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179




Moringa oleifera extract enhances sexual performance in stressed rats*

Thawatchai PRABSATTROO†1,3, Jintanaporn WATTANATHORN†‡2,3, Sitthichai IAMSAARD3,4,
Pichet SOMSAPT5, Opass SRITRAGOOL5, Wipawee THUKHUMMEE3, Supaporn MUCHIMAPURA2,3
(1Graduate School and Department of Physiology (Neuroscience Program), Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand)
(2Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand)
(3Integrative Complementary and Alternative Medicine Research and Development Center, Khon Kaen University, Khon Kaen 40002, Thailand)
(4Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand)
(5Division of Nuclear Medicine, Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand)
†E-mail: backwert@gmail.com; jintanapornw@yahoo.com
Received July 13, 2014; Revision accepted Oct. 31, 2014; Crosschecked Feb. 21, 2015

Abstract: Aphrodisiacs are required to improve male sexual function under stressful conditions. Due to the effects of
oxidative stress and dopamine on male sexual function, we hypothesized that Moringa oleifera leaves might improve
male sexual dysfunction induced by stress. Therefore, the effects on various factors playing important roles in male
sexual behavior, such as antioxidant effects, the suppression of monoamine and phosphodiesterase type 5 (PDE-5)
activities, serum testosterone and corticosterone levels, and histomorphological changes in the testes, of a hy-
droethanolic extract of M. oleifera leaves were investigated. Various doses of extract including 10, 50, and 250 mg/kg
body weight (BW) were given orally to male Wistar rats before exposure to 12 h-immobilization stress for 7 d. The
results demonstrated that the extract showed both antioxidant and monoamine oxidase type B (MAO-B) suppression
activities. At 7 d of treatment, the low dose of extract improved sexual performance in stress-exposed rats by de-
creasing intromission latency and increasing intromission frequency. It also suppressed PDE-5 activity, decreased
serum corticosterone level, but increased serum testosterone, numbers of interstitial cells of Leydig and spermatozoa.
The increased numbers of interstitial cells of Leydig and spermatozoa might have been due to the antioxidant effect of
the extract. The increased sexual performance during the intromission phase might have been due to the suppression
of MAO-B and PDE-5 activities and increased testosterone. Therefore, M. oleifera is a potential aphrodisiac, but further
research concerning the precise underlying mechanisms is still needed.

Key words: Moringa oleifera, Sexual behaviors, Stress
doi:10.1631/jzus.B1400197 Document code: A CLC number: R698


1 Introduction

Sexual feeling and sexual activity are inevitable
parts of life, which play an important role in the sur-
vival of the human race (Kothari, 2001). Since sex is a
most intimate, indispensable, and integral part of
every individual’s life and can be a source of pleasure
and fulfillment, sexual dysfunction can induce de-
pression, anxiety, and debilitating feelings of inade-
quacy (Kennedy et al., 1999; Baldwin, 2001). Alt-
hough male sexual dysfunction is not a life threaten-
ing disorder, it can greatly affect the quality of life. It
has been reported that about 20%–30% of men suffer
from sexual dysfunction (Lewis et al., 2004). Because
of its high impact and high prevalence, a great effort
has been made to search for effective interventions to
protect against sexual dysfunction.

Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
www.zju.edu.cn/jzus; www.springerlink.com
E-mail: jzus@zju.edu.cn


‡ Corresponding author
* Project supported by the Higher Education Research Promotion and
National Research University Project of Thailand, Office of the Higher
Education Commission, through the Food and Functional Food Re-
search Cluster of Khon Kaen University (No. NRU541061) and the
Integrative Complementary Alternative Medicine Research and De-
velopment Center, Khon Kaen University, Thailand
ORCID: Thawatchai PRABSATTROO, http://orcid.org/0000-0001-
6292-9040; Jintanaporn WATTANATHORN, http://orcid.org/0000-
0002-7383-2348
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2015

