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Toxicology in Vitro 22 (2008) 10–17

Antigenotoxic role of Centella asiatica L. extract againstcyproterone acetate induced genotoxic damage in cultured

human lymphocytes

Yasir Hasan Siddique a,*, Gulshan Ara a, Tanveer Beg a, Mohammad Faisal b,Mukhtar Ahmad b, Mohammad Afzal a

a Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University,

Aligarh 202 002, UP, Indiab Forest Entomology Division, Forest Research Institute, Dehradun 248 006, UA, India

Received 14 February 2007; accepted 5 July 2007Available online 18 July 2007

Abstract

The majority of the Indian population use traditional natural preparations derived from plant material for the treatment of variousdiseases, and for that reason it becomes necessary to assess the mutagenic potential or modulating action of plants extract when asso-ciated with other substances. The genotoxicity testing provides human a risk assessment. Earlier in vitro and in vivo studies reveal that theplant extracts from various parts of the plant play a modulating role in xenobiotic effects. Identification and characterization of someactive principles may lead to the development of the strategies to reduce the risk for developing cancer in humans. Cyproterone acetate(CPA), a synthetic progestin is not only a genotoxic agent but also a tumor initiating agent. It is used in oral contraceptives formulationsand also in the treatment of various sexual and metabolic disorders. In this context, the antigenotoxic effect of Centella asiatica L. extractwas studied against the genotoxic effect induced by CPA on human lymphocytes using chromosomal aberrations and sister chromatidexchanges as parameters. The treatment of the two doses of CPA, i.e. 20 and 30 lM was given along with the C. asiatica extract at thedosages of 1.075 · 10�4, 2.125 · 10�4, 3.15 · 10�4 and 4.17 · 0�4 g/ml of culture medium. A clear dose dependent decrease in the geno-toxic damage of CPA was observed, suggesting a protective role of C. asiatica extract during CPA therapy. The results of the presentstudy suggest that the plant extract per se do not have genotoxic potential, but can modulate the genotoxicity of CPA on human lym-phocytes in vitro.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Centella asiatica; Cyproterone acetate; Human lymphocytes; Chromosomal aberrations; Sister chromatid exchange

1. Introduction

Centella asiatica belongs to the family Umbelliferae. It isalso known as mandukparni or Indian Penny Wort. It isfound in swampy area of India, commonly found as a weedcrop fields and other waste places throughout India uptoan altitude of 600 m (Dastur, 1962). The crude extract of

0887-2333/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tiv.2007.07.001

* Corresponding author.E-mail address: yasir_hasansiddique@rediffmail.com (Y.H. Siddique).

C. asiatica and the products derived from glycoside wereused as oral antifertility agents (Dutta and Basu, 1968).The extract of C. asiatica extract possesses antioxidant(Gupta and Flora, 2006), anti-inflammatory (Guo et al.,2004), immunomodulating (Punuree et al., 2005), antitu-mor (Babu et al., 1995), antiproliferative (Yoshida et al.,2005), radioprotective (Sharma and Sharma, 2002) andantigenotoxic (Siddique et al., 2007) properties. The extractof C. asiatica L. has certain bioactive terpene acids such asasiatic acid, madecassic acid and their respective glycoside,asiaticoside and madecassoside (Inamdar et al., 1996).

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 11

Asiatic acid decreased the viability and induced apoptosisin human melanoma SK-MEL-2 cells (Park et al., 2005).Asiaticoside and madecassoside have anti-psoriatic proper-ties (Sampson et al., 2001). Asiaticoside, has wound healingactivity (Shukla et al., 1999), promotes fibroblast prolifera-tion (Lu et al., 2004) and increases the level of enzymaticand non-enzymatic antioxidants (Shukla et al., 1999).There are some phenolic compounds in the extract of C.

asiatica, having the activity same as that of the a-tocoph-erol (Zainol et al., 2003). The crude extract of C. asiatica

was shown to be non-toxic in normal human lymphocytes(Babu et al., 1995) and reduced the genotoxic effects ofmethyl methanesulphonate and cyclophosphamide in cul-tured human lymphocytes (Siddique et al., 2007).

