Indian Journal of Experimental Biology Vol. 43, January 2005, pp. 61-67

Antimutagenic and antioxidant/prooxidant activity of quercetin

Thiraviam Geetha1, Vibha Malhotra 1

, Kanwaljit Chopra2 & Indu Pal Kaur1* 'Department of Pharmaceutics, 2Department of Pharmacology, University Institute of Pharmaceutical Sciences, Pan jab

University, Chandigarh 160014, India

Received 28 July 2004; revised 8 October 2004

The present study has been performed to evaluate the antimutagenic activity of quercetin, ascorbic acid and their combination against an oxidative mutagen. An effort was also made to correlate this activity to the in vitro antioxidant activity of these agents. Antimutagenicity testing was done in Ames Salmonella Assay system using Salmonella typhimurium TA102 against t-butylhydroperoxide as an oxidative mutagen. In vitro antioxidant scavenging activity was tested for DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radical in their specific test systems. Quercetin (0.5-8 nmole/plate) and ascorbic acid (0.1-100 ttmole/plate) showed significant effect. Quercetin (4 and 8 nmole/plate) when combined with ascorbic acid (500 nmole/plate) showed an increase in the antimutagenic activity. In vitro antioxidant activity of quercetin was better than ascorbic acid in all the test systems used. The study indicated that the antimutagenic activity of quercetin was not solely accountable by its antioxidant nature. However, in vitro free radical scavenging activity of quercetin correlated well with the antimutagenic activity.

Keywords: Anti mutagenicity, Antioxidant activity, Ascorbic acid, Ames test, Quercetin.

Molecular oxygen, besides being a terminal oxidant indispensable for the production of metabolic energy can also yield (5% of total oxygen) reactive oxygen species (ROS) which include superoxide radical, hydrogen peroxide and hydroxyl free radical, all of which have one or more unpaired electrons that potentially cause damage to the respiring cells. All these reactive species are highly toxic, mutagenic and reactive'. They can nick DNA, damage essential enzymes and proteins or provoke uncontrolled lipid peroxidation and auto-oxidation reactions.

Quercetin belongs to an extensive class of polyphenolic flavonoid compounds with many beneficial effects that appear to be due to its antioxidant activity. It scavenges oxygen radicals, inhibits xanthine oxidase, protects against lipid peroxidation in vitro, chelates metal ions and forms inert complexes, that cannot take part in the conversion of superoxide radicals and hydrogen peroxide into hydroxyl radicals2

-5

• However, the properties underlying the antioxidant activity of quercetin may also participate in prooxidant reactions. It has been reported that auto-oxidation of quercetin, yields hydroxyl radicals within the cell, which may be

*Correspondent author: Phone no: 91 172 2534107; 2541 142 Fax:9ll72254ll42 e-mail :indupalkaur@yahoo.com

responsible for its cytotoxic, mutagenic and/or biocidal effects6

.

In this study, quercetin was examined alone and in combination with ascorbic acid for its antimutagenic activity against direct acting mutagen (t­butylhydroperoxide) in TA102 strain of Salmonella typhimurium using Ames Salmonella Assay system. T A 102 is a strain especially designed for the detection of oxidative mutagens/antimutagens7

. t-Butyl hydroperoxide is used as the oxidative mutagen for inducing mutations. This test system has been described as suitable for evaluating and establishing the antioxidants as antimutagenic agents8

. Ascorbic acid has been reported to possess flavonoid-protective and flavonoid-enhancing activities probably due to reduction of oxidized flavonoid and regeneration of the flavanols9

. Both quercetin and ascorbic acid are well established antioxidants (the relative antioxidant activity was assessed by us in different test systems) with ascorbic acid acting as a radical scavenger while the proposed mechanism of action for quercetin is metal ion chelation and free radical scavenging5

.

