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Journal of Ethnopharmacology 122 (2009) 579582

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Journal of Ethnopharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ j et hphar m
Ethnopharmacological communication
Aphrodisiac properties of Allium tuberosum seeds extract
Hu Guohua
a,
, Lu Yanhua
b
, Mao Rengang
a
, Wei Dongzhi
b
, Ma Zhengzhi
a
, Zhang Hua
a
a
College of Life and Environment Science, Shanghai Normal University, Guilin Road 100#, Shanghai 200234, PR China
b
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Meilong Road 130#, Shanghai 200237, PR China
a r t i c l e i n f o
Article history:
Received 4 October 2008
Received in revised form 8 December 2008
Accepted 15 January 2009
Available online 7 February 2009
Keywords:
Allium tuberosum
Seed
Herbal medicine
Aphrodisiac
Sexual behavior
a b s t r a c t
Aim of the study: In the present study, we examined the effect of Allium tuberosumseeds extract upon the
expression of male rat sexual behavior, in order to knowwhether Allium tuberosumseeds extract possess
aphrodisiac property.
Materials andmethods: Theaphrodisiac activityof Alliumtuberosumseeds n-BuOHextract was investigated
in male rats. The extract (500 mg/kg body weight/day) and L-dopa (100 mg/kg body weight/day) were
administered orally by gavages for 40 days. Mount latency (ML), intromission latency (IL), ejaculation
latency (EL),mounting frequency (MF), intromission frequency (IF), ejaculation frequency (EF) and post-
ejaculatory interval (PEI) were the parameters observed before and during the sexual behavior study at
day 0, 10, 20, 30 and 40.
Results: The n-BuOH extract reduced signicantly ML, IL, EL and PEI (p < 0.05). The extract also increased
signicantly MF, IF and EF (p < 0.05). These effects were observed in sexually active and inactive male
rats.
Conclusions: Present ndings provide experimental evidence that the n-BuOH extract preparation of
Allium tuberosum seeds possesses aphrodisiac property.
2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Since ancient time, many Allium species, such as onion, garlic,
leek, and chives, have been used as foods, spices, and herbal reme-
dies in widespread areas of the world, especially in the northern
hemisphere. It is well known that the Allium genus, with about
500 species, is a rich source of steroidal saponins, alkaloids, as
well as sulfur-containing compounds (Hostettmann and Marston,
1995). The scientic name of Chinese chive is Allium tuberosum
Rottl. ex Spreng (Chinese chive, Liliaceae). Chinese chive is one of
the daily edible green vegetables for Chinese. Allium tuberosum is
distributed all over mainland China and used not only as food but
also as medicine. Chinese chive is widely cultivated in China, whose
seeds have been reputedly used as a traditional Chinese medicine
for treating both impotence and nocturnal emissions (Jiangsu
New Medicinal College, 1979). With regard to the constituents
of the Allium tuberosum seeds, many new and known steroidal
saponions, alkaloids and amides have been reported from the
seeds (Sang et al., 1999a,b, 2000a,b,c, 2001a,b, 2002, 2003; Zou et
al., 2001). In order to clarify its bioactive compounds, the chemical
constituents of the seeds of Allium tuberosum were analyzed and

Corresponding author. Tel.: +86 21 64322933; fax: +86 21 64322142.


E-mail address: hgh114226@sina.com (H. Guohua).
studied at our research systematically. We reported the fatty
acid compositions of the oil of ten kinds of Allium tuberosum
seed (Guohua Hu et al., 2005). We also reported the chemical
compositions of Allium tuberosum seed (Guohua Hu et al., 2006a,b,
2009). However, no experimental investigations have been carried
out on the diverse pharmacological properties of this traditional
remedy.
