ORIGINAL ARTICLE

Cytoprotective effects of fruit pulp of Eugenia jambolana

on H2O2-induced oxidative stress and apoptosis in rat
Leydig cells in vitro
H. Anand1,2, M. M. Misro1, S. B. Sharma3 & S. Prakash2
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India;
2 Department of Zoology, Dayalbagh Educational Institute, Dayalbagh, India;
3 Department of Biochemistry, University College of Medical Sciences, University of Delhi, India

Keywords
ApoptosisEugenia jambolanaH2O2
Leydig cellsoxidative stress
Correspondence
M. M. Misro, Department of Reproductive
Biomedicine, National Institute of Health and
Family Welfare, Baba Gangnath Marg,
Munirka New Delhi-110067, India.
Tel.: +91 11 26165959;
Fax: +91 11 26101623;
E-mail: mm_misro@yahoo.com
Accepted: May 02, 2012
doi: 10.1111/j.1439-0272.2012.01323.x

Summary
This study was undertaken to investigate the cytoprotective effect of the fruit
pulp of Eugenia jambolana (50250 lg ml 1) against the damage induced by
H2O2 (100 lM) exposure to Leydig cells in vitro. Cell survival with extract was
found comparable to similar effects by N-acetyl-L-cysteine. H2O2-induced rise
in thiobarbituric acid reactive substance formation and decline in the activity
and expression of antioxidant enzymes like superoxide dismutase, catalase and
glutathione-s-transferase were effectively checked. Cellular glutathione and total
antioxidant capacity demonstrated significant improvement. The increase in
expression of inducible nitric oxide (NO) synthase leading to NO production
was successfully countered. Co-treatment of the extract helped in the downregulation of caspase-3 and poly-ADP-ribose polymerase resulting in a significant reduction in Leydig cell apoptosis induced by H2O2. Upstream marker
proteins of extrinsic (caspase-8, Fas, FasL) and intrinsic (caspase-9) pathway of
metazoan apoptosis were identically down-regulated. The Bcl-2 family of proteins, though, remained unaffected. The extract also positively modulated the
other marker proteins like c-Jun NH2-terminal kinase, p38, Akt, nuclear factorjB, c-Fos, cellular FLICE-inhibitory protein, cyclooxygenase-2 and p53. Taken
together, the above-mentioned findings establish the anti-oxidative and antiapoptotic potency of the extract that ameliorates the H2O2-induced adverse
effects on rat Leydig cells in vitro.

Introduction
H2O2 as an oxidant has been recognised as a damaging
entity and a signalling agent mediating various pathogenic
processes in different cells and tissues (Stone & Yang,
2006). As reactive oxygen species (ROS), it is generated
from nearly all sources of oxidative cycle and has the
ability to diffuse in and out of cells (Barbouti et al.,
2002). Leydig cell steroidogenesis is oxygen dependent,
and oxidative stress due to ROS accumulation is shown
to disrupt mitochondria and steroid synthesis in MA-10
cells (Diemer et al., 2000). Besides its indigenous production during steroid synthesis, Leydig cells particularly are
exposed to the risk of more external H2O2 as a secretory
product from the interstitial macrophages with whom
they share a close structural proximity in-vivo. Being an
ROS, H2O2 was reported to affect Leydig cell function
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in vitro, even at physiological concentrations, inducing

oxidative stress and apoptosis (Gautam et al., 2006). ROS
production and accumulation leading to oxidative stress
in Leydig cells may arise due to altered hormonal or
other conditions leading to a decline in cellular function.
Even with persistent stimulation of Leydig cells with
human chorionic gonadotropin (hCG), the decline in
Leydig cell steroidogenesis was as a result of increased
oxidative stress and apoptosis in the target cells (Aggarwal
et al., 2009). Thus, appropriate interventions, which at
present are not many, must be developed so as to provide
the necessary protection to Leydig cells under such
adverse conditions.
In recent years, agents with anti-oxidative and antiapoptotic properties are utilised to counteract H2O2induced DNA damage leading to improved cell survival
through modulation of gene expressions. A synthetic
1

