ISSN (Online) 2249-6084 (Print) 2250-1029
International Journal of Pharmaceutical and
Phytopharmacological Research (eIJPPR)
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Research Article
EVALUATION OF ANTI-GENOTOXIC POTENTIAL OF GINGER
(ZINGIBER OFFICINALE) ON 7, 12-DIMETHYL BENZ (a)
ANTHRACENE INDUCED GENOTOXICITY IN RATS
Sradhasini Rout*a, Bandana Rathb, Sadananda Rathb and Anjan Kumara
a
Roland Institute of Pharmaceutical Sciences, Khodasinghi, Berhampur, Ganjam,
Odisha,India-760010
b
MKCG Medical College and Hospital, Berhampur, Ganjam, Odisha,India-760004
*Corresponding Author
Mrs. Sradhasini Rout
Asst. Professor in Pharmacology
Roland Institute of Pharmaceutical Sciences,
Berhampur, Ganjam,
Odisha, India-760010
E-mail- routsradha@gmail.com
Cellular Phone No- +91-9861420917
1
ABSTRACT:
The antigenotoxic effect of Ginger powder 7,12- Dimethyl benz(a) anthracene induced
genotoxicity in rat bone marrow cells was evaluated.Six to eight weeks old male wistar albino
rats were divided into six groups (n=6). Group I (control) animals were administered with
standard diet. Group III and IV animals were fed with diet mixed with 5% and 10% ginger
powder, for thirty consecutive days. Twenty four hours after the last test dose, Group II, III and
IV rats were injected with 7,12-Dimethyl benz(a)anthracene (DMBA) 30 mg/kg b.w.
intraperitoneally. Group V and VI animals were kept with diet containing 5% and 10% ginger
powder alone respectively. Twenty four hours after DMBA injection, the animals were sacrificed
and were injected with colchicine 2 mM (0.5ml/100gm b.w. ip). Frequency of micronuclei in
polychromatic (P) and normochromatic (N) erythrocytes and scoring of chromosomal aberrations
were done in rat bone marrow cells. Plasma MDA, SOD and liver MDA, SOD and GSH were
also estimated.10% Ginger powder mixed diet produced a significant inhibition of DMBA
induced alteration in P/N ratio and frequency of micronuclei. It decreased the abnormal
metaphases as well as total chromosomal aberrations significantly, inhibited the DMBA induced
chromosomal breaks, gaps, rings, deletions and other abnormalities in bone marrow cells. Also,
it restored the biochemical changes (plasma MDA, SOD and liver MDA, SOD and GSH ) in
DMBA injected rats to a significant extent (p<0.001). Ginger has antigenotoxic activity against
DMBA induced genotoxicity.
Key words: Antioxidants, Chromosomal aberrations, DMBA, Genotoxicity, Zingiber officinale.
2
1. INTRODUCTION:
Various factors are recognized to play a major role in the etiology of different diseases including
cancer.[1] Exposure to environmental pollutants such as poly aromatic hydro carbons (PAH) is
one of the major risk factors responsible for the onset of various diseases associated with
oxidative stress [2], accumulation of reactive oxygen free radicals (ROS).[3, 4]
Oxidative stress is an imbalance between oxidant and antioxidant status, plays a crucial role in
the pathogenesis of several diseases including cancer.[3] DMBA, a prototype of PAH is a potent
pro-carcinogen which on metabolism form diol epoxides and other toxic reactive oxygen species
(ROS).[5,6] These toxic reactive oxygen species can induce chromosomal aberrations and
formation of micronuclei through oxidative base damage and strand breaks in DNA, contributing
to mutagenesis and carcinogenesis.[6]
Plant have the capacity to synthesize a wide variety of phytochemicals they can have
complimentary actions against reactive oxygen species.[7,8] Different food additives like spices
and condiments has been used to enhance the taste and flavor of our preparation since ancient
times. People in many parts of the world often consume several kinds of spices such as
coriander, caraway, chili, pepper, black pepper, garlic, onion, cinnamon, etc. Zingiber officinale
(ginger) family Zingiberaceae, though commonly used as a spice especially in Southeast Asian
countries for a long time, its traditional use for anti-inflammatory[9] anti-rheumatism,
[10]
anti-
oxidant as well as for respiratory condition like bronchitis, common cold cough and ant-emetic
properties helps to prevent nausea and vomiting during motion sickness and pregnancy ect are
well documented. [11]
The important active components of the ginger rhizome are thought to be volatile oils and
pungent principles like
phenolic
compounds such as gingerols [6-gingerol], shogaols,
3
zingerone, and gingiberols.[12,13] several authors have demonstrated that the zinger endowed with
strong in-vitro and in-vivo anti-oxidant properties.[14,15] The anti-genotoxic action of ginger has
been found as one of the possible mechanism of oxygen free radical scavenging followed by
decreased production of reactive oxygen species.[16] The protective role of ginger powder on
DMBA induced carcinogenesis has been evaluated in an in vitro study to explaining its anti –
genotoxic
potential.[17,18] Thus Ginger having anti-mutagenic, anti-oxidant and anti-
inflammatory effects might be a good anti-genotoxicant.
