G2 STUDIES OF ANTIMUTAGENIC POTENTIAL OF CHEMOPREVENTIVE AGENT CURCUMIN IN ALLIUM CEPA ROOT MERISTEM CELLS

FACTA UNIVERSITATIS
Series: Medicine and Biology Vol.15, No 1, 2008, pp. 20 - 23
UC 615.322

G2 STUDIES OF ANTIMUTAGENIC POTENTIAL OF CHEMOPREVENTIVE
AGENT CURCUMIN IN ALLIUM CEPA ROOT MERISTEM CELLS
Loganathan Palani Kumar, Natarajan Paneerselvam
Research Centre and Post Graduate Studies in Botany, The Madura College, Madurai-625 011, S. India
E-mail: npanneer1958@gmail.com
Summary. Turmeric has long been used as a spice and food colouring agent in Asia. Curcumin is one of the active
principles in Turmeric. In this research the antimutagenic potential of curcumin in Allium cepa root meristem cells
was evaluated. The root tip cells were treated with sodium azide at 200µg/ml and 300µg/ml for 1h. Curcumin was
given at 5, 10 and 20 µg/ml for 18h (one cell cycle), prior to sodium azide treatment. The tips were squashed after
colchicine treatment and the cells were analyzed for chromosome damage and mitotic index. Curcumin did not induce
chromosomal aberration in Allium cepa root tip cell. Sodium azide alone induced chromosomal aberrations
significantly with increasing concentrations. The chromosomal aberrations were significantly reduced in root tips
cells pretreated with curcumin. The study reveals the antimutagenic potential of curcumin against sodium azide
induced chromosomal aberrations.
Key words: Curcumin, chromosomal aberrations, sodium azide
Introduction
Materials and Methods
Turmeric is a well-known spice and food colorant
Chemicals
commonly consumed in different parts of the world. Its
Curcumin, Colchicine, Basic Fuchsin was obtained
active principle curcumin is the powdered dry rhizome of
from Sigma chemicals USA. The rest of the chemicals,
Curcuma longa Linn plant of the family Zingiberaceae
(1). It shows anti-mutagenic, anti-genotoxic and protec-
potassium metabi-sulphite Hydrochloric acid, Acetic
acid, Ethanol, Methanol used in this study were of ana-
tive effects on various test systems (2-8). Curcumin pro-
lytical grade purity obtained from Hi-media chemicals
tects cisplatin-induced clastogenesis in vitro and in vivo
India.
assays, by acting as a free radical scavenger (4). It has
been identified to reduce the radiation induced DNA
damage in rat lymphocytes (5), hydrocortisone induced
Test system and treatment

genotoxicity in human lymphocytes (4) and nicotine in-
Healthy onion bulbs (20-25g) were grown in dark in
duced toxicity in wistar rats (7), and lead acetate induced
a cylindrical glass receptacle at room temperature (28oC
genotoxicity in mice (1) and reduced the frequency of
± 0.5oC). Water was changed periodically at every 24h.
micronuclei in polychromatic erythrocytes induced by
When the roots reached 2cm to 3cm in height, they were
gamma radiation (9). Furthermore, it showed a protection
treated with different concentrations of 5, 10, 20 ?g/ml
against ethanol induced cytotoxicity in liver cell culture
of curcumin for 18h (one cell cycle). Following curcu-
in an in vitro condition (10). Curcumin exhibits anti-
min treatment, the bulbs were washed in distilled water
mutagenic effect on cyclophosphamide induced chromo-
and then treated with 200, 300 ?g /ml of sodium azide
somal aberration in wistar rats (2) and benzo(a)pyrene,
for 1h. Then the roots were treated with 0.05% colchi-
cyclophosphamide induced genotoxicity in microbial and
cine for 3h and then fixed in carnoy’s fluid.
mammalian test systems (3). Turmeric has been found to
display inhibition of genotoxic effect of urethane in Dro-
Chromosome analysis
sophila (11). The present study was designed to investi-
gate whether or not curcumin has anti-mutagenic poten-
Microslides were prepared from the treated root tip
tial on sodium azide induced chromosomal aberrations in
cells by Feulgen squash technique. A total of 300 well
Allium cepa root meristem cells.
spread metaphases per concentration were analyzed for
chromosomal aberrations and 3000 cells were scored for
mitotic index. The suppressions percentage of curcumin
on chromosomal aberrations of sodium azide was cal-
culated as