There are numerous reports of aphrodisiac activity attributed
to plants (Emilia Nocerino et al., 2000; Islam et al., 1990;
Subramoniam et al., 1997; Noumi and Zollo, 1998; Bo Lin Zheng
et al., 2000; Gauthaman et al., 2002; Carro-Juareza et al., 2004;
Ramachandran et al., 2004). Allium tuberosum seeds have been
reputedly used as a traditional Chinese medicine for treating
both impotence and nocturnal emissions (Jiangsu New Medicinal
College, 1979). These claims are based largely on subjective opin-
ion rather than scientic observation. In the present study, we
examined the effect of Allium tuberosum seeds extract upon the
expression of male rat sexual behavior, in order to evaluate
the possibility that this plant extract might possess aphrodisiac
property. Experimental investigations on the diverse pharmaco-
logical properties of this plant seed are being actively pursued
in our laboratory. The present study was aimed to search the
active constituents from Allium tuberosum for treating both
impotence and nocturnal emissions. The study evaluated the
effect of this plant seed n-BuOH extract on the male sexual
behavior.
0378-8741/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2009.01.018
580 H. Guohua et al. / Journal of Ethnopharmacology 122 (2009) 579582
2. Materials and methods
2.1. Preparation of Allium tuberosum seeds extract
Allium tuberosum Rottl. seed (791, 791 is furthest widely
cultivated in China in all variety) were purchased from Shanghai
Shun-Feng-Tang Group, Shanghai, China in July 2004. A voucher
specimen (No. 200305), identied by professor Dr. Dongzhi Wei,
was deposited at Institute of Biochemistry, East China University
of Science and Technology, Shanghai, 200237, China.
The powdered seeds of Allium tuberosum (10.0kg) were
extracted successively with petroleum ether (petroleum ether:
seed, 10:1, v/w) 2 and 95% EtOH (EtOH: seed, 10:1, v/w) 3. The
combined EtOHextracts were ltered and evaporated to dryness at
a low temperature under reduced pressure in a rotary evaporator.
After evaporation of ethanol, the residue was suspended in water
and then extracted successively with petroleum ether (petroleum
ether: extract, 1:4, v/v) 3, EtOAc (EtOAc: extract, 1:4, v/v) 3 and
n-BuOH (n-BuOH: extract, 1:4, v/v) 3. The n-BuOH extract was
evaporated to dryness at a low temperature under reduced pres-
sure in a rotary evaporator. The n-BuOH extract (0.8% (w/w) yield)
was suspended in distilled water and used for studying bioactivity.
2.2. Animals
Inbred adult Wistar albino rats of either sex were used for
the study. The animals were fed with standard animal feed and
water. The animals were housed at a temperature of 251

C with
a reversed light dark cycle and relative humidity of 5055%. The
study was performed as per the protocols and recommendation of
the Institutional Animal Ethics Committee, Science and Technology
Commissionof Shanghai Municipality. All animal experiments were
conducted in accordance with the NIHguide for the care and use of
laboratory animals (NIH Publication No. 80-23; revised 1978).
Male rats (n=5/group) were trained for sexual experience. To
provide sexual experience, each male rat was allowed 30min expo-
sure toa female rat inbehavioral estrous, several days before testing
for copulatory performance in a transparent arena. The animals
were tested three times over a 10-day period for copulatory behav-
ior and divided into active and inactive groups (Ramachandran
et al., 2004). Sexually active animals were divided into control,
l-dopa-treated (100mg/kg body weight) and Allium tuberosum
seeds-treated (500mg/kg body weight) groups. The animals that
did not show any sexual interest during training were considered
the inactive group that was administered Allium tuberosum seeds
(500mg/kg body weight). Female rats were ovariectamised using
a standard procedure (Bures et al., 1983). They were allowed to
recover fromthesurgeryfor 10days. Theywerebrought intoestrous
bythe administrationof a single subcutaneous dose of 2g/kgbody
wt. of estrogen benzoate (Sigma Chemical Co., St. Louis, MO, USA)
and500g/kgbodywt. progesterone(SigmaChemical Co., St. Louis,
MO, USA) 48 and 6h before the copulatory study.