Ascorbic acid can enhance the ability of quercetin to rescue cells from the damage induced by oxidative stress, by acting as a free radical acceptor for the prooxidant (semiquinone) form of quercetin. In vitro scavenging of free radicals was done in different test systems to corroborate the postulation that quercetin

62 INDIAN J EXP BIOL, JANUARY 2005

and ascorbic acid are antimutagenic because of their capacity to scavenge ROS.

Materials and Methods Microorganism--TA102 strain of Salmonella

typhimurium was originally obtained as a free gift from Dr. Bruce N. Ames (University of California, Berkeley, USA). Presently, it is being maintained in our own laboratory at the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India.

Chemicals--t-Butylhydroperoxide (70% v/v solution in water) was purchased from Merck­Schuchardt, Hohenbrunn, Germany; hydrogen peroxide was purchased from Qualikems Fine Chemicals Pvt. Ltd., New Delhi; 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co., St. Louis., MO, USA; quercetin and ascorbic acid were from S.D. Fine Chemicals Pvt. Ltd., Boisar, India; agar (extra pure) was pu!chased from Hi Media Laboratories Pvt. Ltd., Mumbai, India and Oxoid nutrient broth No. 2 was purchased from Oxoid Ltd., Basingstoke, Hampshire, England. All other reagents used were of AR grade.

Antimutagenicity test-The plate incorporation procedure as reported by Maron and Ames (1983) was suitably modified 10 for antimutagenicity testing (with the inclusion of pre-incubation step). All the antimutagenicity mutation assays were carried out in duplicate/triplicate and on at least two separate occasions. Results are expressed as mean± SO of his+ revertants per plate for each dose. Negative and positive controls were included in each assay. t­butylhydroperoxide (t-BOOH; 100 ~g/plate in distilled water) was used as the mutagen. Quercetin (0.5-8 nmole/plate in DMSO) and ascorbic acid (0.1-100 JLmole/plate in water) were used as antimutagens. The antimutagenic activity is expressed as % inhibition of mutagenicity vs. dose and the % inhibition of mutagenicity was calculated as:

[

His+ revertants induced/plate by the mutagen] in the presence of Antioxidant

00 1- xl His+ revertants induced/ plate by the mutagen alone

DPPH assay--DPPH is a stable free radical which can be used to determine the antioxidant activities of various compounds 11

• In this assay, 500 J.ll aliquots of

varying concentrations of test compound dissolved in methanol were added to 2 ml ethanolic solution of DPPH (100 J.!M). The reduction in the absorbance at 515 nm was determined spectrophotometrically after 20 minutes of incubation. Degree of DPPH radical scavenging activity was expressed as % inhibition= [(Acontror-Asample)IAcontrot]xlOO; where Acontrol = absorbance of DPPH alone and Asampie = absorbance of DPPH with different dilutions of drug.

Superoxide anion scavenging-Superoxide anion scavenging assay was done by two methods, hydroxylamine auto-oxidation and riboflavin photo­oxidation. When hydroxylamine undergoes auto­oxidation at a pH 10.2, it causes the production of superoxide anions, which are determined by the reduction of nitroblue tetrazolium dye 12

• Quercetin did not show superoxide anion scavenging rather a pro-oxidant activity was indicated. Hence, the superoxide anion scavenging was determined by the riboflavin photo-oxidation method also. In case of riboflavin photo-oxidation method, the photo excitation of riboflavin leads to the generation of riboflavin radical which then auto-oxidizes and generates superoxide anions 13

•

Hydrogen peroxide scavenging-Hydrogen peroxide scavenging was determjned by adding hydrogen peroxide solution (50 JLM) to different concentrations of drug solutions and the reduction in absorbance at 240 nm was measured after subtracting any inherent signal associated with the drug 14

•

Hydroxyl radical scavenging--P-nitrosodimethyl aniline bleaching method was used to determine the hydroxyl radical scavenging15

.