---

G2 STUDIES OF ANTIMUTAGENIC POTENTIAL OF CHEMOPREVENTIVE AGENT CURCUMIN. .
21
40
s
i
o
n
200mg/ml
35
300mg/ml
r
e
s
30
pp 25

f

Su 20
o
ge 15
Statistical analysis
n
ta 10
r
c
e
5
The mean values and SD of different parameters in-
Pe
0
cluding mitotic index and incidence of abnormal cells of
5Cur
10Cur
20Cur
all groups were subjected to statistic comparison using

students t-test p < 0.01 considered significant.
Fig. 1. Percentage of suppression on sodium azide
induced chromosomal aberration.
Results
The G2 effects of sodium azide induced chromosomal
Discussion
aberrations such as break, gap, iso-chromatid break, which
were analyzed in A. cepa root tip cells pretreated with cur-
Considerable emphasis has been laid down on the
cumin (Table 1). In this study curcumin alone did not in-
use of dietary constituents preventing mutagen induced
cytogenetic damage due to their relative non-toxic ef-
duce any chromosomal aberrations at 5, 10, 20 ?g/ml of
fects (12). Since oxidative damage in biological systems
assayed, indicate the anti-clastogenic activity. The number
is considered to cause ageing, degenerative diseases and
of abnormal cells and the number of aberrations were in-
cancers, a particular attention was focused on the possi-
creased with the increasing dosage of sodium azide in
bility of modulating these effects through the use of free
200?g/ml and 300?g/ml tested. The number of abnormal
radical scavengers to minimize cell injury. The benefi-
cells and the number of aberrations were decreased signifi-
cial effects of turmeric were postulated to result from
cantly in all curcumin-pretreated groups. The percentage of
curcumin (13). The biological activities of curcumin are
suppression by curcumin on sodium azide induced chrom-
derived from the antioxidant property of the methoxy
sosmal aberrations increased with increasing concentra-
phenol group and the action of the aryl group in ?-
tions of curcumin in all the concentrations tested (Fig. 1).
diketone (14,15,16). In the present study curcumin was
The results showed that chromatid breaks were more
found to inhibit the incidents of sodium azide induced
common than isochromatid breaks and gaps. This study
chromosomal aberrations in Allium cepa in a dose de-
implies that pretreatment of curcumin has inhibitory po-
pendent manner, suggesting its potential as an an-
tential against the mutagenic action of sodium azide.
timutagenic agent. Similar results were observed in wistar
rats treated with cyclophosphamide (2) and benzo(a)
Table 1.Distribution of different types of chromosomal
pyrene and cyclophosphamide induced genotoxicity in
aberrations in 300 cells analyzed and mitotic
microbial and mammalian test systems (3).
index observed in Allium cepa after treatment
Curcumin has protective effect against radiation-in-
with curcumin and or/ not sodium azide.
duced damage in rat lymphocytes (6) hydrocortisone
induced genotoxicity in human lymphocytes (Ahmed et
al., 2004) and also against the cisplatin induced clasto-
genesis by acting free radical scavenger (5). Curcumin
exerts protective effects by modulating the extent of
lipid per-oxidation and enhancing the antioxidant status
in wister rats (7). Curcumin significantly reduced the
frequencies of micro nucleated polychromatic erythro-
cytes in mice exposed to gamma radiation (9) and it was
also indicated as an antimutagen against environmental
mutagens in vitro and antitumor drug in vivo. The pro-
tective effect of curcumin against cyclophosphamide
induced genotoxicity may be due to one of the follow-
ing: antioxidant action, trapping of free radicals, forma-
tion of complex with mutagens, modulation of mutagen
metabolism or by absorbing the xenobiotics (17). The
modulatory role of curcumin in inhibition of mutage-

necity and carcinogenecity can also be implied to its
Abbrevations: Cur-Curcumin; NaN3-sodium azide; Bre-Break; IsB-
antioxidant activity (18). The antioxidant property of
Isochromatid Break; Exc- Exchange; Supp-suppression
this plant and their products may scavenge peroxides
*Statistically different when compared with sodium azide control
a Statistically different when compared with Curcumin control
and other radical oxygen species as do natural protectors
b Statistically different when compared with Untreated control
against lipid peroxidation such as retinal, ascorbic acid,

