Food and Chemical Toxicology 46 (2008) 3333–3338

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Food and Chemical Toxicology

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Safety assessment of Syzygium aromaticum flower bud (clove) extract with

respect to testicular function in mice
Raghav Kumar Mishra, Shio Kumar Singh *
Reproductive Endocrinology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

article info abstract

Article history: The flower buds of Syzygium aromaticum (clove), a common food flavor, have been used as indigenous medicine for the
Received 20 February 2008 treatment of male sexual disorders in Asian countries. However, the possible mecha-nism(s) by which it acts at testicular level
Accepted 11 August 2008 remain obscure. Therefore, to investigate its effect on testic-ular function, chronic oral exposure of hexane extract of flower
buds of Syzygium aromaticum in three doses (15 mg, 30 mg, and 60 mg/kg BW) were evaluated for a single spermatogenic
cycle (35 days) in Parkes (P) strain mice. The treatment did not induce systemic toxicity at the doses tested. Lower dose (15
Keywords: 5
mg) of the extract increased the activities of D 3 b-HSD and 17 b-HSD, and serum level of testos-terone. The higher doses
Syzygium aromaticum
(30 and 60 mg) of extract inhibited these parameters and induced non-uni-form degenerative changes in the seminiferous
Testis
Flowcytometry tubules associated with decrease in daily sperm production and depletion of 1C (round and elongated spermatids) population.
Spermatogenesis Taken together these results suggest biphasic action of hexane extract of Syzygium aromaticum flower bud on testicular func-
tion, thereby advocating a cautious use of the flower bud as an aphrodisiac in indigenous systems of medicine in Asian
countries.

2008 Elsevier Ltd. All rights reserved.

1. Introduction an ingredient in popular toothpastes and mouth fresheners in India (Banerjee

The World Health Organisation survey suggests that 70–80% of the world
populations rely on non-conventional medicine mainly of herbal sources in
their primary healthcare (Chan, 2003). Such in-crease in popularity has also
brought concerns and fears over the quality, quantity, safety, and efficacy of
these herbal medicines especially of aphrodisiacs. The popularity of
aphrodisiacs are more as males prefer such drugs to reign on bed without
proper knowl-edge of toxicity of such materials. In the last decades, there has
been growing concern over the effects of either synthetic or natural products
on the male reproductive health (Dalsenter et al., 2004).
The flower bud of Syzygium aromaticum (L.) Merr. & Perry. (Fam-ily
Myrtaceae), commonly known as clove, is a well known food flavor for
exotic food preparations and a popular remedy for dental disorders,
respiratory disorders, headache and soar throat in tradi-tional medicines of
Australia, and Asian countries (Domaracky et al., 2007; Gurib-Fakim, 2006;
Sharma, 2001; Kim et al., 1998). In addition to its antimicrobial, antifungal
and antiviral properties, clove oil possesses anti-inflammatory, cytotoxic, and
anesthetic properties (Chaib et al., 2007). Because of its antiseptic and antibi-
otic properties, clove is frequently used to treat toothache and as
* Corresponding author. Tel.: +91 542 6702532; fax: +91 542 2368174.
E-mail addresses: raghavmishra@rediffmail.com (R.K. Mishra), shioks@ rediffmail.com
(S.K. Singh).
et al., 2006).
In Ayurvedic and Unani medicines of Asian countries the Syzyg-ium
aromaticum (clove) is well known for its aphrodisiac property, and used to
treat male sexual disorders (Tajuddin et al., 2003; Sharma, 2001; Jain and
DeFilipps, 1991). It has been reported to produce a sustained increase in the
mounting frequency of normal male rat and mice (Tajuddin et al., 2004,
2003). On the other hand, Eugenol, a chief constituent of clove oil, causes
desquamation of the inner secretory columnar cell layer and exerts adverse
effects on secretory activity of seminal vesicle (Vanitakumari et al., 1998).
The oil of clove also shows spermicidal activity on ejacu-lated human
spermatozoa (Buch et al., 1988).
In spite of its wide use to cure male sexual disorders by the tra-ditional
healers of Asian countries, effects of Syzygium aromaticum on testicular
function has not been investigated so far. Considering the medicinal
importance of flower bud oil and its constituents we have used hexane extract
(to extract fat soluble ingredients) of flower bud of Syzygium aromaticum.
We have evaluated the effect of chronic oral exposure of hexane extract of
flower bud of Syzyg-ium aromaticum (SAx) for 35 days on liver and kidney
functions, testicular sperm production, different germ cell populations in-
volved in spermatogenesis by histology and flow cytometry, serum
5
testosterone level, and testicular enzymes (D 3 b-HSD and 17 b-HSD) assay
as a part of safety evaluation process of this indigenous medicine.

