Journal of Ethnopharmacology 134 (2011) 329333

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Role of Syzygium cumini seed extract in the chemoprevention of in vivo genomic

damage and oxidative stress
Renganathan Arun a , M. Velayutham Dass Prakash a , Suresh K. Abraham b , Kumpati Premkumar a,
a
Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
b
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

a r t i c l e i n f o a b s t r a c t

Article history: Ethanopharmacological relevance: The seeds of Syzygium cumini, Skeels (Jamun) are extensively used in
Received 11 March 2010 India for treatment of diabetes and other ailments.
Received in revised form Aim of the study: The aim of this work was to assess the role of Jamun seed extract (JSE) as a chemopro-
21 November 2010
tective agent against in vivo oxidative stress and genomic damage.
Accepted 14 December 2010
Materials and methods: Experiments were carried out to evaluate in vitro protective effects of JSE against
Available online 21 December 2010
hydroxyl radical induced damage in pBR322 DNA, and in vivo genomic damage and oxidative stress in
mice which received JSE orally for 5 days before exposure to genotoxic carcinogens urethane (URE) and
Keywords:
Syzygium cumini
7,12-dimethyl benz(a)anthracene (DMBA).
Urethane Results: Aqueous and ethanolic extracts of JSE showed signicant protective effects against hydroxyl rad-
DMBA ical induced strand breaks in pBR322 DNA. The in vivo experiments with aqueous JSE showed signicant
Antioxidant protective effects against chromosomal damage induced by the genotoxic carcinogens URE and DMBA.
Micronucleus Biochemical assays registered signicant inhibition of hepatic lipid peroxidation and increase in GSH
Chromosomal aberration level and activity of GST, SOD and CAT.
Oxidative stress Conclusion: Our ndings suggest that JSE can possibly play an important role as a chemopreventive agent
against oxidative stress and genomic damage.
2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction et al., 1996), gastro-protective (Chaturvedi et al., 2007) and radio-

The present study was initiated with the main aim of evaluat-
ing the possible antigenotoxic effects of the medicinal seed extract
obtained from Syzygium cumini, Skeels (Synonym: Eugenia jam-
bolana Lam.; Family Myrtaceae). In India, the deep purple colored,
oblong edible fruit with a large centrally located seed is commonly
known as Jamun. The fruit pulp and seed extract of Jamun have a
long history of medicinal use. Extensive laboratory investigations
carried out during the last three decades have furnished substan-
tial information on the antidiabetic properties of this tropical fruit
(Prince et al., 2003). Besides, there are reports on the antioxidant
(Banerjee et al., 2005; Benherlal and Arumughan, 2007), anti-
inammatory (Chaudhuri et al., 1990), antipyretic (Ghosh et al.,
1985), anti-allergic (De Brito et al., 2007), anti-bacterial (Bhuiyan
protective properties of Jamun seed extract (JSE) (Jagetia and Baliga,
2002).
In view of the paucity of information on the antigenotoxic
effects of this antioxidant-rich medicinal fruit (Veigas et al., 2007;
Reynertson et al., 2008), we initiated the present study to evalu-
ate the possible protective effects of aqueous JSE against in vivo
genomic damage and oxidative stress induced by the genotoxic
carcinogen urethane (URE) and 7,12-dimethylbenz(a)anthracene
(DMBA). Prior to this, in vitro tests were carried out with JSE to
evaluate the free radical induced strand breaks in pBR322 DNA.
2. Materials and methods
2.1. Chemicals

Abbreviations: JSE, Jamun seed extract; URE, urethane; DMBA, 7,12-dimethyl
benz(a)anthracene; LPO, lipid peroxidation; GSH, reduced glutathione; GST, glu-
tathione S-transferase; SOD, superoxide dismutase; CAT, catalase; SD, standard
deviation; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes;
MnPCE, micronucleated PCE.
Corresponding author. Tel.: +91 8056589893; fax: +91 0431 2407045.
E-mail address: pkumpati@hotmail.com (K. Premkumar).
Genotoxic chemicals and stains used for the present study were
obtained from Sigma Chemicals (USA). All the other chemicals were
purchased from Merck (India) and Qualigens (India).
2.2. Plant materials
Syzygium cumini (Jamun) seeds were collected freshly during
JuneAugust from a location in Tiruchirappalli (Tamil Nadu, India).

0378-8741/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.12.014
330 R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329333

The seed coat was removed after partial dryness and shade dried
again for 3 days. The dried seeds were coarsely ground the powder
was stored in a cool and dry place at room temperature. The plant
was identied by Dr. S. John Britto SJ, Director, RAPINET Herbar-
ium & Centre for Molecular Systematics, St. Joseph College Campus,
Tiruchirappalli and a voucher specimen No. RHT. 68473 has been
deposited in the herbarium.