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180
Sexual function is a complex process involving
the brain, hormones, emotions, nerves, muscles, and
blood vessels. It is very sensitive to stress. Therefore,
stressful lifestyles or events usually induce sexual
dysfunction. It has been reported that chronic physical
and psychological stresses modulate neurotransmis-
sion in the median preoptic area and decrease penile
blood flow, resulting in erectile dysfunction (Santosh
et al., 2011). Experimental stress induced by restraint
can suppress testicular steroidogenesis, giving rise to
a reduction in plasma testosterone (Orr and Mann,
1990). Long term exposure to stress destroys inter-
stitial cells of Leydig and decreases serum testos-
terone and spermatogenesis (Rai et al., 2004). Stress
also increases the activity of the hypothalamo-
pituitary-adrenal axis (HPA-axis), leading to the en-
hanced plasma cortisol and increased sympathetic
system function (Carrasco and van de Kar, 2003).
This leads to excessive oxidative stress and stress-
related disorders (Ahmad et al., 2012) including
sexual dysfunction (Orr and Mann, 1990).
To date, most available drugs in the market tar-
get at the intromission phase, the most common phase
of sexual dysfunction. The most popular drugs are
sildenafil citrate, vardenafil and tadalafil citrate,
which target the suppression of phosphodiesterase
type 5 (PDE-5), which in turn increases penile blood
flow and penile tumescence. Unfortunately, these
drugs have serious side effects such as sudden hypo-
tension, hypersensitivity reaction, myalgia, abnormal
vision, and infertility (Fauci et al., 2005). Since the
current therapeutic drugs do not target all phases of
the sexual responsive cycle and produce serious side
effects, a novel strategy is required, which is cheap,
easy to apply, and less toxic.
Various herbs known to be aphrodisiacs have
long been used for enhancing sexual desire and sexual
performance in traditional folklore. They can exert
their actions at various targets such as the neuro-
endocrine system, which plays a crucial role in sexual
motivation and function (Hull et al., 1997; Meston
and Frohlich, 2000), and PDE-5, an important en-
zyme in the signal pathway which regulates penile
erection via the regulation of cavernous smooth
muscle tone, which in turn controls penile blood flow
(Andersson, 2001). Several lines of evidence show
that testosterone and dopamine can control male
sexual function both at the nervous system and at the
penis (Hull et al., 1997). In addition, a recent study
has shown that oxidative stress also plays a role in the
impairment of carvernosal function and the patho-
physiology of erectile dysfunction (Minhas et al.,
2002; Hamed et al., 2003; de Young et al., 2004), and
that antioxidants can improve erectile function
(Zhang et al., 2011).
Moringa oleifera Lam. syn. M. ptreygosperma,
or the drumstick tree, a widely consumed vegetable in
Thailand, belongs to the Moringaceae family. It has
long been used in nutritional, industrial, and medical
fields. M. oleifera leaves are used for treating various
ailments including constipation, headache, fever, and
diabetes (Makonnen et al., 1997). In addition, it has
been demonstrated that M. oleifera leaf extracts in-
hibit 6-β-hydroxylation of testosterone (Monera et al.,
2008). Therefore, the sexual enhancing potential of
this plant has gained attention. On the basis of the
effects of dopamine and oxidative stress on male
sexual function mentioned earlier, we hypothesized
that M. oleifera leaves, which have antioxidant
(Sreelatha and Padma, 2009) and monoamine modu-
lation effects (Ganguly and Guha, 2008), might have
beneficial effects on male sexual dysfunction induced
by stress. Due to the limited supporting scientific data,
we aimed to evaluate the sexual enhancing potential
of M. oleifera by determining the effect of a hy-
droethanolic extract from M. oleifera leaves on var-
ious aspects of male sexual behavior in stressed rats,
and to determine the effect of the extract on various
factors involved in male sexual function, including
the antioxidant effect, monoamine and PDE-5 sup-
pression activities, serum testosterone and corti-
costerone levels, and histomorphological changes in
the testes.