Although the beneficial effects of
ginger have been exploited, little research has been conducted on its anti-genotoxic effect in invivo models. With this background, the present study was undertaken to evaluate the antigenotoxic effect of powder of Zingiber officinale on 7, 12-Dimethyl Benz (a) anthracene
(DMBA) induced genotoxicity in male wistar rats.
2. MATERIAL AND METHODS:
Male Wistar rats of 6-8 weeks old were obtained from the National Institute of Nutrition,
Hyderabad. The animals were housed individually in separate cages in standard laboratory
condition (temperature 22-25˚C, relative humidity 65 ± 5% and 12 h light/dark cycle). They were
fed with standard diet (NIN formula) and water ad libitum. Prior to the experiment, they were
acclimatized to the laboratory conditions for 1 week.16 This study protocol was approved by
Institutional Animal Ethics Committee (Regd. No. 472/2005/CPCSEA) and the experiment was
conducted as per OECD guidelines.
2.1 Drugs and Chemicals:
Rhizome of Zingiber officinale were purchased from local market and authenticated by Prof.
M.K. Mishra, Department of Botany, Berhampur University
4
DMBA (Sigma, USA) Colchicine, May-Grunwald stain, Giemsa stain, fetal bovine serum were
purchased from Hi-media Laboratories, Mumbai, India. All the chemicals were of analytical
grade.
2.2 Preparation of Diet:
Ginger was purchased from local market, peeled, washed, coarsely minced then air dried and
pulverized with a mixture grinder and passed through the mesh to a fine powder. The food
pellets were prepared in 15 kg lots by mixing 5 and 10 % of Zinger powder in two-dimensional
rotating mixture under low light condition, diet were stored at 4 ºC in the dark until use. The
stock diet contained wheat flour 15%, roasted Bengal gram flour 58%, groundnut flour 10%,
skimmed milk powder 5%, refined oil 4%, salt mixture 4% and vitamin mixture 0.2%, casein
4%. The nutritive value of the diets fed to the control and experimental group were identical.
[17,18]
2.3 Experimental protocol:
A total no. of thirty-six male wistar albino rats were divided into 6 groups (n=6).
Group I
(control): Standard diet.
Group II (Disease control) : 7,12- Dimethyl benz(a) anthracene (30 mg/kg ip).
Group III and IV (Test drug): 5% and 10% ginger powder mixed diet (30 days)
[19,20]
+ 7,12-
Dimethyl benz(a) anthracene (30mg/kg i.p.) one hour after the last dose to induce
genotoxicity.[21]
Group V and VI (Test drug): 5% and 10% ginger powder mixed diet alone for 30 days).
(After 30 days there was no more change in their body weight.)
Twenty four hours after the 7,12- Dimethyl benz(a) anthracene injection, all the animals was
injected with 2 mM of colchicine (0.5 ml/100 gm b.w.i.p.).[16,17] After two hours, the animals
5
were anaesthetized with (pentobarbitone 45 mg/kg ip) and blood samples were collected for
estimation of superoxide dismutase (SOD) and malondione aldehyde ( MDA). Then animals
were sacrificed by cervical dislocation and bone marrow of femurs was collected for the
assessment of bone marrow micronucleus frequency
[22]
and chromosomal aberrations.