Effects of Eugenia jambolana on rat Leydig cells

agent, N-acetyl-L-cysteine (NAC), has emerged as an

effective antioxidant and has been reported to successfully mitigate the adverse effects of H2O2 on germ cell
survival by regulation of intrinsic and c-Jun NH2-terminal kinase (JNK) pathway of apoptosis (Maheshwari
et al., 2009). In addition, it has been shown to ameliorate the adverse effects of oxidative stress and apoptosis
in Leydig cells following persistent stimulation with
hCG again mainly through the modulation of extrinsic
and JNK pathways of apoptosis (Aggarwal et al., 2010).
Use of natural products is always considered more
beneficial than chemo-therapeutic or chemo-preventive
agents, and therefore, there is an increasing interest for
the development of phytochemical anti-oxidants as
health benefactors (Krishnaiah et al., 2007). Eugenia
jambolana (Myrtaceae, common name: Black plum/Black
berry in English and Jamun/Jambul in Hindi) seeds have
been reported to possess anti-inflammatory, anti-bacterial, anti-HIV, anti-diarrhoeal (Chaturvedi et al., 2007)
and strong antioxidant properties (Vasi & Austin, 2009).
Hypoglycaemic and hypolipidaemic activity of the aqueous and ethanolic extracts of seeds, at different dose levels, have been tried in animals (Sharma et al., 2006,
2010). The seeds have also shown to have protective
effects on testis of streptozotocin-induced diabetic rats
(Mallick et al., 2007, 2008). The active ingredient from
the fruit pulp of Eugenia jambolana, which reportedly
improved the renal dysfunction in streptozotocininduced diabetic rats, has been chemically characterised
as a-hydroxy succinamic acid by HPLC (Tanwar et al.,
2010) and patented (Sharma et al., 2009). At present,
there is no other product emerged so far, which has
established antioxidative effects comparable with NAC.
The active ingredient is not available in plenty either
commercially or otherwise; therefore, this study was
planned with the purified aqueous extract of Eugenia
jambolana to explore its cytoprotective potential against
H2O2-induced damage of Leydig cells in vitro. The associated molecular mechanisms were also investigated.
Materials and methods
Animals
Adult male albino rats of Holtzman strain, 6090 days
weighing about 200220 g, maintained under controlled
temperature (25 2 C) and constant photoperiodic conditions (12h light/12h dark), were used. The animals were
allowed food and water ad libitum. The animals were sacrificed under strict compliance of the Institutional Guidelines for Animal Care issued by the Committee for the
Purpose of Control and Supervision on Experiments on
Animals, India.
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H. Anand et al.

Leydig cell isolation, viability and treatment

The Leydig cells were isolated as previously described
(Anakwe & Moger, 1986). The cells were incubated with
H2O2 (10250 lM) for different time periods (16 h) at
37 C. The dose of H2O2 (100 lM) showing a cell viability of >75% following 4 h incubation was selected for further analysis. The aqueous extract from the fruit pulp of
Eugenia jambolana has been prepared as described (Tanwar et al., 2010). Briefly, the fruit pulp in dH2O was grinded, filtered, centrifuged and lyophilised in cold and
stored at 20 C until used. The cytoprotective effect of
the extract was then examined at different concentrations
(50250 lg ml 1) following co-incubation of cells with
H2O2 (100 lM). The effective dose (100 lg ml 1) was
selected accordingly, which was later utilised in subsequent experimentation. The beneficial effect of Eugenia
jambola extract (EJE) on Leydig cell viability through trypan blue dye exclusion test (Gautam et al., 2006) was also
assessed in comparison with NAC, the known and recognised antioxidant.
Lipid peroxidation, antioxidant enzyme activity and total
antioxidant capacity (TAC)
Leydig cells untreated and treated with only H2O2 or EJE
or with both were briefly sonicated for 30 s. One part of
the lysate was assayed for lipid peroxidation as described
(Ohkawa et al., 1979). The other part was centrifuged at
10 000 g for 5 min and activities of antioxidant enzymes,
superoxide dismutase (SOD; Das et al., 2000), catalase
(Aebi, 1984) and glutathione-s-transferase (GST; Habig
et al., 1974) were assayed using the supernatant. The protein was assayed using the Bradford method (Bradford,
1976).
Assessment of TAC of Leydig cells, treated or
untreated, was carried out using the commercial kit as
per the manufacturers (Cayman Chemical Company,
Ann Arbor, MI, USA) instructions. The assay exploits the
combined ability of various antioxidants (vitamin, protein, lipids, glutathione, uric acid, etc.) present in the cell
lysate to inhibit the oxidation of 2, 2-Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) by metmyoglobin.
Absorbance at 750 nm was representative of the amount
of the oxidised ABTS produced, and TAC (mM) was calculated using a Trolox standard curve.
Intracellular glutathione levels
Total intracellular cellular glutathione (GSH) levels were
measured using the commercial ApoGSH Glutathione
Detection Kit (Biovision, Mountain view, CA, USA) as per
the manufacturers instructions. Briefly, approximately
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H. Anand et al.