0278-6915/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.08.006
3334 R.K. Mishra, S.K. Singh / Food and Chemical Toxicology 46 (2008) 3333–3338
2. Materials and methods
2.1. Chemicals and reagents
All the chemicals used in the present study were of analytical grade and were purchased
from HiMedia Laboratories, Mumbai or E. Merck India Ltd., Mumbai, un-less stated otherwise.
2.2. Preparation of plant material
The sun dried unopened flower buds of Syzygium aromaticum were collected from Kerala
(South India), and were authenticated by the expert from the medicinal chemistry department of
Banaras Hindu University. The flower buds were coarsely ground. For extraction of fat soluble
ingredients the ground plant material (250 g) was extracted with n-hexane (2.5 l) in a soxhlet
extractor for 24 h (12 2). The sol-vent was evaporated in solvent recovery apparatus to collect
the extract ( 25 g) as dark brown viscous oil. This extract was stored at 4 LC in an air tight
container and dissolved in different concentrations as per doses in olive oil for animal treatment.
Three doses (15, 30, and 60 mg/kg BW) were chosen according to our preliminary observation
and general prescription (1–2 g/dose) by traditional healers in Indian subcontinent (Mishra and
Singh, 2008; Sharma, 2001).
2.3. Animals and treatment
Forty adult (age 14–15 weeks, weight 32–34 g) male mice of Parkes (P) strain were
separated from existing colony of the departmental animal house facility. These animals were
maintained at 24 ± 2 LC with 12 L: 12D photoperiod and were provided standard balanced diet
and drinking water ad libitum. Mice were divided into four groups (10 mice/group) in separate
polypropylene cages (430 270 150 mm) with dry rice husk as bedding material. All procedures
were in conformation to the stipulations of the Animal Ethics Committee of Banaras Hin-du
University. The animals were treated orally by oral feeding tubes for 35 days (duration of one
spermatogenic cycle in mouse) as follows:
Group I: Vehicle controls (Olive oil 200 ll/mouse/day).
Group II: Syzygium aromaticum extract (15 mg/kg body weight/day).
Group III: Syzygium aromaticum extract (30 mg/kg body weight/day).
Group IV: Syzygium aromaticum extract (60 mg/kg body weight/day).
2.4. Autopsy
Animals were killed after 24 h of the last treatment by decapitation. At autopsy the body
weight of all animals were recorded and the trunk blood was collected. The testes were excised,
blotted free of blood and weighed to nearest 0.10 mg. One testis of each mouse was fixed for
histology. The contra lateral testis was immediately used for cell isolation for flow cytometry
(FCM). Testes of remaining five mice were processed for testicular sperm production and
enzymatic studies.
2.5. Histology
Testes were excised, blotted free of blood and then fixed for histological studies in freshly
prepared Bouin’s fluid, dehydrated in graded ethanol series, cleared in benzene and embedded
in paraffin wax. Tissue was sectioned (6 lm) and stained with periodic-acid-Schiff (PAS) and
counter stained with Harris haematoxylin. Stained slides were analysed by light microscope
(Leica, GmbH, Germany).
2.6. Daily sperm production
To observe the effect of SAx treatment on testicular sperm production, sonica-tion resistant
elongated spermatids (Step 14–16 spermatids) were counted accord-ing to Meistrich and van
Beek (1993). Briefly one testis was placed in 1 ml cold distilled water. After homogenizing, the
tissue was sonicated for 1.5 min and sam-ple was counted in hemocytometer under a phase
contrast microscope at 40X. Developing spermatids spend 4.84 days in step 14–16 during
spermatogenesis in mice. Therefore, to calculate daily sperm production the number of step 14–
16 spermatids was divided by 4.84 (Izawa et al., 2007).
2.7. Flow cytometry
For quantitation of cell types involved in spermatogenesis after treatment, flow cytometric
analysis of testicular tissue was performed according to Katoh et al. (2002) with minor
modifications. Briefly the tunica albuginea was removed from testis, placed in ice cold nutrient
medium (Dulbecco’s modified eagle medium) and minced gently with razor. Then the tissue
was transferred to nutrient medium containing 0.25% collagenase (Type IA) (Sigma, St. Louis,
USA) for 30 min in a shak-ing water bath at 32.5 LC. The cell suspension was filtered through a