[23, 24]
Livers were also excised for estimation of SOD, MDA and GSH.[25- 27]
For Bone marrow micronucleus assay the method developed by Schmid (1975) was followed.[22]
Quickly, the bone marrow from femur was collected in a tube containing 1 ml of fetal bovine
serum and centrifuged at 3000 rpm for 5 min
[17]
The collected pellet was re-suspended with
few drops of fresh fetal bovine serum and vortexed. One drop of the suspension was smeared on
coded glass slides, air dried, fixed in methanol, stained with May-Gruenwald stain followed by
Giemsa. After drying, the polychromatic erythrocytes (2000 PCEs) were screened for
micronuclei (MNPCEs) and normochromatic erythrocyte (NCEs) to calculate PCE/NCE ratio. [6]
The method of Adler and Savage was adopted for the chromosomal aberration test. Quickly after
sacrificing the rats, the femoral bone marrow was flushed in to tube containing 6 ml of hypotonic
KCl (0.075M) The marrow suspension was incubated at 37ºC for 15-20 min and centrifuged at
1000 rpm for 10 min. The pellet was mixed with the methanol: acetic acid fixative (3:1). The
suspension was allowed to stand for 30 min and then centrifuged. The pellet was mixed
thoroughly with fresh fixative and 2-3 drops of the suspension was dropped on a clean glass
slides. The slides were flame-dried and stained with 10% Giemsa at pH 6.8 for 15-20 min.
Coded slides were screened for chromosome abnormalities. One hundred well-spread metaphase
plates per animal were scored for structural chromosomal aberrations and recorded.
Concurrently, total chromosomal abnormalities (excluding gaps), number of abnormal
6
metaphase plates per one hundred metaphase plates was counted per rat, and mitotic index was
calculated. [26, 27]
MDA in plasma and liver was estimated by the method of Dhale et al, 1962 [26]. The method of
P. Kakar et al , 1984[25] and Sedlak and Lindsay (1968) [27] was followed for estimation of SOD
and liver GSH respectively.
2.4 Statistical analysis:
The data were expressed as mean ± SEM. Statistical comparison were performed by One Way
ANOVA followed by Duncann’s multiple range test (DMRT). Results with p < 0.05 were
considered statistically significant.
3. RESULTS:
3.1 Effect of Zingiber officinale on bone marrow micronucleus assay:
The frequency of micronuclei, P/N ratio and % PCE are expressed in table-I. Rats treated with
DMBA alone produced a significant increase (p<0.05) in micronucleated cells in polychromatic
erythrocytes and reduced P/N ratio significantly as compared to the control rats. There was
decrease in MNPCE and increase in P/N ratio with pre-treatment of 10% ginger mixed diet to
DMBA injected rats to a significant extent (p<0.05). However, with pre-treatment of 5% ginger
powder mixed diet to DMBA treated rats, no significant change in all the parameters of bone
marrow micronucleus assay was observed. Also ginger powder mixed diet alone in both the
doses did not alter the MNPCE, P/N ratio and % PCE as compared to the control group.
Effect of Zingiber officinale on bone marrow chromosomal aberration assay:
The results of chromosomal analysis in bone marrow cell at metaphase stage are depicted in
Table-II. The mitotic index of DMBA treated rats (gr-II) was reduced significantly along with a
7
significant increase in chromosomal breaks, rings, other chromosomal abnormalities (deletion,
fragmentation, minutes etc.) and total chromosomal abnormalities as compared to the control
rats. Only the 10% ginger powder mixed diet fed rats with DMBA treatment showed a
significant (p<0.05) difference in mitotic index and all the abnormal chromosomal patterns in
comparison to the DMBA treated rats (grp.-II). No significant difference was observed between
control rats and rats fed with 5% and10% ginger powder mixed diet alone with respect to
chromosomal aberrations.
3.2 Effect of Zingiber officinale on biochemical parameters:
Table III shows the effect of ginger on various biochemical changes in Plasma and liver of
different treatment groups. DMBA alone increased MDA level and decreased SOD in Plasma
and liver significantly (p<0.05) as compared to the control rats. Treatment with 10% ginger
powder mixed diet plus DMBA significantly altered the above said enzymatic changes with
significant increase in liver GSH in comparison to the DMBA control group (p<0.05). No
significant change with 5% ginger powder plus DMBA treatment was observed as compared to
DMBA group. Also no significant (p>0.05) difference was observed between ginger alone and
control rats.
Table I: Effects of ginger in the diet on DMBA-induced micronuclei in bone marrow cells.