2.3. Sexual behavior study
The following guidelines (Hart et al., 1983; Islam et al., 1990)
were followed in the study; males were kept individually whereas
females were kept in groups; training of each male for 15min at a
time was performed until sexual behavior was elicited and when
the behavior was noticed, males were exposed to receptive females
(one male with ve females); repeated training to overcome the
lack of sexual response in the presence of observers; the study was
conducted in a single observation cage (0.8m1.0m1.2m) in a
silent roomunder dimred light. Any jerking movement of the mat-
ing area was avoided to enable the rats to chase each other; and
cleaning of the mating area was performed after each trial, since
the urine trails left by one rat might alter the sexual behavior of
the next rat. They were fed commercially available food twice each
day, and water was always available. All sexual behavior tests were
observed for 4h and performed from 09:00 to 13:00h.
Allium tuberosum seeds (500mg/kg body weight/day) and
l-dopa (100mg/kgbodyweight/day) indistilledwater were admin-
istered for 40 days orally by gavage. l-Dopa served as standard
(Greenburg, 1971; Ananthakumar et al., 1994). The control group
received 1ml saline. Each group consisted of six animals (one male
and ve females). The female was then introduced into the cham-
ber and the following sexual behavior parameters were recorded:
(a) mount latency (ML): the time interval between the introduc-
tion of the female and the rst mount by the male; (b) intromission
latency (IL): the interval fromthe time of introduction of the female
to the rst intromissionby the male (characterizedby pelvic thrust-
ing and springing dismount); (c) ejaculation latency (EL): the time
interval between the rst intromission and ejaculation (character-
ized by longer, deeper pelvic thrusting and slowdismount followed
by a period of inactivity); (d) mount frequency (MF): the number
of mounts observed in 30min; (e) intromission frequency (IF): the
number of intromissions observed in 30min; (f) ejaculation fre-
quency (EF): the number of ejaculations observed in 30min; (g)
post-ejaculatory Interval (PEI): the time interval between ejacula-
tion and the rst intromission of the following series.
2.4. Toxicity study
Two groups of six male and six female rats each were used. The
n-BuOH extract (2g/kg body weight) was administered orally by
gavagetotherats inthetest group. Therats inthecontrol groupeach
received 1ml of tap water. Behavioral parameters such as convul-
sions, sedation, hyperactivity, grooming and accelerated breathing
were observed. The animals were observed continuously for 1h for
any behavioral changes or death, then intermittently for 6h and
nally 24h after drug administration.
2.5. Data analysis
Pair-wise statistical comparisons between control and treated
groups were done with Students t-test. Mean differences were
considered statistically signicant if p<0.05.
3. Results
The observations of the sexual behavior study are presented in
Table 1. The present study conrmed the claims of Allium tubero-
sum seeds n-BuOH extract as an aphrodisiac agent. Sexually active
and inactive animals showed increased and improved sexual per-
formance, when Allium tuberosum seeds extract (500mg/kg body
weight) was administered for a period of 3040 days. Alliumtubero-
sumseeds n-BuOHextract reduced ML, IL, EL and PEI signicantly in
both active and inactive male rats. Allium tuberosum seeds n-BuOH
extract also increased MF, IF and EF signicantly in both active and
inactive male rats. All these effects were observed on the 30th and
40th days of the study. The MF and EF was signicantly altered in
Allium tuberosum seeds-treated active rats (20th day), whereas l-
dopa did not alter these parameters on the 20th day. The MF and IF
were signicantly altered in Allium tuberosum seeds-treated inac-
tive rats (20th day), whereas l-dopa did not alter these parameters
on the 20th day. The EL and PEI was signicantly altered in l-dopa
treated active rats (20th day), whereas Allium tuberosum seeds for
both active and inactive male rats did not alter these parameters on
the 20th day. Allium tuberosum seeds for both active and inactive
male rats and l-dopa did not alter any of all these parameters onthe
10th day. In toxicity test to determine any acute toxicity of n-BuOH
extract, no mortality or changes in behavior were observed in the
H. Guohua et al. / Journal of Ethnopharmacology 122 (2009) 579582 581
Table 1
Sexual behavior study of Allium tuberosum seeds extract.
Group Sexual behavior
parameters
MeanS.E.M.