5 9 106 cells were lysed using 200 ll of lysis buffer (provided in the kit) for 10 min at 4 C, centrifuged at
12 000 g and the supernatant filtered through 10 kDa filters (R&D System, Inc., Minneapolis, MN, USA). 40 ll of
the filtrate diluted with lysis buffer (final 100 ll) and GSH
standards (100 ll) were taken in a fluorometric plate to
which 2 ll of monochlorobimane dye was added, shaken
well and incubated for 30 min at 37 C. Fluorescence was
measured using microtitre plate reader (BioTek, Inc., Winooski, VT, USA) at excitation/emission = 380/460 nm.
Total glutathione in each sample was calculated using GSH
standard curve.
Nitric oxide (NO) assay
The levels of NO in Leydig cells were estimated as previously described (Lyle et al., 2009). The assay is based on
the measurement of nitrites in the supernatant using Griess reagent. Briefly, the cells, treated and untreated, were
centrifuged at 10 000 g for 5 min, and 100 ll of the
supernatant was taken in triplicates in a microtitre plate.
Equal volume of Griess Reagent (1% sulphanilamide,
0.1% napthylethylenediamine dihydrochloride and 2.5%
hydrochloric acid) was added, and the plate was incubated
in dark for 15 min at room temperature following which
absorbance was measured at 550 nm using a microtitre
plate reader (BioTek, Inc.). The NO concentrations in the
treated samples were calculated as percentage of the
control.
[TdT]-mediated deoxyuridinetriphosphate nick end
labelling (TUNEL) assay
TUNEL was performed as per the manufacturers (R&D
System, Inc.) instructions. Briefly, treated or untreated
cells were washed with PBS, smeared on poly-L-lysine
coated slides and fixed with 4% formaldehyde for 5 min.
Cytonin treatment was given to the cells for 10 min
followed by quenching with H2O2. TdT was used to
incorporate biotinylated nucleotides into the 3-OH ends
of the DNA fragments and detected by streptavidinhorseradish peroxidase (HRP). DAB was used to develop
the colour in cells that were counterstained with methyl
green. The slides were examined using a Nikon microscope, and the percentage of TUNEL-positive cells was
calculated.
Caspase-3, -8 and -9 activities
Caspase-3, -8 and -9 activities were assayed as per the
protocol supplied by the manufacturer (Biovision, San
Diego, CA, USA). Briefly, approximately 5 9 106 cells
were resuspended in cold lysis buffer and incubated for
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Effects of Eugenia jambolana on rat Leydig cells

10 min. The lysates were centrifuged at 10 000 g for

2 min at 4 C, and an aliquot of the supernatant (100 lg
protein per 50 ll) was added to 50 ll of the reaction buffer containing 200 lM of the chromogen (Ac-DEVDpNA, Ac-IETD-pNA and Ac-LEHD-pNA for caspase-3, -8
and -9, respectively). The reaction mixture was incubated
at 37 C and terminated by addition of stop buffer after
2 h. The release of pNA leads to a change in the absorbance that is measured at 405 nm using a microtitre plate
reader (BioTek, Inc.).
Western blot analysis
Primary antibodies (rabbit polyclonal) anti-MnSOD, antipoly-ADP-ribose polymerase (PARP), anti-caspase-3, anticaspase-9, anti-Fas, anti-FasL, anti-p53, anti-JNK,
anti-c-Jun, anti-Akt, anti-NF-jB, anti-I-jB, anti-c-Fos,
anti-c-Flip and (mouse monoclonal) anti-catalase, anti-caspase-8, anti-phosphorylated form of JNK (pJNK), anti-bactin (Santa Cruz Biotechnology, Santa Cruz, CA, USA),
anti-GST (Bangalore Genei, Bangalore, India) were utilised.
Rabbit monoclonal anti-Cox-2 and anti-p-Akt were from
Cell Signaling Technology, Inc., Danvers, MA, USA, and
rabbit monoclonal anti-inducible nitric oxide synthase
(iNOS) was from Biomeda, Foster City, CA, USA. Goat
anti rabbit/mouse-HRP conjugate secondary antibody was
from Santa Cruz Biotechnology. Whole cell lysates were
prepared in 200 ll lysis buffer, and western blots were carried out as previously described (Maheshwari et al., 2011).
b-actin was used as an internal control to ensure equal protein loading. Densitometric analysis was performed with
the help of Image analysis software (LAB WORKS IMAGE analysis software 4.0; UVP, Upland, CA, USA).
RNA isolation and RT-PCR analysis
Total RNA was extracted using TRI-Reagent (Life Technologies Corp., Ambion , TX, USA). cDNA was synthesised using 2 lg of total RNA by omniscript reverse
transcriptase (RT) kit (Qiagen, Hilden, Germany). PCR
was carried out with 2 ll of RT reaction using the HotStar
HiFidelity DNA polymerase (Qiagen). Specific primers
were obtained from Eurofins MWG Operon (Whitefield,
Bangalore, India). The following temperature profiles were
used for running the PCR reaction: (i) denaturation at
95 C for 15 min; (ii) 30 cycles of 95 C for 30 sec, 55
67 C for 1 min, 72 C for 1 min; (iii) 72 C for 10 min
for final extension. Table 1 shows the sequences, source,
annealing temperatures, Mg2+ concentrations and product
sizes of various primers used. Post-amplification, the products were separated on a 1.5% agarose gel and documented
with the help of a gel documentation system (UVP).
b-actin was used as an internal as well as a nontemplate
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Effects of Eugenia jambolana on rat Leydig cells

H. Anand et al.

Table 1 Primer specific conditions used for PCR amplification of candidate genes

Name

Primer sequence

Reference/accession no.