Statistical analysis-All the data obtained were statistically analyzed by one way ANOV A followed by Tukey's test using a statistical package SIGMASTAT. In the test, the criterion for statistical significance wasP< 0.05.

Results Effect of quercetin on t-BOOH induced

mutagenicity--Quercetin, in a dose range of 0.5-8 nmole/plate was tested for its antimutagenic effects against 100 J.lg/plate t-BOOH. With an increase in the dose of quercetin, there was an increase in the % inhibition of mutagenicity, with the highest activity of 47.5% being expressed at 2 nmole/plate (Fig. 1). At 4 and 8 nmole/plate, there was a decline in % inhibition of mutagenicity even though the inhibition was still significant with respect to the control. There is a

GEETHA et al.: ANTIMUTAGENIC AND ANTIOXIDANT/PROOXIDANT ACTIVITY OF QUERCETIN 63

statistically significant difference with respect to control when the data is analyzed by One Way ANOVA at a P value of 0.001. Apparently at the dose levels of 0.5-2 nmole/plate, an increase in inhibition with an increase in dose was observed, but the corresponding decrease in His+ revertant count with an increase in dose was found to be insignificant (r ~ 0.8). Quercetin did not show any bactericidal/inhibitory effect on the growth of the tester strain at the tested dose levels nor did it show any mutagenic effect at these doses (when tested in the absence of t-BOOH).

Effect of ascorbic acid on t-BOOH induced mutagenicity--Antimutagenic effect of ascorbic acid was tested in the dose range of 0.1-100 Jlmole/plate against 100 Jlg/plate dose of t-BOOH. Decrease in mutagenicity was observed with an increase in the dose of ascorbic acid with the highest effect of almost 76% inhibition at 100 J.tmole/plate (Table 1). But, the dose-response relationship was insignificant (r ~ 0.8). There was a statistically significant difference with respect to control when the data was analyzed by One Way ANOVA at P value of 0.001.

Effect of combination of quercetin with ascorbic acid on t-BOOH induced mutagenicity--Effect of 500 nmole/plate of ascorbic acid on 4 and 8 nmole/plate of quercetin was studied. Inhibition(%) of mutagenicity achieved with these combinations was higher compared to that of quercetin or ascorbic acid alone (Fig. 2). QCl+AA and QC2+AA were significantly different from AA, when the data was analyzed by One Way ANOVA followed by Tukey's test.

60

>. 55

= 50 .2 i 45

' 40

I 35 - 3) 0 c 25 .S! = :10 .a :c 15 .. ·. .s 10 ~ 5

0 0.5 2 4 8

Quercetin (nmolealplllte)

Fig. !-Antimutagenic effects of quercetin on t­butylhydroperoxide induced mutagenicity in TA102 strain of Salmonella typhimurium. There is a statistically significant difference P = <0.001) for all dose levels with respect to the control, when analyzing the data by one way ANOV A.

Scavenging of free radicals by quercetin--DPPH assay method was used for the generation of free radicals. Quercetin showed a high scavenging effect to the tune of 94.71% in DPPH assay. This in vitro antioxidant activity of quercetin was also compared with ascorbic acid and the study indicated quercetin to be two times more active than ascorbic acid. However, in case of superoxide anion scavenging by hydroxylamine auto-oxidation method, quercetin induced an increase in the production of superoxide anions. This may be due to higher pH (i.e., 10.2) employed for the study, and the latter may cause the drug to act as a pro-oxidant16

• In case of riboflavin photo-oxidation method, ascorbic acid did not show any activity probably because of the oxidation of ascorbic acid by riboflavin in the presence of light to ascorbyl semiquinone radicals. Photo-excited riboflavin can perform one electron oxidation of ascorbic acid generating a riboflavin radical 17

. While in case of riboflavin photo-oxidation method, quercetin exhibited a maximum of 79% activity at 50 nmole. In case of hydrogen peroxide scavenging, quercetin showed three times and in hydroxyl radical scavenging eight times better activity than ascorbic acid. The data for these in vitro test systems have been shown in Tables 2, 3.

