the potential to be

used toward the control of leishmanial infection. This

prompted us to evaluate the function of quercetin in

combination with SAG in the control of both anemia and

infection in hamsters infected with L. donovani (Table 2).

In cellular oxidation reactions, O2S� is usually found at the

initial stage and subsequently produces other kinds of cell-

damaging free radicals and oxidizing agents among which

the damaging action ofSOH seems to be the strongest [38].

ROS are implicated in the etiology of the development of

anemia associated with the progress of infection in VL [39].

Radical-mediated protein oxidation (Fig. 3) may contribute

to the progressive membrane degradation (Fig. 4) and

destabilization, which eventually alter membrane perme-

ability, resulting in osmotic damage (Fig. 5) with consequent

lysis of the affected red cells. The combined treatment with

quercetin and SAG yielded better results than the treatment

with either of the drugs alone, thereby suggesting an additive

effect of the combination therapy. Recently, quercetin has

been reported to interact with human serum albumin, which

seems to be the principal carrier protein for this flavonoid in

human blood plasma [40,41]. Interestingly, SAG treatment

showed a tendency toward the rectification of hypoalbuminia

(Fig. 6) observed during the leishmanial infection and may

play a promotive role toward the outcome of the simulta-

neous treatment with quercetin. Taken together, our result

prompts us to suggest the protein–flavonoid interaction in

favor of the better efficacy of the combination treatment.

Quercetin is an important member of the flavonoid family

and its high intake in our daily diet is considered to be

advantageous for such treatment. Finally the data presented

in this study suggest the combination therapy utilizing

quercetin to be a strong candidate against the infection and

the anemia associated with VL.

Acknowledgments

This work was supported by grants from the Indian

Council of Medical Research, the Government of India, and

the Department of Science and Technology, Government of

India. We thank Mr. Goutam Chandra of the Neurobiology

Division at the Indian Institute of Chemical Biology, for his

help in the measurement of hydroxyl radical using the

HPLC system.

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