Mg2+ conc.
(mM)

Catalase

F-CCGACGAGATGGCACACTTTGACA
R-CGCGAGCACGGTAGGGACAGTTC
F-CTTCAGCCTGCACTGAAGTTCAAT
R-CTGAAGATAGTAAGCGTGCTCCC
F-CTCTCCGCGGTGGCACAG
R-CCACCACCGGGTCGGACATAC
F-TTGGGTCTTGTTAGCCTAGTC
R-TGTGCAGTCCCAGTGAGGAAC
F-CTGGGAAGGATCGACGATTA
R-CATGTCCTGCATTTTGATGG
F-GCAATGCTTCTCTCTGTGACCACT
R-GCTGTTGTGCTCGATCTCATCG
F-GAATGGGAAGACACATATGGAACTGC
R-CATATCTGGCCAGTAGTGCAGTAATTC
F-AGCCAGATGCTGTCCCATAC
R-CAGGAGACAAAACCTGGGAA
F-GGCCATCTACAAGAAGTCAC
R-CCAGAAGATTCCCACTGGAG
F-CTGTGCCCATCTATGAGGGTTAC
R-AATCCACACAGAGTACTTGCGCT

Maheshwari et al. (2009)

2.5

62

972

Maheshwari et al. (2009)

2.5

65

326

Bhor et al. (2004)

3.5

62

290

Hierholzer et al. (1998)

2.5

59

264

Maheshwari et al. (2009)

4.0

65

123

Maheshwari et al. (2009)

3.5

65

351

Maheshwari et al. (2009)

1.5

65

238

Maheshwari et al. (2009)

2.5

65

132

Maheshwari et al. (2011)

2.5

55

317

Maheshwari et al. (2009)

2.5

60

539

Mn SOD
GPx
Inducible nitric
oxide synthase
Caspase-8
Fas
FasL
Caspase-9
p53
b-Actin

control, and densitometric analysis was performed as previously described.

Statistical analysis
All the figures (for Western Blotting and RT-PCR) are
representative of three independent experiments with
similar results. The results have been shown as the
mean standard deviation (SDs) of three experiments.
Statistical analysis was performed using one-way ANOVA
followed by Tukeys test using GRAPH PAD PRISM Software.
P < 0.05 was considered statistically significant.
Results
Eugenia jambolana extract counters the decrease in
Leydig cell viability induced by H2O2
To determine the beneficial effects of EJE, a systematic
analysis of Leydig cell viability through trypan blue dye
exclusion was carried out with different doses of H2O2
under incubation for 4 h and with or without EJE supplementation. As compared to controls, a significant decline
in viability (~13%) was observed with 100 lM H2O2 exposure that increased further (~25%) with the increase in
H2O2 (250 lM) concentration (Fig. 1a). The cell viability
was found unaffected when examined for 13 h even
with the highest concentration of H2O2 used (data not
shown). Accordingly, the dose (100 lM) and duration
(4 h) of H2O2 exposure was maintained for all subsequent
4

Annealing
temp. (C)

Product
size (bp)

investigations. A significant (P < 0.001) improvement in

cell viability (88.51 2.023%) was seen following EJE
(100 lg ml 1) supplementation (Fig. 1b). The effect of
EJE (100 lg ml 1) on cell survival was found comparable
with NAC (5 mM), a known but synthetic antioxidant
(Fig. 1c). Therefore, this particular dose of EJE
(100 lg ml 1) was used in all subsequent experiments.
EJE co-treatment counteracts H2O2-induced oxidative
stress
H2O2 exposure induced a two-fold rise in lipid peroxidation in the target cells as compared to unexposed controls.
EJE intervention, however, completely prevented this rise
(Fig. 2a). It also helped to restore the decline in the activities of antioxidant enzymes, SOD, catalase and GST to
normal levels (Fig. 2bd). When the protein and transcript levels of these enzymes were further examined, identical up-regulation in the expressions was observed for
catalase (Fig. 2e,f). Up-regulation only in the transcript
(Fig. 2f) or protein levels (Fig. 2e) was seen for MnSOD,
glutathione peroxidase (GPx) and GST, respectively. A significant improvement in the GSH levels (P < 0.001) and
TAC (P < 0.001) was observed (Fig. 3a,b).
EJE supplementation counters H2O2-induced rise in
nitric oxide
A significant rise (P < 0.001) in NO levels was seen in
Leydig cells following H2O2 exposure that was successfully
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(b)