2 Materials and methods
2.1 Chemicals and reagents
Folin-Ciocalteu reagent, sodium carbonate,
sodium acetate, FeCl3·6H2O, aluminum chloride,
potassium acetate, bovine serum albumin, tannic acid,
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tris
(2-pyridyl)-s-triazine (TPTZ) (Fluka), clorgyline
(selective MAO-A-I), tyramine, vanillic acid, and
4-aminoantipyrine were obtained from the Sigma-
Aldrich Corporation, Ltd., Thailand. Gallic acid,
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181
ascorbic acid, and quercetin used were of analytical
grade. A PDE-Glo™ phosphodiesterase Assay Kit
was purchased from the Promega Corporation, Thai-
land. Sildenafil citrate used in this study was from the
Pfizer Company. All other substances were of ana-
lytical grade.
2.2 Plant collection and extraction
Fresh leaves of M. oleifera were harvested
during November and December, 2011 from the
Khon Kaen Province in Thailand. The plant specimen
was authenticated by Assoc. Prof. Dr. Panee
SIRISA-ARD, Faculty of Pharmaceutical Sciences,
Chiangmai University, Thailand.
The leaves were homogenized as powder after
being cleaned and dried at 60 °C. The powder was
extracted in 50% hydro-ethanolic solution using a
maceration technique for 72 h. The non-soluble part
was separated using Whatman No. 1 filter paper. The
filtrated extract was evaporated using a rotary evap-
orator. The yielded extract of 17.49% dry plant tissue
was kept at −20 °C until used. A voucher specimen
was deposited at the Integrative Complementary Al-
ternative Medicine Research and Development Cen-
ter, Khon Kaen University, Thailand.
2.3 Evaluation of total phenolic compounds and
flavonoid content
The total content of phenolic compounds was
determined using the Folin-Ciocalteau method
(Basma et al., 2011). In brief, the mixture, which
contained 500 µl of Folin-Ciocalteau reagent (10%),
0.8 ml of sodium carbonate (7.5%, 0.075 g/ml) and
0.5 ml distilled water (DW), was mixed with 0.2 ml of
1 mg/ml M. oleifera leaf extract and left at room
temperature in the dark for 45 min. The optical den-
sity was read with an ultraviolet-visible spectropho-
tometer at 765 nm. DW was used as a blank. The
accuracy of data analysis was enhanced by using a
triplicate-sample method. The total phenolic content
(TPC) is expressed as mg of gallic acid equivalent
(GAE)/g of dry weight, using the standard calibration
line of gallic acid.
The total flavonoid content (TFC) was measured
by aluminum chloride colorimetry (Pourmorad et al.,
2006). A mixture containing 50% alcohol, 0.1 ml of
10% aluminum chloride, 0.1 ml of 1 mol/L potassium
acetate, and 2.8 ml of DW was mixed with 0.5 ml of
the plant extract (2 mg/ml) and left at room temper-
ature for 40 min in the dark. The absorbance at
415 nm was read using an ultraviolet-visible spec-
trophotometer. DW containing all the chemicals
mentioned earlier except the plant extract, was used
as the blank and each sample was prepared in tripli-
cate. The mean value of the triplicate was used for
analysis. The TFC is presented as mg of quercetin
equivalent (QE)/g of dry weight, using the standard
calibration line of quercetin.
2.4 Antioxidant assays
Both DPPH and ferric reducing antioxidant
power (FRAP) assays were carried out to determine
the antioxidant activity of the extract. Stable free
radical scavenging capacity was measured via the
DPPH method (de Ancos et al., 2002) with a slight
modification. In brief, 2.96 ml of a 0.1 mmol/L solu-
tion of DPPH in methanol was incubated with 40 μl of
various concentrations of extract (1.0, 2.0, 5.0, 10.0,
20.0, and 25.0 mg/ml) at room temperature for 30 min.
The decrease in DPPH radicals was evaluated by
measurement of optical density at 515 nm. The stable
free radical scavenging capacity is presented as the
percentage of inhibition of DPPH radicals (IDPPH),
calculated according to the following equation:
IDPPH=(Ac−As)/Ac×100%, where Ac and As are ab-
sorbances of control and sample, respectively.
To determine the FRAP, the reaction mixture
containing FRAP solution (300 mmol/L sodium ace-
tate buffer (pH 3.6), 10 mmol/L TPTZ solution in
40 mmol/L HCl, and 20 mmol/L FeCl3·6H2O solution
at a ratio of 10:1:1 (v/v)) (Thaipong et al., 2006) and
0.15 ml of the dilution of the sample (0.5 mg/ml), was
incubated at 37 °C in the dark for 30 min. The optical
density of the colored product (ferrous tripyridyltria-
zine complex) was measured at 593 nm. Data are
presented as µmol ascorbic acid equivalent (AAE)/g
extract.
2.5 Amino acid determination
Amino acid evaluation was performed according
to the method of Nwidu et al. (2012). To hydrolyze
peptide bonds, 1 ml of 6 mol/L HCl and 0.08 ml
of 5% (v/v) phenol/water solution were mixed with
5 mg of extract and heated in a Pyrex tube with plastic
Teflon-coated screw caps (13 cm×1 cm) at 110 °C for
72 h. The solution was reheated in an oven at 70 °C,
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182
diluted with 1.0 ml of sodium citrate buffer (pH 2.2)
and filtered through a GV Millex Unity filter (Milli-
pore). The amino acid profile analysis was carried out
by cation-exchange chromatography using an auto-
mated amino acid analyzer, Shimadzu LC-10A/C47A.
The sample was eluted by sodium, and post-column
derivatization was performed using o-phthaldialdehyde
(OPA). The retention time and area obtained by each
amino acid were compared with those of standards to
identify and quantify the amino acid profile.
2.6 Determination of monoamine oxidase type B
(MAO-B) inhibition
The MAO-B suppression effect was determined
by spectrophotometry (Schurr and Livne, 1976). In
brief, the rats were sacrificed by cervical dislocation
and the cerebral cortices of healthy rats were removed
and prepared as an homogenate with RIPA buffer
(50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl,
2 mmol/L ethylene diamine tetraacetic acid (EDTA),
0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate (SDS), and 1% Triton X-100) and centrifuged
at 14 000g at 4 °C for 20 min. After the centrifugation,
the supernatant was harvested and served as the source
of MAO-B. To initiate the enzyme reaction, 50 μl of
brain homogenate, preincubated with 50 µmol/L
clorgyline (a selective inhibitor of MAO-A) at a ratio
of 100:1 (v/v), was added to the solution containing
2.75 ml Tris buffer (0.1 mol/L, pH 7.4) and 100 μl of