---

22
L. Palani Kumar, N. Paneerselvam
tocopherol, glutathione and antioxidant enzymes such as
timutagenic and anticarcinogenic activity. Furthermore,
catalase, superoxide dismutase, and glutathione peroxi-
curcumin acts as a potent anticarcinogenic compound
dase which have the capacity to scavenge reactive oxy-
through various mechanisms. The induction of apop-
gen species and lipid free radicals (19,20).The antioxi-
tosis plays an important role in its anticarcinogenic ef-
dant effects of these compounds mainly depend on the
fect (25) and also inhibits cancer at initiation, promotion
stabilization of the formed phenoxy free radical. The p-
and progression stages of development against
hydroxy phenyl moiety is very essential to produce the
benzo(a)pyrene induced skin tumors in female Swiss
henoxy free radical responsible for free radical scav-
mice (22). The results of present investigation conclude
enging effect (21). Hydroxylated derivatives of curcu-
a dose dependent antimutagenic potential of curcumin
min are known to be antimutagenic and it has been pro-
on sodium azide induced clastogenic damage in A. cepa.
posed that p-hydroxy group is also essential for its
The molecular mechanisms of antimutagenic or anti-
chemo preventive-effect (22,23). Curcumin has two p-
genotoxicity of curcumin and its definite role in Allium
hydroxy groups and scavenges free radical DNA dam-
cepa root meristem cells need further investigations.
age there by acting as a potent antioxidant (23,24).

Since mutations induced at the cytogenetic levels are
Acknowledgement. The authors kindly acknowledge the receipt
probable causes of cancer, therefore the inhibition of
of research grant from Department of Science and Technology,
chromosomal aberration by curcumin suggests the an-
Government of India, New Delhi.
References
1. EL-Ashmawy IM, Ashry, KM, EL-Nahas, AF, Salama OM.
13. Antunes LM, Araujo MC, Darin JD, Bianchi ML. Effects of the
Protection by turmeric and myrrh against liver oxidative dam-
antioxidants curcumin and vitamin C on cisplatin-induced
age and genotoxicity induced by lead acetate in mice. Basic &
clastogenesis in wistar rat bone marrow cells. Mutat Res 2000:
clinical Pharmacology & Toxicology 2006; 98: 32-37.
16: 131-137.
2. Shukla Y, Arora A, Taneja P. Antimutagenic potential of curcu-
14. Jovanovic SV, Boone CW, Steenken S, Trinoga M, Karskey
min on chromosomal aberrations in wistar rats. Mutat Res
RB. How curcumin works preferentially with water soluble an-
2002; 515: 197-202.
tioxidants. J Am Chem Soc 2001; 123: 3064-3068.
3. Shukla Y, Arora A, Taneja P, Antigenotoxic potential of certain
15. Sun YM, Zhang HY, Chen DZ, Liu CB. Theoretical elucidation
dietary constituents. Teratog Carcinog Mutagen Suppl 2003; 1:
on the antioxidant mechanism of curcumin a DFT study. Or-
323-35.
ganic Lett, 2002; 4: 2909-2911.
4. Ahmad MS, Sheeba,. Afzal M. Amelioration of genotoxic dam-
16. Anto RJA, Mukhopadhyay A, Denning K, Aggarwal BB.
age by certain phytoproducts in human lymphocyte cultures.
Curcumin (diferuloymethane) induces apoptosis through acti-
Chem Biol Interact 2004;149: 107-15.
vation of caspase-8, Bid cleavage and cytochrome c release: its
5. Antunes LM, Araujo,MC, Darin JD, Bianchi ML. Effects of the
suppression by ectopic expression of Bcl-2 and Bcl-xl. Car-
antioxidants curcumin and vitamin C on cisplatin-induced
cinogenesi, 2002; 123; 143-150.
clastogenesis in wistar rat bone marrow cells. Mutat Res 2000;
17. Premkumar K, Kavitha S, Santhiya ST, Ramesh A. Interactive
16: 131-7.
effects of saffron with garlic and curcumin against cyclophos-
6. Thresiamma KC, George J, Kuttan R, Protective effect of
phamide induced genotoxicity in mice. Asia Pac.J Clin Nutr,
curcumin, elleagic acid and bixin on radiation-induced
2004; 13: 292- 294.
genotoxicity. J Exp Clin Cancer Res 1998; 17: 431-446.
18. Nagabhusan M, Amonkar AJ, Bhide SV. In vitro antimutagenicity
7. Kalpana C, Menon VP, Inhibition of nicotine induced toxicity
of curcumin against environmental mutagens. Food Chem. Toxicol,
by curcumin and curcumin analog: a comparative study. J Med
1987; 25: 545-547.
Food 2004; 7: 467-71.
19. Srinivas L, Shalini LK, Shylaja M. Turmerin; A water soluble
8. Araujo MC, Antunes LM, Takahashi, C.S., Protective effect of
antioxidant peptide from Turmeric (Curcuma longa). Arch.Bio-
thiourea, a hydroxyl radical scavenger, on curcumin induced
chem Biophys 1992; 292; 617-623.
chromosomal aberrations in an in vitro mammalian cell system.
20. Burtis M, Bukar F. Antioxidant activity of Nigells sativa essen-
Teratog Carcinog Mutagen 2001; 21: 175-80.
tial oil. Phytother Res 2000; 14: 323-328.
9. Abraham SK, Sarma L, Kesavan PC, Protective effects of
21. Youssef KM, El-Sherbony MA. Synthesis and antitumor activ-
chlorogenic acid, curcumin, and betacarotene against gamma
ity of some curcumin analogs.. Arch. Pharm. Chem.Life Sci,
radiation induced in vivo chromosomal damage. Mutat Res
2005; 338: 181-189.
1993; 303: 109-12.
22. Nagabhusan M, Bhide SV. Curcumin as an inhibitor of cancer.
10. Naik RS, Mujummdar AM, Ghaskadbi S. Protection of liver
J Am Coll Nutr, 1992; 11: 192-8.
cells from ethanol cytotoxicity by curcumin in liver slice cul-
23. Kunchandy E, Rao MNA. Oxygen radical scavenging activity
ture in vitro. J Ethnopharmacol 2004; 95: 31-7.
of curcumin. Int J Pharmacol 1990; 58: 237-240.
11. El-Hamss R, Analla M, Campos-sanchez J, Alonso-Moraga A,
24. Subramanian M, Sreejayan Rao MNA, Devasagam TPA, Singh
Munoz-Serrano A, Idaommar M, A dose dependent anti-
BB. Diminution of singlet oxygen induced DNA damage by
genotoxic effect of turmeric. Mutat Res, 1999; 446: 135-139.
curcumin and related antioxidants. Mutat Res 1994; 311: 249-
12. Reddy, A.C.P., Effects of dietary turmeric (Curcuma longa) on
255.
iron induced lipid peroxidation in the rat liver. Food Chem.
25. Chen HW, Huang HC. Effect of curcumin on cell cycle
Toxicol 1994; 32: 279-283.
progression and apoptosis in vascular smooth e cells. Br J
Pharmacol 1998; 124: 1029-1040.