(a)

Fig. 1 Leydig cell viability (%) as determined

by trypan blue dye exclusion. (a) Viability was
assessed after 4 h of incubation with different
doses of H2O2 (10250 lM). 100 lM H2O2
was selected for further studies as it maintained the cell viability >75%. (b) Eugenia
jambolana extract (EJE) (100 lg ml 1) intervention with 100 lM H2O2 significantly
improved cell viability comparable with that of
(c) 5 mM N-acetyl-L-cysteine (NAC). **P < 0.01
and ***P < 0.001 compared with untreated
controls. ###P < 0.001 compared with H2O2,
DDD
P < 0.001 compared with 100 lg ml 1
EJE, $P < 0.05 compared with 100 lg ml 1
EJE + 100 lM H2O2, aaaP < 0.001 compared
with 1 mM NAC, bbbP < 0.001 compared with
1 mM NAC + 100 lM H2O2 treatment.

(c)

brought down by EJE co-treatment (Fig. 4a). The protein/mRNA levels of iNOS found simultaneously
(P < 0.01; P < 0.05, respectively) up-regulated as a result
of H2O2 treatment were restored to original levels following EJE supplementation (Fig. 4b,c).

Fig. 2 Evaluation of oxidative stress in H2O2treated isolated rat Leydig cells with or without Eugenia jambolana extract (EJE). (a) Lipid
peroxidation after H2O2 treatment as measured by thiobarbituric acid reactive substances formation was significantly brought
down with EJE co-incubation. Restoration in
the activities of the antioxidant enzymes, (b)
superoxide dismutase (SOD), (c) catalase and
(d) glutathione-s-transferase (GST) was also
evident. A similar trend was observed in (e)
western blot analysis for catalase, MnSOD and
GST, as well as (f) RT-PCR analysis for catalase,
MnSOD and glutathione peroxidase (GPx).
*P < 0.05 and ***P < 0.001 compared with
untreated controls. #P < 0.05 and ##P < 0.01
compared with H2O2 treatment.

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Inhibition of H2O2-induced apoptosis in Leydig cells by

EJE
H2O2 as an oxidant is a recognised apoptosis inducer
as TUNEL positivity in the exposed cell population

(a)

(b)

(c)

(d)

(e)

(f)

Effects of Eugenia jambolana on rat Leydig cells

(a)

H. Anand et al.

(Fig. 8a) and expression (Fig. 8b,c) was similarly downregulated by EJE supplementation in the H2O2-treated
cells. However, EJE was seen to have very little influence
on the other intrinsic, apoptotic protein markers such as
Bax, Bid, Bak and Bad and similarly the anti-apoptotic
Bcl-2 (data not shown).
c-Jun NH2-terminal Kinase (JNK) modulation by EJE

(b)

Fig. 3 Assessment of (a) total glutathione (b) total antioxidant

capacity (TAC) in H2O2 exposed Leydig cells with or without Eugenia jambolana extract (EJE). EJE supplementation restored the cellular glutathione levels and improved the TAC by ~45% compared
with untreated controls. *P < 0.05, **P < 0.01, and ***P < 0.001
compared with untreated controls, ###P < 0.001 compared with
H2O2 treatment.

demonstrated a significant (P < 0.001) increase (Fig. 5b,

g). While EJE alone showed no adverse effect (Fig. 5c,g),
co-treatment with H2O2 prevented this apoptotic transformation in the target cells (Fig. 5d,g). The inhibition of
apoptotic induction was very much comparable to the
effect when EJE was replaced with NAC (Fig. 5e,f,g). EJE
was also able to counter the rise in the expression and
activity of caspase-3 in H2O2-treated cells (Fig. 6a,b).
Similarly, it prevented the H2O2-induced over-expression
of PARP cleavage in the target cells (Fig. 6b).
EJE mediated regulation of apoptotic pathway
To explore the molecular mechanisms of EJE mediated
regulation of Leydig cell apoptosis induced by H2O2, the
expression of upstream markers in the extrinsic and
intrinsic pathway of apoptosis was examined. Raised caspase-8 activity (Fig. 7a) and expression (Fig. 7b,c) in the
H2O2 exposed cells was found effectively checked by EJE
intervention. EJE modulation of expression of the associated markers Fas and FasL followed an identical trend
(Fig. 7b,c). In the intrinsic pathway, caspase-9 activity
6