0 day 10th day 20th day 30th day 40th day
Active (Control) ML 65.16 5.15 62.32 4.95 66.87 5.12 65.28 4.00 63.29 5.36
IL 179.28 15.85 174.37 15.54 175.61 14.70 171.06 15.81 168.12 17.46
EL 8.16 0.70 7.93 0.65 8.19 0.48 7.43 0.39 8.03 0.35
MF 21.29 3.00 21.18 2.86 22.37 3.07 23.64 3.28 22.76 3.14
IF 7.50 1.65 7.32 1.47 7.3 1.28 7.81 1.75 7.70 1.98
EF 2.74 0.33 2.82 0.41 2.91 0.45 2.70 0.53 3.01 0.67
PEI 8.44 0.68 8.15 0.67 8.18 0.59 8.29 0.56 7.92 0.72
Active (Allium tuberosum seeds
extract 500mg/kg body wt.)
ML 69.39 4.51 64.62 4.67 59.45 4.10 39.87 2.65
*
32.34 1.89
*
IL 167.21 15.28 155.67 16.27 146.31 17.21 101.73 8.69
*
84.25 6.63
*
EL 8.05 0.78 7.62 0.73 7.40 0.75 5.94 0.42
*
4.12 0.37
*
MF 29.56 2.90 32.02 3.20 39.55 2.60
*
43.28 2.90
*
52.71 4.10
*
IF 15.20 1.68 15.30 1.89 15.50 1.82 16.9 1.56
*
19.8 1.78
*
EF 3.09 0.36 3.32 0.40 3.99 0.31
*
4.05 0.35
*
4.23 0.33
*
PEI 7.81 0.59 7.30 0.63 7.15 0.61 5.61 0.35
*
4.93 0.42
*
Inactive (Allium tuberosum seeds
extract 500mg/kg body wt.)
ML 130.61 10.18 117.36 11.19 113.72 12.08 91.34 7.06
*
76.66 7.38
*
IL 298.09 28.14 268.98 29.31 237.46 26.36 185.49 15.45
*
141.25 10.33
*
EL 6.13 0.48 5.68 0.52 5.57 0.47 4.76 0.40
*
4.10 0.39
*
MF 19.85 1.91 19.82 1.95 22.96 1.81
*
24.15 2.00
*
25.68 1.97
*
IF 8.22 0.78 8.65 0.80 11.57 0.89
*
13.31 0.81
*
15.57 1.28
*
EF 2.61 0.34 3.14 0.45 3.22 0.53 4.39 0.42
*
5.91 0.57
*
PEI 9.25 0.40 9.18 0.41 8.74 0.36 7.52 0.32
*
5.31 0.38
*
Active (l-dopa 100mg/kg body wt.) ML 64.58 5.61 54.73 6.60 52.31 6.01 35.38 3.58
*
26.65 2.12
*
IL 198.26 19.23 177.36 16.18 171.08 19.49 104.51 10.81
*
75.86 6.53
*
EL 7.21 0.50 7.02 0.48 6.00 0.32
*
5.74 0.35
*
3.96 0.29
*
MF 21.36 1.50 23.20 1.79 23.97 1.56 29.48 1.41
*
34.54 2.02
*
IF 7.39 1.21 7.62 1.18 8.48 1.30 10.49 1.30
*
12.32 1.58
*
EF 2.46 0.29 2.48 0.26 2.56 0.28 3.22 0.31
*
4.19 0.30
*
PEI 5.72 0.40 5.65 0.45 4.27 0.35
*
4.13 0.36
*
4.02 0.32
*
ML, mount latency; IL, intromission latency; EL, ejaculation latency; MF, mount frequency; IF, intromission frequency; EF, ejaculation frequency; PEI, post-ejaculatory interval.
*
Signicance level: p<0.05.
control group or in the group of male and female rats treated at the
dose of 2g/kg body weight of n-BuOH extract.