Synthetic progestins are used in the treatment of sexualand metabolic disorders, and also in oral contraceptives(IARC, 1999). Prolonged use of oral contraceptives hasbeen shown to develop various types of malignancies inhuman and experimental animals (IARC, 1999). Earlierstudies reveal that synthetic progestins have DNA damag-ing potential (Joosten et al., 2004; Siddique and Afzal,2004a; Siddique and Afzal, 2004b). The genotoxic effectsof synthetic progestins can be reduced by the use of antiox-idants (Siddique et al., 2006; Siddique et al., 2005; Ahmadet al., 2002) and natural plants products (Siddique andAfzal, 2005a; Siddique et al., 2006; Siddique et al., inpress). Cyproterone acetate (CPA) is a tumor initiatingagent in the liver of female rats (Deml et al., 1993; Martelliet al., 1996). It induced micronucleus in rat liver cells (Mar-telli et al., 1996), chromosomal aberrations in V79 cells(Kasper et al., 1995), and human peripheral blood lympho-cytes (Reimann et al., 1996; Siddique and Afzal, 2005b),and also sister chromatid exchanges in human peripheralblood lymphocytes in vitro (Siddique and Afzal, 2005b).Studies of genotoxicity and antigenotoxicity of naturalplant extracts can help us to evaluate the safety and effec-tiveness of herbal health products (Romero-Jimenezet al., 2005). The herbal preparations are traditionally usedin the treatment of cancer therapy but there may be bio-activated components that may be responsible to promotecancer. In India, the majority of population uses tradi-tional natural preparation derived from the plant materialfor the treatment of various diseases, and for that reason itbecomes necessary to assess the mutagenic potential ormodulating action of plant extract when associated withother substances. The genotoxicity testing provides humana risk assessment. An increase in the frequency of chromo-somal aberrations in peripheral blood lymphocytes is asso-ciated with an increased overall risk of cancer (Hagmaret al., 1994; Hagmar et al., 1998). The ready quantifiablenature of sister chromatid exchanges with high sensitivityfor revealing toxicant-DNA interaction and the demon-strated ability of genotoxic chemicals to induce significantincrease in sister chromatid exchanges in cultured cellshas resulted this endpoint being used as indicator ofDNA damage in blood lymphocytes of individuals exposedto genotoxic carcinogens (Albertini et al., 2000). The above

genotoxic end-points are well known markers of genotoxi-city and any reduction in the frequency of these genotoxicend-points gives an indication of the antigenotoxicity of aparticular compound or plant infusion/extract, while anincrease is associated with the possibility of carcinogenesis(Albertini et al., 2000).

Synthetic progestins after oral administration readilyabsorbed through the intestinal lining with maximumplasma concentration being reached within 30–60 min,then they undergo first pass metabolism in liver (Martelliet al., 2003). However, markedly higher blood concentra-tions may be reached in some clinical conditions (Martelliet al., 2003), hence the present study was performed on cul-tured human lymphocytes, using two different concentra-tions of CPA. Since the plant extract have compounds,that may enhance or reduce the genotoxic effect of a partic-ular compound, the knowledge of a particular plant extractwill contribute us to form the basis of herbal medicine(Roncada et al., 2004). Earlier in vitro and in vivo studiesreveal that the plant extract from the various parts of theplant plays an important role in xenobiotic effects (Renet al., 2001). The objective of the present work was to studythe effect of C. asiatica L. extract against the genotoxicdoses of CPA, on human lymphocytes in vitro.

2. Material and methods

2.1. Chemicals

Cyproterone acetate (CAS No: 427-1-0, Sigma); RPMI1640, Fetal calf serum, Phytohaemagglutinin-M, Antibi-otic-antimycotic mixture (Gibco); Dimethylsulphoxide, 5-Bromo-2-deoxyuridine, Colchicine (SRL, India); Giemsastain (Merk).

2.2. Extract peparation

C. asiatica L. leaves were collected from the nursery ofForest Research Institute (FRI), Dehradun (UA) and wereair dried and ground to fine powder. Extraction was per-formed by soaking samples (30 gm of dry weight) in300 ml of acetone for 8–10 h at 40–60 �C in soxhlet’s appa-ratus. After filtration, the excess of solvent was removed byrotatory evaporator. The extract concentrations of1.075 · 10�4, 2.127 · 10�4, 3.15 · 10�4 and 4.17 · 10�4

g/ml of culture medium were established (Siddique et al.,2007).

2.3. Human lymphocyte culture

Duplicate peripheral blood cultures were preparedaccording to Carballo et al. (1993). Briefly, heparinizedblood samples (0.5 ml), were obtained from healthy femaledonors and were placed in a sterile culture bottle containing7 ml of RPMI-1640 medium, supplemented with fetal calfserum (1.5 ml), antibiotic–antimycotic mixture (1.0 ml)

12 Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

and phytohaemagglutinin (0.1 ml). The culture bottles werekept in an incubator at 37 �C for 24 h.