Treatment
Group
% of MNPCE PCE/NCE % of PCE*
I
Std. Diet
0.07±0.02a
1.04±0.02 a
50.93
b
b
b
I
DMBA(30mg/kg)
0.66±0.05
0.41 ±0.01
28.94
III
5 %ZP+DMBA
0.51±0.06 b
0.49±0.01 b
32.69
c
c
IV
10% ZP+DMBA
0.27±0.04
0.69±0.03
40.85
V
5% ZP
0.05±0.04 a
1.07±0.02 a
49.06
a
a
VI
10% ZP
0.06±0.02
1.06±0.03
51.44
Values are expressed as mean ± SE (n=6). Data analyzed by one-way ANOVA with post hoc
Duncan’s multiple range test (DMRT). The values not showing common super script differ
significantly (p < 0.05)*Percentage of polychromatic erythrocytes was calculated as follows:
[PCEs / (PCEs+NCEs) × 100.
8
Table II:-Effect of Zingiber officinale on bone marrow chromosomal aberration assay.
Chromosomal aberration per rat
Total
Group
Drug & dose
aberrations
Other
Gap
Break
Ring
per rat
aberration
a
a
a
a
a
I
Std.Diet
4.74 ±0.31
0.5 ±0.22
1.17 ±.40
0.17±0.17
1.00 ±0.36
2.33 ±0.67 a
b
b
b
b
b
II
DMBA30mg/k 1.07±0.19
12.17±0.95
14±1.07
5.17±0.79
12±0.93
31.17±1.62 b
b
b
b
b
b
g ZP+DMBA 1.92±0.39
III
5%
10.67±0.67
1.3±0.73
4.83±0.48
10.5±0.43
28.33±1.18 b
IV
10%ZP+DMB 3.20±0.36 c 5.83±0.60 c
5.33±0.42 c 2.5±0.43 c
7.33±0.96 c 15.17±1.02 c
a
a
a
a
A
V
5% ZP
3.98±0.19
0.45 ±0.31
1.55 ±0.33
0.7±0.29
1.21 ±0.33 a 3.54±0.40 a
VI
10% ZP
4.47±0.45 a
0.50±0.22 a 1.67±0.42 a
0.5±0.34 a
1.17±0.31 a 3.33±0.42 a
Values are expressed as mean ± SEM where n=6. Data analyzed by one-way ANOVA with post
hoc Duncan’s multiple range test (DMRT). The values not showing common super script differ
significantly (p < 0.05)
*
Mitotic index has been calculated by analyzing 1000 cells/animal (for a total of 6000
cells/treatment) and percentage of the mitotic cells calculated for each treatment group.
**
Frequency per 100 cells. Each chromosomal aberration has been counted by analyzing 100
cells/animal (6 animals/group, for a total of 600 cells/treatment) and mean ±SE were calculated
per treatment group.#Gaps were not included in total chromosomal aberrations.
*Mitotic
index (%)
Table -III:- Effect of Zingiber officinale on biochemical parameters.
Plasma
Liver
Group
Drug & dose
SOD
MDA
SOD
MDA
GSH
n=6
U/ml
nmol/ml
U/100mg
nmol/100m µmol/100mg
c
g
I
Std.Diet
32.37±0.95 c
3.25±0.26 c
25.71±1.05 c
6.14±0.43
15±0.05 c
a
a
a
a
II
DMBA(30mg/kg) 20.51±0.73
6.46±0.35
18.59±1.24
11.92±0.72
7.58±0.87 a
a
a
a
a
III
5%ZP +DMBA
24.75±1.10
5.83±0.19
19.58±0.89
10.38±0.49
10.42±0.77 a
IV
10% ZP +DMBA 30.94±1.25 b,c 4.38±0.27 b,c 22.91±1.46 b,c 7.25±0.32 b,c 14.50±0.82 b, c
V
5% ZP
30.55 ±0.99 c 2.65 ±1.04 c 25.85 ±0.79 c 5.68 ±0.75 c 15.21 ±0.84 c
VI
10% ZP
31.42±0.95 c
2.88±0.20 c
24.28±0.64 c
5.96±0.30 c
15.42±0.77 c
VI Values are expressed as mean ± SE (n=6). Data analyzed by one-way ANOVA with post hoc
Duncan’s multiple range test (DMRT). The values not showing common super script differ
significantly (p < 0.05)
III
9
a
c
b
d
Fig.1: a- Chromosomal break, b-Ring chromosome, c-Minute, d- Fragments
4. DISCUSSION:
A lot of interest for studying the genotoxic and antigenotoxic potential of some dietary
constituents has been taken since last decades. In a number of studies, emphasis has been laid
down on use of certain dietary constituents for prevention of many drug and chemical induced
genotoxicity (Ferulic acid, Adhatoda vasaca, lipoic acid, Aloe Vera garlic, curcumin, gingiber,
piperin, carotinoidsand vit-C and E).[17,28-31] With such a background the present study has been
carried out to evaluate any anti-mutagenic activity against 7,12-Dimethylbenz(a)anthracene
(DMBA) induced genotoxity
DMBA present in the environment as a product of incomplete combustion of complex
hydrocarbons is an indirect carcinogen. It forms DNA attacking toxic reactive oxygen species
which cause chromosomal aberrations. (Christou et al).[32] Both in vivo and in vitro studies have
demonstrated polyploidy and sister chromatid exchanges in DMBA induced genotoxicity. An
10
increase in micronucleus frequency and chromosomal aberrations in bone marrow of DMBA