100 lm and 40 lm nylon mesh (BD Falcon, Bedford, USA) in order to discard tissue debris and
to obtain single cell suspension, respectively. The suspension was centrifuged at 300 Xg for 5
min and the pellet was washed twice with medium. One aliquot of each cell suspension was
stained with 0.2% trypan blue for the determination of via-bility. The cells were fixed in ice
cold 70% methanol and stored at 20L C for min-imum 24 h. After fixation cells were incubated
with 0.25% RNase (Bangalore Genei, India) (37 LC, 20 min). After washing twice with PBS,
DNA staining was carried out by Propidium Iodide (PI; Sigma, St. Louis, USA) solution (50
lg/ml in PBS), and placed in dark for 30 min at 4 LC. Enumerations of cells on the basis of their
DNA content was carried out in a Flow Cytometer (FACS Calibure, Becton-Dickinson, USA)
equipped with air-cooled 15 mW Argon ion laser at 488 nm and the emitted light from cells was
detected at 580 ± 30 nm on FL2 (ten thousand fluorescent events were recorded at a rate of
approximately 150 fluorescent events per second). The DNA histograms were analysed by Cell
Quest (Becton-Dickinson, USA) software, Version 3.1s.
2.8. Testicular enzyme assay
5
To observe the effect of SAx treatment on steroidogenic pathway testicular D 3 b
hydroxysteroid dehydrogenase (HSD) and 17 b hydroxysteroid dehydrogenase (HSD) were
measured according to the method of Talalay (1962) and Jarabek et al. (1962), respectively with
minor modifications. Briefly, one testis was homog-enized in 20% spectroscopic grade glycerol
containing 5 mM potassium phosphate and 1 mM EDTA at a tissue concentration of 100 mg/ml
and was centrifuged at 10,000g for 30 min. at 4 LC. The supernatant ( 950 ll) was used for
enzyme assay.
3 b hydroxysteroid dehydrogenase (HSD) was measured by mixing 250 ll of the
supernatant with 250 ll of 100 lM sodium pyrophosphate buffer, pH 8.9, 10 ll eth-anol
containing 30 lg DHEA (Sigma, St. Louis, USA) and 240 ll of 25 mg% BSA (Ban-galore
Genei, India). Enzyme activity was measured after addition of 50 ll of 0.5 lM NAD to the
mixture in a spectrophotometer at 340 nm against a blank (without NAD). One unit of enzyme
activity was the amount causing a change in absorbance of .001/min at 340 nm.
17 b HSD was measured by mixing 250 ll of the supernatant with 250 ll of
440 lM sodium pyrophosphate buffer (pH 10.2), 10 ll ethanol containing 0.3 lM testosterone
(Sigma, St. Louis, USA) and 240 ll of 25 mg% BSA. Enzyme activity was measured after
addition of 50 ll of 0.5 lM NAD to the mixture in a spectropho-tometer at 340 nm against a
blank (without NAD). One unit of enzyme activity was the amount causing a change in
absorbance of .001/min at 340 nm.
2.9. Serum testosterone level
After decapitation, blood was collected for determination of serum testosterone levels.
Serum was separated after centrifugation (4500 rpm, 20 min, 4 LC) and fro-zen at 20 LC. Level
of testosterone in the serum (100 ll) was measured by RIA kit (Immunotech, Marseille, France)
as per the manufacturer instructions. Intra and inter assay coefficient of variation were 12% and
15%, respectively.
2.10. Toxicological investigations
To observe the effect of SAx treatment on liver and kidney function, serum ala-nine
aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine were determined
using kits (Span Diagnostic Ltd., Surat, India) as per the manufacturer instructions. The enzyme
activity is expressed in units where one unit denotes lmoles of the product formed per hour.
2.11. Statistical analyses
All data, except body weight were analysed by one-way analyses of vari-ance (ANOVA)
followed by Newman–Keuls’s multiple range test. Body weight data were analysed by Student’s
t-test. Differences were considered significant at P < 0.05.
3. Results
3.1. Body weight and testis weight
The SAx treatment had no effect on the body weight of animals, and all
the animals maintained a healthy appearance throughout the investigation.
However, higher doses (30 mg and 60 mg) of SAx adversely affected the
weight of testis as compare to control (Table 1).
3.2. Daily sperm production
Testicular sperm production in animals treated with lower
dose (15 mg) of the SAx did not differ significantly from the
controls,
 R.K. Mishra, S.K. Singh / Food and Chemical Toxicology 46 (2008) 3333–3338 3335