2.4. Chromosomal aberration analysis

After 24 h, 20 lM of CPA treatment (dissolved indimethyl sulphoxide, 5 ll/ml) was given separatelywith 1.075 · 10�4, 2.125 · 10�4 3.15 · 10�4 and 4.17 ·10�4 g/ml of Centella asiatica L. extract. Similarly, 30 lMof CPA treatment was also given with the same four dos-ages of C. asiatica extract. The culture bottles were keptfor another 48 h in an incubator at 37 �C. After 47 h,0.2 ml of colchicine (0.2 lg/ml) was added to the culturebottle. Cells were centrifuged at 800 g for 10 min. Thesupernatant was removed and 5 ml of prewarmed (37 �C)KCl hypotonic solution (0.075 M) was added. Cells wereresuspended and incubated at 37 �C for 15 min. The super-natant was removed by centrifugation at 800 g for 10 min,and 5 ml of chilled fixative (methanol: glacial acetic acid;3:1) was added. The fixative was removed by centrifugationand the procedure was repeated twice. The slides werestained in 3% Giemsa solution in phosphate buffer (pH6.8) for 15 min. Three-hundred metaphases were examinedfor the occurrence of different types of abnormality. Crite-ria to classify the different types of aberrations were inaccordance with recommendation of EHC 46 for environ-mental monitoring of human Population (IPCS, 1985).

2.5. Sister chromatid exchange analysis

For sister chromatid exchange analysis, bromodeoxyuri-dine (10 lg/ml) was added at the beginning of the culture.

Table 1Effect of Centella asiatica L. extract on chromosomal aberrations (CA) induce

Treatment Abnormal metaphases without gaps Chr

Number Mean% ± SE Gap

CPA (lM)

20 11 3.67 ± 1.08a 630 16 5.33 ± 1.29a 10

CPA (lM) + CAE (g/ml)

20 + 1.075 · 10�4 9 3.00 ± 0.98b 430 + 1.075 · 10�4 12 4.00 ± 1.13b 820 + 2.125 · 10�4 8 2.67 ± 0.93b 330 + 2.125 · 10�4 10 3.33 ± 1.03b 720 + 3.15 · 10�4 6 2.00 ± 0.80b 230 + 3.15 · 10�4 7 2.33 ± 0.87b 620 + 4.17 · 10�4 5 1.67 ± 0.73b 230 + 4.17 · 10�4 5 1.67 ± 0.73b 5

Untreated CAE (g/ml) 2 0.66 ± 0.46 11.075 · 10�4 3 1.00 ± 0.57 12.125 · 10�4 3 1.00 ± 0.57 13.15 · 10�4 4 1.33 ± 0.56 24.17 · 10�4 4 1.33 ± 0.56 2

Negative control

(DMSO, 5 ll/ml) 3 1.00 ± 0.57 2

CPA: Cyproterone acetate; CAE: Centella asiatica extract; DMSO: Dimethylsa P < 0.01 Significant with respect to untreated.b P < 0.05 Significant with respect to CPA treatment.

After 24 h, 20 lM of CPA (dissolved in dimethylsulphox-ide, 5 ll/ml) treatment was given separately with 1.075 ·10�4, 2.125 · 10�4, 3.15 · 10�4 and 4.17 · 10�4 g/ml of C.

asiatica L. extract respectively. Similar treatments weregiven with 30 lM of CPA and kept for another 48 h inan incubator. Mitotic arrest was performed by adding0.2 ml of colchicine (0.2 lg/ml). Hypotonic treatment andfixation were performed in the same way as for the chro-mosomal aberration analysis. The sister chromatidexchange average was taken from an analysis of metaphaseduring second cycle of division (Perry and Wolff, 1974).

2.6. Statistical analysis

Student ‘t’-test was used for analysis of CAs and SCEs.Regression analysis was performed using statistica soft Inc.