Discussion Extent of protectiveness provided by phenolic

antioxidants is related to their permeability into the biomembranes of cells. It has been shown that quercetin is an effective inhibitor of auto-oxidation of rat cerebral membranes2

. Effectiveness of quercetin is related to its deeper interaction with phospholipidic bilayers. Further, quercetin has also been reported to be a better suppressor of active oxygen induced cytotoxicity in comparison to catechin 18

• This higher

Table )-Inhibition of t-BOOH (100 J..Lg/plate) induced mutagenicity by different doses of ascorbic acid in T A 102 strain of Salmonella typhimurium

[Values are mean± SE of 4 replicates] .

Ascorbic acid (JLmole/plate)

0 0.1

0.25 0.5 1

10 100

% Inhibition of mutagenicity

0 14.4 ± 4.8 22.8 ± 3.6 23.6 ±4.7 24.5 ± 2.5 62.6 ± 1.1 76.0 + 1.9

ID so (JLmole)

8.7

64 INDIAN I EXP BIOL, JANUARY 2005

response of quercetin is related to its structural features. All these literature reports indicate that quercetin is an effective antioxidant. In our studies, it

was found to be a better antimutagen (in comparison to ascorbic acid) as well. However, recently oxidizing abilities of flavonoids have been indicated and this has been reported to affect their physiological activity 19

• These authors have concluded that e-aq scavenging activity of quercetin is quite high20

•21

.

50

l:' 45 t-BOOH has been reported to cause single strand

breaks in the DNA of a strain of Salmonella typhimurium similar to that of TA10222

• Alkoxyl and alkyl radicals are produced by t-BOOH which are responsible for its mutagenicity in S. typhi TA10223

.

Since quercetin reacts strongly with hydroperoxide radicals and hydrogen peroxide as indicated by our in vitro data (Table 2), the antimutagenic activity of quercetin against t-BOOH is also probably due to its radical scavenging activitl4

. Further, quercetin has been reported to show an inhibitory effect on the 4-hydroxy-2-nonenol (HNE) induced activation of stress signaling pathways25

• HNEs are strong alkylating agents that can cause the inhibition of DNA, RNA and protein synthesis25

. Presence of hydroperoxides can convert them to even more reactive compounds which form stable adducts with guanine bases of DNA26

. Based on these reports, it may be said that quercetin might prevent the reaction of HNEs with hydroperoxides (e.g., t-BOOH in our

:2 40 c: & 35 C1l -30 :::J E .... 25 0 c:

20 0 :0:: :ii 15 :c .5 10 ~

5

0

...-0 0

1 N 0 0

Fig. 2-Effect of 500 nmole/plate of ascorbic acid (AA) on the antimutagenic activity of quercetin (QC1: 4 nmole/plate; QC2: 8 nmole/plate). There was statistically significant difference (P = <0.001) between AA, QC1+AA and QC2+AA when the data is analyzed by one way ANOVA followed by Tukey's test.

Table 2-Scavenging effect of quercetin on DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radicals [Values are mean± SE of 6 replicates]

Concentration % Inhibition of (nmole) DPPH free radical Superoxide anion (Riboflavin Hydrogen peroxide Hydroxyl radical

photooxidation)

50 94.71 ± 0.8 79.75 ± 1.1 50.92 ± 5.0 53.52 ± 3.8 25 89.29 ± 0.8 55.71 ± 3.2 28.96 ±4.0 49.32 ± 3.3 5 41.84 ± 1.3 45 .44 ± 0.1 20.02 ± 2.5 45.21 ± 3.1

2.5 34.05 ± 1.9 35.89 ± 0.3 9.33 ±4.0 40.96 ± 3.8 0.5 22.08 ± 1.1 30.21 ± 0.5 2.62 ± 3.8 35.38 ± 4.7