0.1 mol/L benzylamine (Holt et al., 1997; Dhingra
and Goyal, 2008). The absorbance changes in the
tested samples in the double beam spectrophotometer
at a wavelength of 249.5 nm within 5 min were rec-
orded against blanks containing Tris buffer and
5-hydroxytryptamine (Xu et al., 2005). The MAO-B
suppression effect of Selegiline, a standard MAO-B
inhibitor, was also studied using the same procedure,
except that Selegiline was assayed instead of the plant
extract.
2.7 Stress exposure
Since immobilization or restraint stress has been
recognized as a valid test for studying stress-induced
physical and psychological alterations and stress-
related disorders (Al-Mohaisen et al., 2000; Xu et al.,
2006; Zaidi et al., 2014), it was used as the experi-
mental model in this study. Immobilization stress was
induced using a modified method of Retana-Márquez
et al. (2003) and Ahmad et al. (2012). In brief, im-
mobilization stress was applied between 6:00 a.m.
and 6:00 p.m. The rats were put into a transparent
perforated plastic tube (20 cm long and 7 cm in di-
ameter). Since the animal fitted tightly in the tube, it
could not move or turn around.
2.8 Male sexual behavior evaluation
Fertile male Wistar rats were used as experi-
mental animals. They were randomly divided into
seven groups each with six animals. (1) Group I:
naive control (non-stress group); rats received no
treatment. (2) Group II: vehicle plus stress; rats were
given the vehicle (DW) orally, 45 min before being
subjected to 12 h-restraint stress exposure for 7 d.
(3) Group III: sildenafil; this group served as a posi-
tive control based on the sexual enhancing effect of
sildenafil citrate. All rats in this group were admin-
istered sildenafil citrate at a dose of 5 mg/kg 45 min
before being subjected to 12 h-restraint stress expo-
sure for 7 d. Since this drug has a maximum mode of
action of 4 h after administration, an oral suspension
of this drug at a dose of 5 mg/kg was given to all rats
1 h before the mating behavior assessment. (4) Group
IV: tianeptine; this group also served as a positive
control based on the dopamine enhancing effect of
this drug (Invernizzi et al., 1992) and the positive
modulation effect of dopamine on sexual dysfunction
(El-Shafey et al., 2006). All rats in this group were
given tianeptine orally at a dose of 15 mg/kg 45 min
before being subjected to 12 h-restraint stress expo-
sure for 7 d. (5) Groups V–VII: M. oleifera leaf ex-
tract treated groups; rats in these groups were given
orally various doses of M. oleifera leaf extract, in-
cluding 10, 50, and 250 mg/kg 45 min before being
subjected to 12 h-restraint stress exposure for 7 d.
The treatments and the stress-exposure were
carried out once daily. After being exposed to stress,
the animals were given a 3 h-refreshment period be-
fore their sexual behavior evaluation (Prabsattroo et
al., 2012). The sexual behavior assessments were
performed by an experienced observer blind to the
treatments, at room temperature between 9:00 p.m.
and 12:00 midnight after a single intervention and
after one week of interventions.
Male rats subjected to single or repeated doses of
the plant extract were paired with estrous female rats
induced by estradiol benzoate (Sigma, St. Louis, MO,
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183
USA) at a dose of 2 μg/kg body weight (BW) and
progesterone (Sigma, St. Louis, MO) at a dose of
500 μg/kg BW subcutaneously at 48 and 6 h, respec-
tively, before the determination of copulatory be-
havior. The sexual behavior of the rats in a clear
plastic box was monitored for 3 h using video re-
cording (Gauthaman et al., 2002). The assessed
sexual parameters were latencies and frequencies of
mounting, intromission, and ejaculation.
2.9 Determination of PDE-5
The level of PDE-5 was determined using a
PDE-Glo™ Phosphodiesterase Assay Kit (Promega
Corp., Thailand). The penis was washed with phos-
phate buffer solution (PBS), cut into small pieces, and
prepared as homogenate using lysate RIPA buffer.
The sample was centrifuged at 14 000g at 4 °C for
15 min. The supernatant was separated and served for
the determination of PDE-5 activity. The PDE-Glo™
phosphodiesterase assay was carried out in a 96-well
plate. The assay was performed according to the
guidelines of the kit. In brief, the penis was incubated
with cyclic guanosine monophosphate (cGMP) sub-
strate in reaction buffer until the phosphodiesterse
reaction was complete. PDE-Glo™ termination
buffer was incubated with PDE detection solution
containing adenosine triphosphate (ATP) and protein
kinase A (PKA). The amount of ATP consumed by
this reaction, which is directly correlated with the
cGMP level, was evaluated using the luciferase-based
Kinase-Glo reagent. After a 10-min incubation pe-
riod at room temperature, the optical density of the
sample was determined using a SpectraMax® L mi-
croplate luminometer (MDS AT (US) Inc.) and ex-
pressed as relative light units (RLUs) and as a per-
centage of the control.
2.10 Determination of testosterone and corti-
costerone levels
At the end of study, the venous blood of each
animal was prepared as serum by centrifugation at
2000g at 4 °C for 15 min. The serum was stored at
−80 °C until used. Testosterone levels were measured
using a radioimmunoassay (RIA) Kit (TESTO-CT2,
Cisbio International, France) and corticosterone lev-
els were measured using a Corticosterone Double
Antibody Radioimmunoassay Kit (MP Biomedicals)
for the quantitative determination of corticosterone in
rat and mice serum. The results are expressed as ng/ml.
Both assays were performed at the Radiology De-
partment, Srinakarindhra Hospital, Faculty of Medi-
cine, Khon Kaen University, Thailand.
2.11 Histological study
The testes were dissected out, freed from sur-
rounding tissues and weighed quickly on a sensitive
balance. They were fixed, embedded in paraffin, cut
into 10 µm thick sections, and stained using hema-
toxylin and eosin (H&E). Histomorphological analy-
sis was performed using a light microscope.
2.12 Statistical analysis
The experimental data are presented as mean±
standard error of the mean (SEM). The statistical
significance was evaluated using analysis of variance
(ANOVA) followed by Duncan’s test. A P-value of
<0.05 was considered to be statistically significant.