3. Results

Cyproterone acetate (CPA) induced a significant increaseof abnormal metaphases as compared to the untreated. Asignificant dose dependent decrease in number of abnormalmetaphase was observed when 20 and 30 lM of CPA wastreated, separately, with the different dosages of C. asiatica

extract, i.e. 1.075 · 10�4, 2.125 · 10�4, 3.15 · 10�4 and4.17 · 10�4 g/ml (Table 1; Fig. 1). For sister chromatidexchange analysis, a significant increase was observed atboth the dosages of CPA, i.e. 20 and 30 lM (Table 2;Fig. 2). A significant decrease in sister chromatid exchangesper cell was observed when 20 and 30 lM of CPA was trea-ted, separately, with the different dosages of C. asiatica

extract, i.e. 1.075 · 10�4, 2.125 · 10�4, 3.15 · 10�4 and

d by cyproterone acetate (N = 2)

omosome aberrations

s number Fragments number CTB number CSB number

3 9 37 14 5

2 7 25 10 31 5 13 8 1– 2 12 3 1– 2 –1 2 1

– 2 –– 2 1– 2 1– 2 1– 3 1

– 2 1

ulphoxide; CTB: Chromatid break; CSB: Chromosome break.

0

2

4

6

8

10

12

14

16

18

CPA

1C

PA2

CPA

1+C

AE1

CPA

1+C

AE2

CPA

1+C

AE3

CPA

1+C

AE4

CPA

2+C

AE1

CPA

2+C

AE2

CPA

2+C

AE3

CPA

2+C

AE4

CAE

1C

AE2

CAE

3C

AE4 U NC

No

. of

Ab

no

rmal

met

aph

ases

wit

ho

ut

gap

s

Fig. 1. Effect of Centella asiatica L. extract on abnormal metaphases induced by cyproterone acetate (N = 2).

CPA = Cyproterone acetate CAE = Centella asiatica extractCPA1 = 20 lM; CPA2 = 30 lM U = Untreated NC = Negative Control (DMSO 5 ll/ml)CPA1 + CAE1 = 20 lM + 1.075 l10�4 g/ml CPA2 + CAE1 = 30 lM + 1.075 l10�4 g/ml CAE1 = 1.075 l10�4 g/mlCPA1 + CAE2 = 20 lM + 2.125 l10�4 g/ml CPA2 + CAE2 = 30 lM + 2.125 l10�4 g/ml CAE2 = 2.125 l10�4 g/mlCPA1 + CAE3 = 20 lM + 3.15 l10�4 g/ml CPA2 + CAE3 = 30 lM + 3.15 l10�4 g/ml CAE3 = 3.15 l10�4 g/mlCPA1 + CAE4 = 20 lM + 4.17 l10�4 g/ml CPA2 + CAE4 = 30 lM + 4.17 l10�4 g/ml CAE4 = 4.17 l10�4 g/ml

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 13

4.17 · 10�4 g/ml (Table 2; Fig. 2). Regression analysis wasalso performed to determine the dose effect of C. asiatica

extract on 20 and 30 lM of CPA, for number of abnormalmetaphases and sister chromatid exchanges. A decrease inthe slope of linear regression lines was observed as the doseof the extract was increase, in each of the treatment. For

Table 2Effect of Centella asiatica L. extract on sister chromatid exchanges (SCE)induced by cyproterone acetate (N = 2)

Treatment SCEs/Cell (Mean ± SE) Range

CPA (lM)

20 6.02 ± 0.43a 2–730 8.30 ± 0.62a 2–9

CPA (lM) + CA (g/ml)

20 + 1.075 · 10�4 4.22 ± 0.32b 2–530 + 1.075 · 10�4 6.34 ± 0.44b 2–820 + 2.125 · 10�4 3.32 ± 0.33b 2–530 + 2.125 · 10�4 4.26 ± 0.38b 2–520 + 3.15 · 10�4 2.88 ± 0.17b 2–530 + 3.15 · 10�4 3.36 ± 0.25b 2–520 + 4.17 · 10�4 2.52 ± 0.16 2–530 + 4.17 · 10�4 3.02 ± 0.22 2–5

Untreated

CAE (g/ml) 1.24 ± 0.11 0–51.075 · 10�4 2.12 ± 0.18 0–52.125 · 10�4 2.32 ± 0.21 0–53.15 · 10�4 2.64 ± 0.23 0–54.17 · 10�4 2.70 ± 0.26 0–5Negative control

(DMSO, 5 ll/ml) 1.82 ± 0.15 0–5

CPA: Cyproterone acetate; CAE: Centella asiatica extract; DMSO:Dimethylsulphoxide.

a P < 0.01 Significant with respect to untreated.b P < 0.05 Significant with respect to CPA treatment.

abnormal metaphases the treatment of 20 lM (F = 95.14;P < 0.001) and 30 lM (F = 273.3; P < 0.001) of CPA, withthe increase in the dosages of C. asiatica extract result inthe decrease in slope of the linear regression lines (Figs. 3and 4). For sister chromatid exchange analysis, the treat-ment of 20 lM (F = 40.18; P < 0.002) and 30 lM(F = 15.60; P < 0.01) of CPA, with the increase in the dos-ages of C. asiatica extract, the decrease in slope of linearregression lines was observed (Figs. 5 and 6).