Table 1 In case of severe damage the seminifereous tubules were lined by Sertoli cells
Effect of Syzygium aromaticum extract on body weight, weight of the testis, daily sperm
and few germ cells only (Fig. 1c and d).
production, and on percentage of affected seminiferous tubules in mice

Groups Body weight (g) Testis weight Daily Affected 3.4. Flowcytometry
(mg) sperm seminiferous
Initial Final
production tubules (%)
On the basis of DNA content, three main germ cell peaks could be
per testis
6 identified in control animals: 1C (round and elongated sperma-tids), 2C
( 10 )
(spermatogonia), and 4C (primary spermatocytes). The re-gion between 2C
I 32.00 ± 0.00 34.00 ± 1.21 105.51 ± 5.07 2.64 ± 0.26 14.27 ± 3.73
II 34.00 ± 0.00 34.00 ± 0.41 99.27 ± 4.18 2.34 ± 0.31 21.55 ± 5.90 and 4C peaks is composed of cells actively synthesizing DNA and is termed
III 34.00 ± 0.00 33.00 ± 1.73
*
97.28 ± 2.11
*
1.38 ± 0.34
*
64.64 ± 6.71 as S-phase (synthetic phase).
IV 34.00 ± 0.00 35.00 ± 1.21
*
93.13 ± 3.11
*
1.26 ± 0.22
*
80.83 ± 10.33 Treatment with different doses of SAx resulted in germ cell depletion as
Values are mean ± S.D. for five animals.
there was a gradual loss in 1C population concomitant with a parallel rise in
*
Significantly different from controls (p < 0.05). the 2C, S-phase, and 4C populations. Further administration of different doses
of SAx also resulted in significant alterations in various germ cells ratios (Fig.
2; Table 2).
whereas those treated with higher doses of the SAx showed signif-icant
decrease as compare to control (Table 1). 3.5. Serum testosterone level

3.3. Histology
In histologic examination, no changes in testis were observed in the
controls. (Fig. 1a) In lower dose (15 mg) group, majority of seminiferous
tubules exhibited active spermatogenesis, but there were loosening of
germinal epithelium or intraepithelial vacuola-tion in some seminiferous
tubules (Fig. 1b). By contrast, marked alterations were noticed in testis of
mice treated with higher doses of the SAx. There were significant increases in
percentage of af-fected seminiferous tubules in higher dose groups, and
severity of the testicular damage showed dose dependency (Fig. 1c and d;
Table 1). Elongated and round spermatids were the most strongly depleted
cells in these treated groups. Intraepithelial vacuolation, loosening of
germinal epithelium, marginal condensation of chro-matin in round
spermatids, and degeneration of other cell types were also observed in the
seminiferous tubules of treated animals.
Serum level of testosterone in mice treated with lower dose (15 mg) of
SAx showed significant increase, whereas marked de-cline was noticed in
testosterone level in higher dose groups as compare to control (Table 3).
3.6. Testicular enzyme assay
5
Testicular D 3 b-HSD and 17 b-HSD activities were increased in animals
treated with lower dose (15 mg) of the SAx, while these parameters were
decreased significantly in higher dose groups as compare to control (Table 3).
3.7. Toxicological investigations
No differences were detected in ALT, AST and creatinine levels in serum
of mice treated with SAx as compare to control (Table 3).

Fig. 1. a–d are PAS-Haematoxylin stained sections of mouse testis. X 175. (a). Control testis showing normal appearance of seminiferous tubules. (b) Treated (SAx, 15 mg/kg BW/day for 35 days)
testis showing loosening of germinal epithelium and appearance of intraepithelial vacuoles in a seminiferous tubule ( ). (c) Treated (SAx, 30 mg/kg BW/ day for 35 days) testis showing marked
alterations in seminiferous tubules. Arrow indicates marginal condensation of chromatin of round spermatids. (d) Treated (SAx, 60 mg/kg BW/day for 35 days) testis showing atrophic appearance of
seminiferous tubules.
3336 R.K. Mishra, S.K. Singh / Food and Chemical Toxicology 46 (2008) 3333–3338

Fig. 2. Representative DNA histograms of testicular germ cells of mice treated with Syzygium aromaticum extract.