Mitogen Activated Protein Kinase (MAPK) modulation

by EJE was also investigated in the H2O2 exposed Leydig
cells that demonstrated a significantly up-regulated
expression of JNK and its phosphorylated form (pJNK).
EJE co-treatment was able to down-regulate these expressions back to the control levels (Fig. 9a). Protein blots
indicating c-Jun, c-Fos and pP38 expressions demonstrated an identical pattern of regulation by EJE (Fig. 9a).
On the other hand, EJE induced up-regulation of the
expression of anti-apoptotic marker proteins like nuclear
factor-jB (NF-jB), cellular FLICE-inhibitory protein
(c-Flip) and cyclooxygenase-2 (Cox-2) in the H2O2treated cells (Fig. 9b). The expression of I-jB, the inhibitor of NF-jB, followed an opposite trend and was seen
completely reversed by EJE treatment (Fig. 9b).
Regulation of p53 and Akt expression
While p53 was over-expressed in the H2O2-treated Leydig
cells, the anti-apoptotic Akt and pAkt were significantly
down-regulated. EJE was seen to modulate the expression
of these proteins favourably back to control levels
(Fig. 9c,d,e).
Discussion
The findings from this study establish that the active
ingredient from the fruit pulp of Eugenia jambolana
(EJE) supplementation significantly ameliorates the
adverse effects of H2O2 on rat Leydig cells in vitro and
promotes cell survival by mitigating oxidative stress and
apoptosis. The underlying molecular mechanism includes
favourable modulation of up- and down-stream marker
proteins in the extrinsic/intrinsic and other associated
pathways of metazoan apoptosis.
The onset of oxidative stress in any cellular system may
be mediated by an excessive production of ROS or exposure to ROS/ROS stimulating agents that may eventually
affect its normal functioning. We had earlier reported
that hCG-stimulated testosterone production in Leydig
cells was severely impacted following exposure to H2O2
even in low concentrations (Gautam et al., 2006). As
impaired Leydig cell function is closely associated with
testicular dysgenesis syndrome that includes a range of
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H. Anand et al.

(a)

Fig. 4 Estimation of (a) nitric oxide (NO) and

(b) protein and (c) transcript expression of
inducible nitric oxide synthase (iNOS) in Leydig
cells with or without Eugenia jambolana extract
(EJE) intervention. EJE supplementation helped
to reduce NO levels (expressed as percentage
of untreated control) significantly and the
over-expression of iNOS protein and mRNA.
*P < 0.05, and ***P < 0.001 compared with
untreated controls, ##P < 0.01 compared with
H2O2 treatment.

(b)

male reproductive disorders (hypospodiasis and cryptorchidism) leading to genital malformations, hypospermatogenesis and even testicular cancer (Joensen et al., 2008),
the implication of oxidative stress in all these conditions
cannot be overemphasised. All these clinical problems are

Fig. 5 TUNEL of H2O2-treated Leydig cells

with or without Eugenia jambolana extract
(EJE) supplementation. The rise in TUNEL-positive cells (?) after H2O2 treatment was significantly brought down by EJE, (a, g) control,
(b, g) H2O2, (c, g) EJE only, (d, g) H2O2 + EJE,
(e, g) 5 mM N-acetyl-L-cysteine (NAC) only,
and (f, g) H2O2 + 5 mM NAC showing percentage of TUNEL-positive cells under different incubating conditions. The data from the
graph were collected from four different
fields each from three separate experiments.
*P < 0.05, ***P < 0.001 compared with
untreated controls, ###P < 0.001 compared
with H2O2 treatment.

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(c)

the outcome of an irreversible developmental disorder

and Leydig cell dysfunction originating in early foetal life
(Anderson et al., 2004). An association between compromised Leydig cell function and testicular cancer has been
indicated due to the fact that significantly increased

(a)

(c)

(e)

(b)

(d)

(f)

(g)

Effects of Eugenia jambolana on rat Leydig cells

H. Anand et al.

(a)

(b)

Fig. 6 (a) Activity and expression (b) of caspase-3 was down-regulated along with cleaved poly-ADP-ribose polymerase after Eugenia
jambolana extract co-treatment. **P < 0.01 and ***P < 0.001 compared with untreated controls, ###P < 0.001 compared with H2O2
treatment.

luteinising hormone (LH) levels and low testosterone

were found in men with carcinoma testis in situ (Petersen
et al., 1999). Besides, there are a host of other clinical

conditions in adult hood like liver disease, alcoholism,

chronic renal disease, metabolic cardiovascular syndrome,
diabetes and rheumatic disease, in which Leydig cell function is also found to be affected (Karagiannis & Harsoulis, 2005). However, interventions aimed to improve or
restore the functions of Leydig cells under such conditions are not many. A recent finding indicates that oral
administration of sodium tungstate to adult male streptozotocin-diabetic rats not only normalised serum levels of
glucose but also Leydig cell function leading to restored
LH levels in circulation and improved testosterone production (Ballester et al., 2005). As all synthetic products
have the risk of bio-incompatibility leading to adverse
reactions, in this work, we preferred to use a natural
plant product, active ingredient from the fruit pulp of
Eugenia jambolana, under oxidant-induced stressed
conditions in Leydig cells in vitro to explore its beneficial
cytoprotective effects first before examining its potency
on cellular function in-vivo.
Testicular oxidative stress has long been implicated in
several conditions of male infertility including toxicant
exposure (Samanta & Chainy, 1997; Han et al., 2004;
McClusky et al., 2007), chemotherapy (Arnon et al.,
2001), ionising radiation (Sohal et al., 1995; Manda et al.,
2007), inflammation (Allen et al., 2004), varicocele
(Santoro & Romeo, 2001), cryptorchidism (Misro et al.,
2005; Li et al., 2006), ageing (Syntin et al., 2001; Cao
et al., 2004; Luo et al., 2006) and testicular torsion
(Lysiak et al., 2001, 2007). All these conditions whether
therapeutical or pathological end up generating more