4. Discussion

The results of the study reveal that the selected dosagesof the plant extract were not genotoxic per se but reducedthe genotoxic damage of cyproterone acetate (CPA) onhuman lymphocytes in vitro. In our earlier study, fourdoses of CPA (5, 10, 20 and 30 lM) were studied (Siddiqueand Afzal, 2005b). CPA was found genotoxic at 20 and30 lM. The International Agency on Cancer (IARC),mainly on the basis of epidemiological studies classifies ste-roidal estrogens and estrogen progestin combinationsamong agents carcinogenic to humans (Group 1), proges-tins as possibly carcinogenic (Group 2) and androgenicanabolic steroids, as probably carcinogenic (Group 2A)(Martelli et al., 2003). Carcinogenicity to humans of sexsteroids has been evaluated, and is reported that high doseof estrogen–progestin combinations can cause liver cancerto humans (IARC, 1999). In a very recent ‘‘Multi centrestudy’’ on oral contraceptives and liver cancer the ‘‘ProjectTeam’’ came to the conclusion that oral contraceptivesmay enhance the risk of liver carcinomas (Martelli et al.,2003). CPA is not only a tumor promoting agent but alsoa genotoxic chemical (Joosten et al., 2004). Sister chromatid

0

1

2

3

4

5

6

7

8

9

10

CPA

1C

PA2

CPA

1+C

AE1

CPA

1+C

AE2

CPA

1+C

AE3

CPA

1+C

AE4

CPA

2+C

AE1

CPA

2+C

AE2

CPA

2+C

AE3

CPA

2+C

AE4

CAE

1C

AE2

CAE

3C

AE4 U NC

Sis

ter

chro

mat

id e

xch

ang

es/c

ell

Fig. 2. Effect of Centella asiatica L. extract on sister chromatid exchanges induced by cyproterone acetate (N = 2).

CPA = Cyproterone acetate CAE = Centella asiatica extractCPA1 = 20 lM; CPA2 = 30 lM U = Untreated NC = Negative Control (DMSO 5 ll/ml)CPA1 + CAE1 = 20 lM + 1.075 l10�4 g/ml CPA2 + CAE1 = 30 lM + 1.075 l10�4 g/ml CAE1 = 1.075 l10�4 g/mlCPA1 + CAE2 = 20 lM + 2.125 l10�4 g/ml CPA2 + CAE2 = 30 lM + 2.125 l10�4 g/ml CAE2 = 2.125 l10�4 g/mlCPA1 + CAE3 = 20 lM + 3.15 l10�4 g/ml CPA2 + CAE3 = 30 lM + 3.15 l10�4 g/ml CAE3 = 3.15 l10�4 g/mlCPA1 + CAE4 = 20 lM + 4.17 l10�4 g/ml CPA2 + CAE4 = 30 lM + 4.17 l10�4 g/ml CAE4 = 4.17 l10�4 g/ml

Regression95% confid.

Y = 10.570 - 1.357 * X

Correlation: r = -.9897

Concentration of extract

Nu

mb

er o

f ab

no

rmal

met

aph

ases

4.5

5.5

6.5

7.5

8.5

9.5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Fig. 3. Regression analysis for the dose effect of C. asiatica extract onabnormal metaphases induced by cyproterone acetate (20 lM). Concen-tration of extract is in (· 10�4 g/ml).

Regression95% confid.

Y = 14.621 - 2.327 * X

Correlation: r = -.9964

Concentration of extract

Nu

mb

er o

f ab

no

rmal

met

aph

ases

4

5

6

7

8

9

10

11

12

13

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Fig. 4. Regression analysis for the dose effect of C. asiatica extract onabnormal metaphases induced by cyproterone acetate (30 lM). Concen-tration of extract is in (· 10�4 g/ml).