painted rodents have been reported. (Husain et al, 1989).[33] In many research studies DMBA has
been proofed to be a mutagenic and carcinogenic due to generation of excess ROS and induce
lipid peroxidation .[17]
In the present study, single intraperitoneal injection of DMBA ( 30 mg/kg B.W.) showed a
significant increase in frequency of MNPCEs and decrease in P/N ratio as well as %PCEs in
comparison to standard diet treatment group (Table-I). These findings corroborate with that of
Pachaiappan and Shanmugam et al, 2009. [6]
In chromosomal aberration test DMBA even reduced mitotic index, increased frequency of
chromosomal break(figure- a), ring(figure- b) and other multiple aberrations like minutes and
fragments (figure- c,d) to a highly significant extent (Table-II). A similar observation was
recorded by Kuppusamy et al, 2008,[5] On biochemical parameters, DMBA decreased plasma
SOD, liver SOD, liver GSH but increased the lipid peroxidation marker that is plasma MDA and
liver MDA to a highly significant extent.(Table-III). Our results thus suggest that the observed
increase in the frequency of MnPCEs and chromosomal aberrations in DMBA treated rats are
due to excessive generation of reactive oxygen species. Subramanian et al, 2008 and
Pachaiappan et al, 2009 also observed similar biochemical changes following DMBA injection.
[17, 6]
There was no modification of DMBA induced changes in the frequency of MNPCEs, P/N ratio
and % PCEs on bone marrow smear , incident chromosomal aberrations and any of the
biochemical parameters in plasma and liver was observed with pretreatment of 5% ZP mixed
standard diet (Table-III)
11
Administration of 10% ZP mixed diet for 30 days prior to DMBA injection, significantly
reversed the alteration in %PCEs and P/N ratio on bone marrow micronucleus assay.(Table-I)
Also this dose of ZP modified the DMBA induced changes in mitotic index, incidence
chromosomal break(figure- a), ring(figure- b) and other multiple aberrations to a significant
extent as compared to that of DMBA treated group alone (Table-II) All these above parameters
of the chromosomal aberration test and micronucleus test following 10% ZP treatment in diet
were comparable to that of standard diet treatment group alone. A similar observation was found
in the study of K. Nirmala et al, 2007 who have reported the in vivo antimutagenic potential of
Ginger on formation and excretion of urinary mutagens in rats using an in vitro assay method. [15]
With respect to biochemical estimations, pretreatment with 10% ZP mixed diet restored the
DMBA induced decrease in plasma and liver SOD, liver GSH to a normal level (comparable to
control animal) (Table-III). Again DMBA induced rise in lipid peroxidants, measured from
plasma and liver was reduced to near normal by 10% ZP mixed diet pretreatment (Table-III).
Our observations corroborate with that of Sekiwa et al 2000 and Zancan et al, 2002. [34, 35]
The strong antioxidant activity of ginger is well documented in earlier studies.[2-4] Considering
the observations in our study, the anti- genotoxic activity exerted by Gingiber could be explained
through its anti oxidant activity against DMBA induced oxidative stress and subsequent nuclear
damage in erythropoetic cells.
5. CONCLUSION:
Thus in our in vivo study, Ginger possessed anti-genotoxic effect along with antioxidant activity
in wistar albino rats The regular consumption could be therefore beneficial to counteract the
adverse effects by enhancing antioxidant defense mechanism and neutralizing the toxic effect of
12
reactive oxygen species generated by environmentally present genotoxicants. More research
studies on other genotoxic models are suggested to establish the role of ginger against the
genotoxicants.
6. ACKNOWLEDGEMENT:
Author is thankful to Dr. Satpal Singh Bist and Mr. Ghanashyam Panigrahi for his kind guidance
and technical support for the research work.
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