2.3. Sample preparation

100 g of seed powder was extracted with different solvents

(ethanol, acetone, ethyl acetate and aqueous) in material to solvent
ratio of 1:2 (w/v) at ambient temperature (2530 C) under contin-
uous stirring for 5 h and repeated three times. After each extraction,
the residue was ltered through muslin cloth and the ltrate was
pooled and stored at 4 C until further use. The clear ltrate was
concentrated in a rotary evaporator at low temperature (<40 C)
under vacuum. The concentrated extracts were made to dry in oven
at 60 C and stored at 20 C till further analysis.

2.4. Qualitative phytochemical screening of extracts

Chemical tests were carried out using the different extracts from
plants and/or the powdered specimens, using standard procedures
to identify the constituents as described by Sofowora (1993), Trease
and Evans (1989) and Harborne (1973).
2.5. Determination of total phenolic and avonoid content of
extract
Total phenolic and avonoid content of Jamun seed extracts
(JSEs) was determined colorimetrically by the method of McDonald
et al. (2001) and Chang et al. (2002), respectively.
Fig. 1. Protective effects of ethanol and aqueous JSE on hydroxyl radical (OH )
induced pBR322 DNA strand breaks. (A) Lane 1: DNA + potassium phosphate
buffer (PPB; 20 mM), Lane 2: DNA + Fentons reagent (FR), Lane 3: DNA + EtOH JSE
(250 g/ml) + FR, Lane 4: DNA + EtOH JSE (500 g/ml) + FR, Lane 5: DNA + Aq. JSE
(250 g/ml) + FR, Lane 6: DNA + Aq. JSE (500 g/ml) + FR, Lane 7: DNA + EtOH JSE
(500 g/ml), Lane 8: DNA + Aq. JSE (500 g/ml). (B) Total protective percentage of
ethanol and aqueous JSE (250 and 500 g/ml) on hydroxyl radical (OH ) induced
plasmid DNA strand breakage. Data are presented as mean SD of three experi-
ments.
2.9. Animals

2.6. Determination of free radical scavenging activity and
reducing power of JSE
The DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging
activity and reducing power of JSEs was determined by the method
of Koleva et al. (2002) and Oyaizu (1986).
2.7. DNA nicking assay
DNA cleavage assay was performed using supercoiled pBR322
DNA (Bangalore Genei Ltd., Bangalore, India) by the method of Lee
et al. (2002).
2.8. HPTLC determination of avonoids and phenolic acids
All the experiments were carried out using 1012 weeks old
male and female Swiss albino mice maintained in the University
animal house on standard mouse pellets and water ad libitum in
accordance with CPCSEA, India guidelines. Approval for this work
was obtained from the University animal ethical committee.
2.10. In vivo assays for determining genomic damage
2.10.1. Mouse bone marrow micronucleus test
Genotoxic effects were evaluated in the mouse bone marrow
micronucleus test according to Schmid (1975). 2500 polychromatic
erythrocytes (PCEs) were scored per animal per slide to determine
the frequency of micronucleated polychromatic erythrocytes (Mn
PCEs). All the slides were scored by the same observer.

Preliminary qualitative phytochemical screening of JSE tested
for the presence of avonoids and phenols. The HPTLC quan-
tication was performed at Indian Institute of Crop Processing
Technology (IICPT), Ministry of Food Processing Industries, Govt. of
India, Tanjavur, Tamil Nadu, India with the standardized protocol.
Briey, aqueous extract of Jamun seed and mixed standards (gallic
acid, rutin, ferulic acid, caffeic acid and quercetin) were dissolved in
HPLC grade methanol and applied to pre washed silica gel 60 F254
HP-TLC plates (10 cm 10 cm, silica-gel thickness 2 mm, Merck)
with an automator applicator (Linomat IV; CAMAG). The samples
were then separated (migration distance 75 mm) using HPLC grade
solvents. Then the plates were scanned in CAMAG TLC scanner and
the peaks were recorded at a wavelength of 366 nm. The Rf values
and concentration of separated compounds were determined with
WINCATS planar chromatography Manager Software.
2.10.2. Mouse bone marrow chromosome aberration test
The in vivo chromosomal aberration analysis was performed
according to the protocol of Kilian et al. (1977). One hundred-well
spread and uniformly stained metaphases, having 40 1 chromo-
somes, per animal and 600 metaphases per dose were observed for
the presence of chromosomal aberrations.
2.11. Biochemical assays
Hepatic reduced glutathione (GSH) level and glutathione S-
transferase (GST) activity was determined by the method of Moron
et al. (1979) and Habig et al. (1974). Activities of superoxide oxide
(SOD) and catalase (CAT) were assayed by the method of Marklund
and Marklund (1974) and Sinha (1971), respectively. Lipid peroxi-
dation (LPO) in liver was estimated colorimetrically by the method
 R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329333 331