(a)

(b)

(c)
Fig. 7 Role of Eugenia jambolana extract (EJE)
modulation of extrinsic pathway of apoptosis.
EJE co-treatment with H2O2 significantly contained the rise in (a) activity and (b, c) expression (protein and transcript levels) of caspase-8.
Identical down-regulation in (b, c) Fas, and (b,
c) FasL expression was also seen. **P < 0.01,
***P < 0.001 compared with untreated
controls, ###P < 0.001 compared with H2O2
treatment.

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Effects of Eugenia jambolana on rat Leydig cells

H. Anand et al.

(a)

(b)

Fig. 8 Role of Eugenia jambolana extract

(EJE) modulation of intrinsic pathway of apoptosis. EJE co-treatment restored the (a) activity
and expression of (b) protein and (c) transcript
levels of caspase-9 back to control levels.
**P < 0.01 compared with untreated control,
###
P < 0.001 compared with H2O2 treatment.

ROS associated with reduced intracellular antioxidant

activity unable to counter the ROS mediated detrimental
effect. ROS, in general, include superoxide anions and
hydroxyl radicals, reactive oxygen, as well as species of
H2O2, NO and peroxynitrite anion that are not radicals
characteristically but possess the ability of forming
radicals in cellular environments (Chang et al., 2008).
Either produced indigenously or following exposure to

(c)

extracellular sources, these oxidants can cause tissue damage by a variety of mechanisms including DNA damage,
lipid peroxidation, protein oxidation, depletion of cellular
thiols and activation of pro-inflammatory cytokine
release. Significant increase in Leydig cell death due to
apoptosis was reported from this laboratory following
H2O2 exposure at 100 lM concentrations although
hCG-induced testosterone production was affected at

(a)

(b)

(d)

(c)

Fig. 9 Role of Eugenia jambolana extract

(EJE) modulation of other pathways of apoptosis. EJE supplementation was seen favourably modulating (a) JNK, p-JNK, c-Jun, c-Fos,
pP38 and (b) NF-jB, I-jB, c-Flip and Cox-2
expression in the target cells. A significant
down-regulation of p53 (c) protein and (d)
transcripts along with up-regulation of antiapoptotic (e) Akt and p-Akt expression was
also observed.

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Andrologia 2012, XX, 113

(e)

Effects of Eugenia jambolana on rat Leydig cells

concentrations even less than 30 lM (Gautam et al.,

2006). Simultaneous supplementation with antioxidants is
the only solution to salvage the situation that at present
is limited to the use of recognised antioxidants like NAC;
the exact molecular mechanism of its counteraction
against apoptotic cell death is still very much debated
(Lum et al., 2002). This opens up the quest for identifying other sources or ingredients that could be used as
antioxidants and cyto-protectants under identical conditions.
As plants possess natural antioxidant properties to
counter these toxic oxygen derivatives, in this work we
utilised a plant product, the active ingredient from the
fruit pulp of Eugenia jambolana, the seeds of which have
been recognised to possess antioxidant properties (Vasi &
Austin, 2009). This is for the first time we tested the antioxidant potential of the fruit pulp preparation under the
oxidant-exposed conditions in Leydig cells in vitro and
found that the preparation has remarkable cytoprotective
effects that promote cell survival under adverse conditions. Simultaneous exposure of EJE (100 lg ml 1)
+ H2O2 (100 lM) significantly improved the Leydig cell
viability over a period of 4 h comparable only to similar
supplementation with 5 mM of NAC (Fig. 1c). It helped
in reducing the formation of thiobarbituric acid reactive
substances while improving the activities of antioxidant
enzymes, SOD, catalase and GST (Fig. 2ad). Even the
gene expressions of these antioxidant enzymes are found
favourably augmented (Fig. 2e,f). Increase in NO levels
has been recognised as an indication of oxidative stress
leading to inhibition of testosterone production (Mehta
et al., 2002). There is evidence that H2O2 and NO besides
acting as independent signalling molecules may interrelate to form an oxidative death cycle. Addition of
10 mM H2O2 to a leaf protein preparation of mung bean
caused an 8.3-fold increase in the NO activity suggesting
the role of H2O2 as an upstream signal leading to NO
production (Turner & Lysiak, 2008). H2O2 exposure, in
this study, does increase the NO levels, but EJE was able
to regulate iNOS expression leading to NO depletion in
the target cells (Fig. 4); which further confirmed the antioxidant potency of the preparation utilised.
The onset of oxidative stress is potentially accompanied
by cell apoptosis in the testis that results from the activation of numerous molecular pathways (Mehta et al.,
2002; Maheshwari et al., 2009). Downstream caspase-3
up-regulation and the FasL activation were implicated in
the H2O2-mediated Leydig cell apoptosis (Gautam et al.,
2006). This work confirms the earlier findings and
extends to demonstrate the increase in PARP cleavage in
the target cells, which was successfully attenuated by EJE
supplementation (Fig. 6). EJE co-incubation also effectively checked the increase in caspase-8 activity and
10