IC 50 (nmole) 16.13±0.40 17.26 ± 1.08 48.5 ±4.80 22.90 ± 3.7

Table 3--Scav~nging effect of ascorbic acid on DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radicals

[Values are mean± SE of 6 replicates]

Concentration % Inhibition of (nmole) DPPH free radical Superoxide anion Hydrogen Hydroxyl radical

(Hydroxylamine auto-oxidation) peroxide

500 95.65 ±0.2 75.75 ± 2.1 72.31 ± 0.3 57.23 ± 2.6 250 79.81 ± 0.7 63.75 ± 2.8 66.34 ±0.5 53.97 ± 3.4 125 49.18±1.2 52.71 ± 3.2 44.62 ± 0.5 46.91 ± 2.7 50 38.29 ± 0.7 42.76 ±0.8 36.13 ± 0.6 39.39 ± 3.2 10 27.86 ± 1.37 34.69 ± 1.9 29.78 ± 1.7 33.10 ± 3.0 5 19.99 ± 0.5 18.43 ± 2.5 29.10±2.1 25.38 ± 4.7

IC 50 (nmole) 37.6 ± 1.65 103.4 ± 2.75 146.56 ± 4.08 189.38 ± 2.7

GEETHA et al.: ANTIMUTAGENIC AND ANTIOXIDANT/PROOXIDANT ACTIVITY OF QUERCETIN 65

test system) by effectively scavenging the latter. The observed antimutagenic activity could be due to the direct inhibition of lipid peroxidation (decreased production of HNEs) or indirectly through scavenging of hydroperoxides and hence inability of HNEs to form stable DNA adducts resulting in mutations and/or cancer.

Considering the low IC50 values in the in vitro tests and an inhibition of t-BOOH induced mutagenicity in T A 102, it may simply be concluded that quercetin is an effective antimutagenic agent. The same fact has been reported by other workers27

• But looking at the results more closely makes the above statement ambiguous because of the two facts: (i) beyond the dose level of 2 nmole, the extent of inhibition of quercetin was found to decrease (Fig. 2), even though these doses (i.e., 4 and 8 nmole) were priorly established to be non-mutagenic and non-toxic. (ii) the antimutagenic action did not go beyond 48-49% (iii) there was an increase in inhibition with an increase in dose but a linear dose-response relationship was absent (r:S0.8).

These results can be explained to an extent in terms of pro-oxidant activity exhibited by quercetin. It has been shown that flavonoids auto-oxidize readily to free radicals like hydroxyl radical and the semiquinone radical. Such species are known to be toxic and are reported to bind irreversibly to various cell constituents by the formation of covalent binding with sulfhydryl groups and/or other essential groups producing secondary free radicals28

'29

• The formation of these secondary free radicals is responsible for the pro-oxidant activity of flavonoids. The latter depends on the concentration of the flavonoid and the free radical source. 0-semiquinone and o-quinone are the enzymatically catalyzed oxidative degradation products of quercetin and o-semiquinone may facilitate the formation of superoxide and depletion of GSH, which could confer a specific pro-oxidative action in situ. One electron reduction of o-quinone leads to the generation of o-semiquinone. This intracellular metabolic activation of quercetin to o­quinone is partially associated with the observed concentration dependent cytotoxic effect of quercetin30

. Quercetin has also been reported to be a direct acting mutagen31

•32

. This could again be a consequence of its pro-oxidant activity. Thus, an increase in His+ revertants, at the dose level of 4 and 8 nmole, observed in our study can be attributed to the pro-oxidant nature of quercetin. The pro-oxidant

actlVlty of quercetin may deplete the nuclear antioxidant defense and lead to oxidative DNA damage which may be responsible for its mutagenicit/3

. Sahu and Gray have explained that this genotoxicity can have a result of the production of ROS by redox cycling34

• Quercetin gives superoxide anion by auto-oxidation which leads to the formation of hydrogen peroxide and ultimately to DNA damage16

•35

. An increase in the production of superoxide anion radicals (hydroxylamine auto­oxidation method) i.e., a pro-oxidant effect of quercetin was also observed in our in vitro studies as discussed earlier.