Table 2
Effect of Syzygium aromaticum extract on relative percentage of testicular germ cell populations, and on germ cell ratio of mice

Groups Spermatids (1C) Spermatogonia (2C) Primary spermatocytes (4C) S-phase 1C:2C 1C:4C 4C:2C 4C:S-ph
I 60.96 ± 5.48 20.4 ± 1.75 13.07 ± 2.67 3.82 ± 1.26 3.01 ± 0.49 4.90 ± 1.53 0.64 ± 0.12 3.59 ± 0.71
* * * * * * *
II 32.99 ± 1.28 37.08 ± 0.97 15.32 ± 2.33 12.24 ± 0.70 0.88 ± 0.01 2.19 ± 0.38 0.41 ± 0.07 1.26 ± 0.26
* * * * * * *
III 34.31 ± 1.12 41.95 ± 1.46 11.4 ± 2.71 9.42 ± 1.21 0.81 ± 0.03 3.12 ± 0.62 0.27 ± 0.07 1.24 ± 0.42
* * * * * * *
IV 9.02 ± 1.25 65.39 ± 1.40 11.25 ± 1.93 11.80 ± 0.86 0.13 ± 0.02 0.82 0.24 0.17 ± 0.03 0.96 ± 0.23
Values are mean ± S.D. for five animals.
*
Significantly different from controls (p < 0.05).

Table 3
5
Effect of Syzygium aromaticum extract on testicular D 3 b-HSD and 17 b-HSD and on serum level of testosterone, ALT, AST, and creatinine in mice
5
Groups D 3 b-HSD 17 b-HSD Serum Testosterone ALT AST Creatinine
Unit/mg of tissue/h Unit/mg of tissue/h ng/ml U/ml U/ml mg/100 ml
I 15.70 ± 1.53 19.34 ± 3.21 2.56 ± 0.28 24.10 ± 2.39 18.60 ± 3.63 2.61 ± 0.15
* *
II 17.16 ± 0.84 23.72 ± 3.79 3.23 ± 0.36 24.80 ± 1.88 18.64 ± 2.39 2.31 ± 0.47
* * *
III 6.65 ± 1.08 12.02 ± 1.41 1.56 ± 0.05 24.94 ± 1.56 17.37 ± 1.84 2.54 ± 0.45
* * *
IV 5.62 ± 1.30 8.00 ± 1.53 1.61 ± 0.18 24.82 ± 1.48 20.23 ± 2.91 3.02 ± 0.54
Values are mean ± S.D. for five animals.
*
Significantly different from controls (p < 0.05).