4. Discussion
Present ndings provide experimental evidence that the n-
BuOH extract preparation of Allium tuberosum seeds, used as a
traditional remedy, possesses aphrodisiac property. The present
study provides evidence that the extract of Allium tuberosum is a
potent stimulator of sexual behavior, particularly of sexual arousal
in male rats, and that it promotes the expression of masculine sex-
ual behavior. It has been reported that EGb 761 (a standardized
extract of Ginkgo biloba leaves, especially at the dose of 50mg/kg)
enhances the copulatory behavior of male rats and suggest that the
dopaminergic system, which regulates prolactin secretion, may be
involved in the facilitatory effect of EGb 761 (Kuei-Ying Yeh et al.,
2008). Generally sexual behavior is enhanced by elevated testos-
terone levels. Drug-induced changes in neurotransmitter levels or
their action in the cells could also change sexual behavior. The
brain area most associated with sexual behavior is the limbic sys-
tem. Research with various animal and human models indicates
a relationship between brain dopamine, 5HT and sexual behav-
ior (Ramachandran et al., 2004; Singh and Mukerjee, 1998). Both
dopamine and 5HT are implicated in depression. The relationship
of dopamine to human sexual behavior is supported by reports of
persexuality behavior induced by l-dopa in parkinsonian patients.
Stimulants andantidepressants are knownto affect libido, erection,
ejaculation and orgasm.
Chinese men have always been interested in increasing virility
and sexual satisfaction to the extent of using any means possi-
ble, an aspect that has been well documented (Krishna Kaphle et
al., 2006). Allium tuberosum is widely cultivated in China, whose
seeds have been reputedly used as a traditional Chinese medicine
for treating both impotence and nocturnal emissions (Jiangsu New
Medicinal College, 1979). According to the dictionary of Chinese
drugs (Jiangsu New Medical College, 1986), the leaves have been
used for treatment of abdominal pain, diarrhea, hematemesis,
snakebite and asthma, while the seeds of this plant have been used
in Chinese traditional medicine as a tonic and aphrodisiac. Ancient
Chinesemanuscripts arefull of recipes for aphrodisiacs andphilters,
while in the modern era anything from rhino horn to mice testes
ends up as medicine (Krishna Kaphle et al., 2006). Aphrodisiacs
are substances that stimulate/increase sexual desire and perfor-
mance. Active investigations are in progress in our laboratory to
explore these possible mechanisms of action of Allium tuberosum
seeds. The present investigation reveals the rst time that the n-
BuOH extract of Allium tuberosum seeds can enhance male sexual
activity in normal mice. There is a possibility of developing the
n-BuOHextract of Alliumtuberosumseeds for stimulating male sex-
ual activity in cases where there are moderate sexual deciencies.
With regard to the constituents of the Allium tuberosum seed n-
BuOH extract, about thirty new and known steroidal saponions,
nucleoside, alkaloids and amides have been reported (Sang et al.,
1999a,b, 2000c, 2001a,b, 2002, 2003; Zou et al., 2001; Guohua Hu
et al., 2006b, 2009). Among the constituents reported of the Allium
tuberosumseedn-BuOHextract, Steroidal saponions andnucleoside
are the main constituents of the Allium tuberosum seeds n-BuOH
extract.
Further investigations are in progress to ascertain the mecha-
nism by which chemical constituents in Allium tuberosum seeds
n-BuOH extract exerts potent aphrodisiac activity in both sexually
582 H. Guohua et al. / Journal of Ethnopharmacology 122 (2009) 579582
active and inactive males. The effect of the drug on female sexual
behavior and fertility remains to be studied.
5. Conclusion
Sexual behavior was studied in normal rats to further under-
stand the role of Allium tuberosum seeds as an aphrodisiac. There
was an overall increase in the sexual behavior parameters in the
Allium tuberosum seeds n-BuOH extract treated groups of rats as
reected in MF, IF and EF, and reduction in ML, IL, EL and PEI. These
results were statistically signicant. It is concluded that Allium
tuberosum seeds n-BuOH extract appears to possess aphrodisiac
activity.
Acknowledgments
The work is supported by Shanghai Municipal Education Com-
mission. The authors are grateful for the assistance of Zhou Jiachun
and Zhuo zhixiang of the department of Food Science and Engi-
neering, East China University of Science and Technology, Shanghai,
China; Yang Xiaotong, Wu Shifu and Cao Tong of College of Life
and Environment Science, Shanghai Normal University, Shanghai,
China.
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