Fig. 2. Effects of aqueous JSE on urethane induced oxidative stress in liver. Effects of aqueous JSE on (A) lipid peroxidation (LPO) expressed as nmol of MDA formed per mg
protein. (B) Superoxide dismutase (SOD) activity expressed as 50% inhibition of pyrogallol auto-oxidation/min/mg protein. (C) Catalase (CAT) activity expressed as moles
of H2 O2 consumed/min/mg protein. (D) Glutathione S-transferase (GST) activity expressed as nM of CDNB conjugated min1 mg1 protein. (E) Reduced glutathione (GSH)
levels expressed as g of GSH formed mg1 protein. All values are expressed in mean SD with six mice in each group. ### p < 0.001; ## p < 0.01 compared with normal control
group; ***p < 0.001; **p < 0.01compared with URE and aqueous JSE treated groups.

of Ohkawa et al. (1979). The total protein was determined by the
method of Bradford (1976).
2.12. Statistical analysis
All data were expressed as means SD of number of experi-
ments (n = 6). The statistical signicance was evaluated by one-way
analysis of variance (ANOVA) using SPSS version 11.5 (SPSS, Cary,
NC, USA) and the individual comparison were obtained by Duncans
Multiple Range Test (DMRT). A value of p < 0.05 was considered to
indicate a signicant difference between groups.
3. Results
Preliminary phytochemical screening of aqueous, ethanol, ace-
tone and ethyl acetate extracts of Jamun seed have shown
positive results for carbohydrates, phytosterol, phenols, tannins,
phlobatannins and avonoids. Terpenoids were detected in the
ethanol, acetone and ethyl acetate extracts. The total phenolic
content in the aqueous (168.33 7.64), ethanol (471.67 29.3),
acetone (230 25) and ethyl acetate (375 40.93) extracts were
determined with respect to gallic acid equivalent (mg/g of
extract). The avonoids content in the aqueous (65.31 1.77),
ethanol (114.88 5.36), acetone (103.86 3.67) and ethyl acetate
(138.26 6.58) extracts were determined with respect to quercetin
equivalent (mg/g of extract).
The DPPH radical scavenging activity of different JSEs at 5, 10,
25, 50 and 100 g/ml was less when compared to gallic acid. How-
ever, with 100 g/ml of different JSEs, the antioxidant activity was
similar to that of gallic acid. The IC50 values for antioxidant activity
of gallic acid, aqueous, ethanol, acetone and ethyl acetate extracts
are 25.33, 25.02, 24.53, 24.29 and 24.42 g/ml, respectively. The
reductive potential of different JSEs at concentrations 20, 40, 60, 80,
100 g/ml were approximately one-third of that observed for the
positive control ascorbic acid. The IC50 values for reductive poten-
tial of ascorbic acid, aqueous, ethanol, acetone and ethyl acetate
332 R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329333

Fig. 4. Effects of aqueous JSE on urethane induce chromosomal aberrations in bone