3 Results
3.1 Phenolic compound and flavonoid contents
The total phenolic compounds in a 50% (v/v)
hydro-ethanolic extract of M. oleifera leaves were
determined by the Folin-Ciocalteau method and ex-
pressed as GAE. The concentration of phenolics
found in the M. oleifera leaf extract used in this study
was (62.333±0.008) mg GAE/g extract. The flavo-
noid concentration of the extract was also determined
using the aluminum chloride colorimetric method and
was found to be (29.900±0.001) mg QE/g extract.
3.2 DPPH radical scavenging activity and FRAP
In this study, the 50% hydro-ethanolic extract of
M. oleifera leaves was tested for its antioxidant
scavenging effects on DPPH radicals. The results
obtained at different concentrations of extract are
given in Fig. 1. The results show that the extract
showed dose-dependent activity. The concentration
required for a 50% (v/v) DPPH reduction (inhibitory
concentration 50%; IC50) by the extract was deter-
mined using ascorbic acid as reference. The IC50
of ascorbic acid was (0.124±0.009) mg/ml, whereas
that of the M. oleifera leaf extract was (8.270±
0.023) mg/ml.
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184













Reducing power has been reported to be a vali-
dated indicator reflecting antioxidant activity (Oktay
et al., 2003). The reducing power of M. oleifera leaf
extract was assessed via an FRAP assay. The reduc-
ing power of the extract at various concentrations is
shown in Fig. 2. The IC50 of the extract evaluated
using the FRAP assay was (399.00±0.010) μmol/L
AAE/mg extract.
