14 Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

exchanges (SCE) have been commonly used to evaluatecytogenetic responses to chemical exposure, and an excel-lent dose response relationship has been established forhundred of chemicals in a wide variety of in vivo andin vitro short term experiments (Tucker and Preston,1996). Chromosomal aberrations are changes in chromo-some structure resulting from a break or an exchange ofchromosomal material. Most of the chromosomal aberra-tions observed in the cells are lethal, but there are many cor-responding aberration that are viable and cause geneticeffects, either somatic or inherited (Swierenga et al., 1991).These events lead to the loss of chromosomal material atmitosis or due to the inhibition of accurate chromosome

segregation at anaphase. SCE is generally a more sensitiveindicator of genotoxic effects than structural aberrations(Tucker and Preston, 1996). There is a correlation betweenthe carcinogenicity and SCE inducing ability of large num-ber of chemicals (Gebhart, 1981). Concerning our earlierstudy on CPA it was found genotoxic by generating freeradicals in the test system (Siddique and Afzal, 2005b).An excess of reactive oxygen species (ROS) leads to theDNA damage. Many plant products protect against xeno-biotics either by inducing detoxifying enzymes or by inhib-iting oxidative enzymes (Morse and Stoner, 1993). Theverification of the possible mutagenic and/or anti-muta-genic effects of medicinal plants infusion/extracts is another

Regression95% confid.

Y = 4.6503 - .5382 * X

Correlation: r = -.9764

Concentration of extract

Sis

ter

chro

mat

id e

xch

ang

es /

cell

2.4

2.8

3.2

3.6

4.0

4.4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Fig. 5. Regression analysis for the dose effect of C. asiatica extract onsister chromatid exchanges induced by cyproterone acetate (20 lM).Concentration of extract is in (· 10�4 g/ml).

Regression95% confid.

Y = 7.0169 - 1.055 * X

Correlation: r = -.9415

Concentration of extract

Sis

ter

chro

mat

id e

xch

ang

es /

cell

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Fig. 6. Regression analysis for the dose effect of C. asiatica extract onsister chromatid exchanges induced by cyproterone acetate (30 lM).Concentration of extract is in (· 10�4 g/ml).

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 15

important factor in studies. Such effects have been eluci-dated in some plant species by using various test systems(Roncada et al., 2004). Some plants may possess substancesthat can modulate the genotoxicity of other compounds.The data obtained in the present study suggests that thecompound present in the extract of C. asiatica, are notmutagenic on their own or when associated with the CPA.The extract of C. asiatica is capable of reducing the geno-toxic effect of CPA. The protective effect observed in thepresent study, i.e. significant reduction in the frequency ofcells with chromosomal damage and sister chromatidexchanges may be due to the direct action of the compoundspresent in the extract on CPA by inactivating it enzymati-cally or chemically. The genotoxic effects of CPA in livercells have been attributed to the bio-activation of CPA byhydroxyl steroid sulfotransferase (HST) to reactive metab-olites (Kasper and Mueller, 1999). In our earlier study with

cyclophosphamide, the extract of C. asiatica reduced thegenotoxic damage this is due to the possible prevention ofmetabolic activation of cyclophosphamide by the extract(Siddique et al., 2007). The compounds present in the extractmay also scavenge electrophiles/nucleophiles (Maurichet al., 2004). In the present study, the extract may possiblyscavenge free radicals generated by CPA via nucleophilicreaction (Siddique and Afzal, 2005b) and reduced the geno-toxic damage. The compounds may also enhance the DNArepair system or DNA synthesis or even may prevent thebio-activation of certain chemicals (Kuroda et al., 1992).The treatment of extract reduced the frequency of SCEand chromosomal aberrations in the test system, therebyindicating the possibility of reducing the chances of carcino-genesis during the CPA therapy in patients. The antigeno-toxic potential of the plant extracts have been attributedto their total phenolic content (Maurich et al., 2004). Medic-inal herbs contain complex mixtures of thousand of com-pounds that can exert their antioxidant and free radicalscavenging effect either separately or in synergistic ways(Romero-Jimenez et al., 2005). Identification and character-ization of these active principles in the plant extract may leadto the strategies to reduce the risk for developing cancer inhumans (Dearfield et al., 2002). The identification and char-acterization of the compounds present in the C. asiatica

extract to determine their particular functions will be thepart of our future study, however at present it can be con-cluded from the study that C. asiatica extract has the poten-tial to reduced the genotoxic damage induced by CPA incultured human lymphocytes.

Acknowledgements

Thanks are due to the CSIR, New Delhi for awardingthe Fellowship No. 9/112(355)/2003-EMR to the author(YHS) and to the Chairman, Department of Zoology,A.M.U., Aligarh, U.P., for laboratory facilities.

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