H. Anand et al.

expression of caspase-8, Fas and FasL marker proteins of

the extrinsic pathway of metazoan apoptosis (Fig. 7).
However, upstream, intrinsic, Bcl-2 family of proteins
remained unaffected following H2O2 exposure to Leydig
cells (data not shown). Rise in activity and expression of
caspase-9 was noticed which was restored back to control
levels by EJE intervention (Fig. 8). The dichotomy in the
expression of the above-mentioned marker proteins may
be explained by the fact that pro-caspase-9 can act as a
substrate for caspase-12, and the activation of caspase-9
may be initiated independent of the cytochrome-c release
from the mitochondria, thereby, bypassing the involvement of the Bcl-2 family of proteins (Morishima et al.,
2002). On the basis of these findings, it is difficult to verify the above-mentioned proposition that can only be
ascertained in future studies.
Exposure to H2O2 has also been implicated in the activation of MAP kinases (MAPK) that are important mediators of stress-inducing signals (Maheshwari et al., 2009).
JNK that phosphorylates and regulates the activity of
transcription factors, c-Jun and p53, and the activation of
JNK, in turn, is regulated by scaffold proteins such as
NF-jB (Liu & Lin, 2005). JNK and c-Jun activation has
been recently shown to be involved in Burkitts Lymphoma cell line (BJAB) cell death after 2 h of exposure to
H2O2 (Son et al., 2009). c-Fos, a nuclear protein that
dimerises with c-Jun to form the transcription factor
complex activator protein-1 (AP-1), has been shown to
be regulated during c-Myc-induced apoptosis of serumdeprived hepatoma cells involving the activation of P38
MAP kinase cascade (Kalra & Kumar, 2004). In this
work, H2O2-induced cell death in Leydig cells demonstrated identical over-expression of JNK, pJNK, c-Jun,
c-Fos, pP38, I-jB and p53 (Fig. 9ad). Being anti-apoptotic, NF-jB, c-Flip, cox-2, Akt and pAkt were found
down-regulated (Fig. 9b,e). Introducing EJE along with
H2O2 favourably modulated the expression of all these
proteins promoting cell survival and inhibiting cell death
by apoptosis (Fig. 5). Based on the above-mentioned
findings, a model showing the molecular mechanisms of
EJE against H2O2-induced Leydig cell apoptosis is proposed (Fig. 10).
The threat of the long-term side effects of synthetic
antioxidants has increasingly brought back the interest of
the use of natural resources for health and medicine
(Krishnaiah et al., 2007). The roots of Morinda officinalis
have been shown to have a cytoprotective effect on
H2O2-induced oxidative stress in Leydig TM3 cells
(Chang et al., 2008). Green tea polyphenols have been
found to have an anti-oxidative and anti-apoptotic effect
against azothioprine-induced liver injury in rats
(El-Beshbishy et al., 2011). Orthosiphon stamineus Benth
has anti-oxidative and anti-apoptotic effect on
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H. Anand et al.

Effects of Eugenia jambolana on rat Leydig cells

Fig. 10 A model showing Eugenia jambolana

extract modulation of H2O2-induced Leydig
cell apoptosis in vitro is proposed. Up-regulation (), down-regulation () and inhibition ()
of expression of markers in different pathways of apoptosis are indicated.

H2O2-induced cellular damage in MDA-M231 cells (Abdelwahab et al., 2011). Identical to the above-mentioned
findings, the fruit pulp of Eugenia jambolana is shown
here for the first time to possess antioxidant properties
having the capacity to mitigate the oxidative stress in Leydig cells induced by H2O2. The preparation is very much
cytoprotective and needs to be explored further for therapeutical use to alleviate Leydig cell dysfunction.
Acknowledgements
The generous gift of Eugenia jambolana extract (EJE)
from Dr Suman Bala Sharma, Professor, University College of Medical Sciences, Delhi, is greatly acknowledged.
The study was funded by NIHFW.
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