---

G2 STUDIES OF ANTIMUTAGENIC POTENTIAL OF CHEMOPREVENTIVE AGENT CURCUMIN. .
23
G2 STUDIJE ANTIMUTAGENOG POTENCIJALA
HEMOPREVENTIVNOG AGENSA KURKUMINA U ?ELIJAMA
POKORI?NOG TKIVA KORENA VRSTE ALLIUM CEPA
Loganathan Palani Kumar, Natarajan Paneerselvam
Istraživa?ki centar i postdiplomske studije botanike, Madura koledž, Madurai-625 011, Indija
E-mail: npanneer1958@gmail.com
Kratak sadržaj: Indijski šafran (turmerik) se dugo koristi kao za?in i agens za bojenje hrane u Aziji. Kurkumin je jedna
od aktivnih osnova šafrana. U istraživanjima je procenjen antimutageni potencijal kurkumina u ?elijama pokori?nog
tkiva korena vrste Allium cepa. ?elije vrha korena tretirane su natrijum azidom 200µg/ml i 300µg/ml sat vremena.
Dodato je 5, 10 i 20 µg/ml kurkumina tokom 18 ?asova (jedan ?elijski ciklus), pre tretmana natrijum azidom. Vrhovi
su iscedjeni posle tretmana kolcihinom i analizirano je ošte?enje hromozoma i mitoti?ni indeks ?elija. Kurkumin nije
izazvao aberaciju hromozoma ?elija vrha korena Allium cepa. Samo sa pove?anjem koncentracije natrijum azida
pove?avale su se aberacije hromozoma. Hromozomne aberacije su bile zna?ajno manje kod ?elija vrha korena koje su
prethodno bile tretirane kurkuminom. Studija je dokazala antimutageni potencijal kurkumina kod aberacija
hromozoma izazvanih natrijum azidom.
Klju?ne re?i: Kurkumin, aberacije hromozoma, natrijum azid

---