Another intriguing factor in the study was the absence of any dose response relationship. This fact is being reported by us for the first time. Recent reports indicate quercetin to be an effective inhibitor in a similar test27

; but the authors have tested only two widely placed doses (0.02 and 0.2 mg/plate). The doses selected correspond to 59 and 591 nmole/plate, while a similar extent of inhibition was achieved by us at a much lesser dose range of 0.5-2 nmole/plate. Even though, a significant inhibition of t-BOOH induced mutagenicity was observed at all the dose levels, there was no significant increase in the inhibition of mutagenicity with an increase in the concentration of quercetin. This absence of a dose response relationship indicated that the limiting step in the expression of antimutagenic action was not the concentration of quercetin.

Strong antioxidant effect of quercetin is indicated by its low IC50 values in all in vitro test systems used. Prompted by the fall in the antimutagenic action of quercetin at higher doses and absence of a linear dose­response effect and also considering the pro-oxidant nature of flavonoids with the generation of semiquinone radical, we tried to visualize the effect of ascorbic acid on quercetin. We expected a mutual effect between ascorbic acid and quercetin. Since both of these are pro-/antioxidants, we hypothesized that any unstable free radical of quercetin generated by scavenging of the oxidative mutagen will be further scavenged by ascorbic acid. So an additive or synergistic effect of ascorbic acid on the antimutagenic action of quercetin was apprehended by us. Such co-operative effects of ascorbic acid on quercetin (protection of cutaneous tissue from oxidative stress) and rutin (lipid peroxidation of liposomes) are also reported in the literature36

. High doses of quercetin (at which the antimutagenic action

66 INDIAN J EXP BIOL, JANUARY 2005

was found to decrease; indicating the generation of prooxidant species) and a middle dose of ascorbic acid showing a similar inhibition was selected, with a view that the two inhibitions would at least add up to 50% inhibition. Even though, an increase in the antimutagenicity of quercetin was observed when ·combined with ascorbic acid, the increase was not additive or synergistic (Fig. 3). The low antimutagenic activity, coupled with a failure to sufficiently increase the activity upon addition of ascorbic acid, once again point towards factors other than the antioxidant nature being responsible for the antimutagenicity of quercetin.

So, it seems probable that quercetin concentrations were changed reversibly to its semiquinone, where the latter acted as a depot for constant regeneration of a limited concentration of quercetin, which produced an almost similar response at all the dose levels. Furthermore, penetrability into the cell could also have a limiting effect. The mechanism of antimutagenic action of flavonoids is quite complex. While their antioxidant nature is the main driving force, other limiting factors like passage through the biomembranes, in situ oxidation to semiquinone, in vitro interaction with oxidizing species and regeneration of flavonoid moiety from oxidized (pro­oxidant) semiquinone radical cannot be ruled out. Quercetin interacts with the polar heads of phospholipids by concentrating at the membrane surface at physiological pH37

. However, more elaborate in vitro and in vivo mechanistic studies are required to confirm this.

Selection of a proper dose for extension of biological effects of quercetin and/or other flavonoids to clinical use thus becomes a very important factor to be monitored and needs to be stressed upon the scientists involved in the study of these agents. A cautious use of excessive flavonoid intake and uncertainty regarding the conditions and the levels of flavonoid which may pose a potential health hazard have been mentioned in the literature33

•34

. In future, there is a need to extrapolate these studies at clinical level with a special stress on dosage standardization; and a comprehensive review of other antioxidant molecules with an effort to identify a safe agent with minimal pro-oxidant effect.

Acknowledgement We are thankful for the financial assistance

provided by University Grants Commission, New Delhi, India.

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