marrow cells of mice. All bars are expressed in mean SD of total chromosomal
aberrations per cell with six mice in each group. ### p < 0.001 compared with normal
control group; ***p < 0.001 compared with URE and aqueous JSE treated groups.
Fig. 3. Effects of aqueous JSE on URE or DMBA induced micronucleated polychro-
matic erythrocytes (MnPCEs) in bone marrow cells of mice. All bars are expressed
in mean SD of total number of MnPCEs per 2500 PCEs with six mice in each group.
(a) ### p < 0.001; compared with normal and JSE alone groups; (b) ***p < 0.001; com-
The results from our present study have demonstrated for the
pared with urethane and aqueous JSE treated groups; (c) ***p < 0.001; compared
with DMBA and aqueous JSE treated groups.
rst time that aqueous JSE can exert signicant in vivo antigeno-
toxic effects against the indirectly acting carcinogens URE and
DMBA, and protect against in vitro DNA damage induced by free
radicals. DNA damage resulting from free radical induced oxida-
extracts are 40.10, 60.10, 59.10, 59.10 and 59.95 g/ml, respec-
tive stress can lead to gene mutation and chromosome aberrations.
tively. The IC50 values obtained for ascorbic acid was lower when
In this study, we observed signicant reductions in chromosome
compared to that for the different JSEs.
aberrations in metaphase cells and micronuclei in polychromatic
From Fig. 1, it is evident that the presence of aqueous and
erythrocytes formed as a result of chromosomal damage. Many
ethanol JSEs along with plasmid pBR322 DNA during Fentons reac-
phytochemicals with antioxidant activity can either minimize or
tion protected DNA from OH radical induced strand breaks. At a
prevent this potentially harmful genomic damage mediated by
concentration of 500 g/ml, the aqueous and ethanol extract pro-
oxidative stress. From the biochemical assays carried out with the
tected the DNA from OH radicals to the extent of 90% and 99%,
liver samples of these animals, there is evidence for the role of JSE
respectively.
pre-treatment in inhibiting LPO and enhancing the level of GSH and
The results of the biochemical assays for determining the hep-
activity of the antioxidant enzymes GST, SOD and CAT. These results
atic LPO, GSH level and activity of SOD, CAT and GST in the control
suggest that the antioxidant property of JSE is responsible for the
and treated mice have been presented in Fig. 2. Increase in LPO
observed chemopreventive effect.
level and a concomitant decrease in GSH, GST SOD and CAT were
URE, the genotoxin used in our study is found in trace amounts
observed in mice treated with the genotoxin URE alone. Adminis-
in fermented foods and beverages and it is not genotoxic or car-
tration of three doses of aqueous JSE to the experimental animals for
cinogenic per se (Kemper et al., 1995). However, vinyl carbamate
ve consecutive days before exposure to URE showed a reduction
epoxide is believed to be the metabolite responsible for its geno-
in LPO and a dose-related increase in GSH, GST, SOD and CAT.
toxic and carcinogenic effects. Conjugation of vinyl carbamate
Fig. 3 illustrates the results of the in vivo assays for evaluating
epoxide with GSH can lead to detoxication and this process is
genomic damage. It shows that pre-treatment of mice with three
catalyzed by GST (Kemper et al., 1995). From our results it is
doses (500, 1000 and 1500 mg/kg b.w.) of aqueous JSE for ve con-
evident that pre-treatment of mice with JSE has increased the
secutive days resulted in signicant reductions in the frequencies
level of GSH and activity of GST. These results indicate that the
of micronuclei induced by the genotoxic carcinogen either URE or
inducible GST defense system has possibly played an important
DMBA. This protective effect was not always dose-related. A similar
role in reducing the reactive form of URE which can cause DNA
trend was observed when the frequencies of chromosome aberra-
lesions.
tions were evaluated in the metaphase cells of mice pretreated with
A search for the bioactive constituents of JSE would lead to sev-
three doses of aqueous JSE and challenged with URE (Fig. 4). The
eral potential candidates. HPTLC analysis of aqueous JSE used in
incidence of aberrations/cell was signicantly reduced when com-
the present study showed the presence of rutin, quercetin, gallic
pared to that of URE alone. Treatment of mice with the combination
acid, ferulic acid and caffeic acid (data not shown). When a com-
of URE/DMBA with JSEs did not show any signicant change in the
plex mixture of bioactive phytochemicals is used for evaluating
percentage of PCEs. There was no signicant increase in genotoxic-
antigenotoxic effects, the results would largely depend on the syn-
ity following pre-treatment of mice for 5 days with the highest test
ergistic/additive interaction between the above chemopreventive
dose (1500 mg/kg b.w) of aqueous JSE.
phytochemicals (Liu, 2004).

4. Discussion
Today, a wide range of antioxidant phytochemicals present in
food stuffs and beverages are known to exert chemopreventive
effects. This prompted us to investigate whether the traditional
phytomedicine JSE which has shown antioxidant activity can
protect against DNA damage induced by environmental genotox-
ins/carcinogens.
5. Conclusion
In conclusion, our present study has demonstrated for the rst
time that oral administration of the phytomedicine aqueous JSE
to mice can enhance the activity of the antioxidant defense and
lead to protective effects against genomic damage induced by the
carcinogens DMBA and URE.
 R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329333
Acknowledgements
The authors thank Bharathidasan University, Tiruchirappalli,
Tamil Nadu, India for providing the Financial Support to Dr. K.
Premkumar as a startup fund. The authors would like to thank Mr.
S. Kumaravel, Senior Scientist and Mr. R. Paranthaman, Chemist,
IICPT, Thanjavur, Tamil Nadu, India for the technical help rendered
for HPTLC analysis.
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