4. Discussion appearance of animals throughout experiment as well as lack of adverse effect

Flower buds of Syzygium aromaticum (clove) have been used for
treatment of male sexual disorders in Ayurvedic and Unani medi-cines of
Asian countries. Although few studies have been carried out to find out the
effect of Syzygium aromaticum flower bud on male sexual behavior
(Tajuddin et al., 2004, 2003), its effect on functional physiology of male
reproductive organs has not been studied so far. Therefore, the present study
was designed to evalu-ate the testicular effects of chronic exposure of hexane
extract of Syzygium aromaticum (SAx) in mice. Three doses of SAx (15 mg,
30 mg, and 60 mg/Kg BW) were administered orally over 35 days (one
spermatogenic cycle in mouse) to adult male mice in order to ascertain
possible testicular effects.
Serum levels of transaminases (ALT and AST), and creatinine are good
indicators of liver and kidney functions, respectively. Healthy
on the body weight or liver and kidney function sug-gest that SAx did not
induce any systemic toxicity at the doses tested. In mice treated with lower
dose (15 mg) of SAx, the semi-niferous tubules presented almost normal
appearance, whereas higher doses (30 and 60 mg) of SAx caused non-uniform
degener-ative changes in the seminiferous tubules; also with increasing dose
of the treatment, the alterations in the seminiferous tubules increased in
severity. Similar observations have also been reported in testes of P mice after
treatment with neem leaf extract (Mishra and Singh, 2005) and several
antispermatogenic agents (Singh and Chakravarty, 2003; Singh and Dominic,
1995).Seminiferous tu-bules make up about 90% of the wet
weight of the normal testis, the testicular weight loss in
higher doses groups may be attributed to the spermatogenic
disruption and degeneration of germinal elements.
 R.K. Mishra, S.K. Singh / Food and Chemical Toxicology 46 (2008) 3333–3338
The major disadvantage of histopathologic evaluation is that it allows
limited number of seminiferous tubules to be evaluated (Suter et al., 1998).
Recently flow cytometry (FCM) has become a useful tool for objective
quantitation of the cell types involved in spermatogenesis and supplies
therefore very valuable information for the detection of testicular toxicity
(Katoh et al., 2002; Suter et al., 1998). Treatment with different doses of SAx
resulted in germ cell depletion as there was a gradual loss in 1C population
concomitant with a parallel rise in the 2C and S-phase populations. A similar
reduction in 1C population and increase in 2C and S-phase cells has been
reported in the testicular cells of rat treated with different antispermatogenic
agents such as estradiol benzo-ate, gossypol, and CDRI 84/35 (Ojha et al.,
2008). Decline in 1C (round and elongated spermatids) population after
treatment in the present study indicates the sensitivity of post meiotic cells to
the treatment (Russell et al., 1990). The rise in 2C (spermatogonia) and S-
phase cells in treated mice may not indicate an increase in actual cell number
but represent the proportional increase in 2C and S-phase population (per
10,000 cell counted) over other cell types.
The perturbations in the relative percentage of different germ cell
populations at various dose levels after SAx treatment were manifested as
alterations in the ratio of different germ cell popula-tions. The reduction in
1C:2C, 1C:4C and 4C: S-phase ratio in dose dependent manner indicates
inhibition of transformation of sper-matogonia into round spermatids (Jyothi
et al., 2001). Further, a decrease in daily sperm production per testis was
noticed which correlates well with the present data of FCM indicating
perturba-tion in the process of spermatogenesis.
The data of present study indicate the biphasic nature of lower (15 mg)
and higher (30 and 60 mg) doses of SAx on testosterone biosynthesis. Several
plants having traditional medicinal impor-tance have been reported to regulate
the serum testosterone level (Kamal et al., 2003). In testicular steroidogenic
5 Aspects Med. 27, 1–93.
events D 3 b-HSD and 17 b-HSD play a key regulatory role as these are the
prime en-zymes in testicular androgenesis (Jana et al., 2006; Strauss, 2004).
5 1069–1078.
Lower dose (15 mg) of SAx increased the activities of D 3 b-HSD and 17 b-
HSD, whereas higher doses (30 and 60 mg) of SAx inhib-ited the activities of
these enzymes. In the present study, an in-crease or decrease in serum level of
testosterone with respect to doses may occur due to alterations in the activities
of these en-zymes by the treatment. Pituitary gonadotropins are prime regula-
tors of testicular androgenic enzyme activities (Shimomura et al., 2005). In
5 Jarow, J.P., Wright, W.W., Brown, T.R., Yan, X., Zirkin, B.R., 2005. Bioactivity of androgens
the present study alterations in the activities of testicular D 3 b-HSD and 17 b-
HSD may be due to the effect of treatment on pituitary gonadotropins secretion
(Ghosh et al., 2002; Jana et al., 2006).
The findings of this experiment also indicate that SAx affects the process
of spermatogenesis by altering the testosterone biosynthe-sis. Testosterone
exerts a biphasic effect on spermatogenesis by both inhibiting and promoting
the process in vivo (Mc Lachlan et al., 2002). Intratesticular testosterone
concentration is 30–50 times higher than serum testosterone level to maintain
normal spermatogenesis (Jarow et al., 2005).The higher serum testoster-one
level is thought to suppress testicular testosterone level, which ultimately
affects the process of spermatogenesis (Shimamura et al., 2005). This may be
the reason for loosening of germinal epi-thelium in some seminiferous tubules
and decrease in 1C cell types in testis of lower dose (15 mg) group in the
present study, in spite of high serum testosterone level.
In conclusion, data of the present study suggest the biphasic nature of
lower (15 mg) and higher (30 and 60 mg) doses of the flower bud extract on
testis. The lower dose increased testosterone production, while higher doses
caused reduction in testosterone production thereby perturbation in
spermatogenesis of mice. Hence, data of the present study advocate cautious
use of this flow-
3337
er bud as an aphrodisiac in indigenous systems of medicine in Asian
countries.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
Authors would like to thank Director C.D.R.I., Lucknow, India for
permission for flow cytometry usage. This work was supported by the
Department of Science and Technology (DST), New Delhi, in the form of
SERC FAST Track Project (No. SR/FT/L-72/2005) to Raghav Kumar Mishra.
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