3.3 Amino acid composition of M. oleifera leaf
extract
The amino acid composition of the leaf extract
of M. oleifera is shown in Table 1. Phenylalanine
showed the highest concentration (2981.00 mg/100 g).
Serine, glutamic acid, leucine, lysine, isoleucine,
tyrosine, histidine, valine, alanine, aspartic acid, pro-
line, tryptophan, glycine, cysteine, methionine, thre-
onine, arginine, hydroxylysine, and hydroxyproline
were also observed.














3.4 MAO-B suppression effect of M. oleifera
The MAO-B suppression effect of the extract
was determined and used as an indicator to reflect the
available dopamine, an important neurotransmitter
which plays a pivotal role in male sexual function.
The IC50 of MAO-B was (0.255±0.008) mg/ml
while that of selegiline, a standard MAO-B inhibitor,
used for enhancing the dopamine level, was (0.005±
0.001) mg/ml.
3.5 Male sexual enhancing effect of M. oleifera
leaf extract
The effects of M. oleifera leaf extract on sexual
behavior are presented in Table 2. The current data
confirm the reputation of M. oleifera as a sexual
stimulating agent. Rats that received either tianeptine
(Group IV) or sildenafil citrate (Group III) and were
subjected to the 12-h immobilization stress showed
significantly decreased mounting latency (P<0.05,
compared to vehicle control plus stress treated group
(Group II) after a single intervention. However, only
those rats in Group III showed a decreased intromis-
sion latency after the 7-d intervention period (P<0.05,
compared to Group II). Unfortunately, no changes in
latencies or the numbers of mounting, intromission, or
ejaculation events were observed in rats of Group IV.
After a single administration, rats treated with the
extract at a dose of 50 mg/kg and exposed to immo-
bilization stress (Group VI) showed a significantly
decreased mounting latency (P<0.05, compared to
Group II). Only rats subjected to the extract at a dose
of 10 mg/kg and exposed to immobilization stress
(Group V) significantly increased mounting numbers
Fig. 1 DPPH radical scavenging activities of M. oleifera
leaf extract and ascorbic acid (standard control)
Table 1 Amino acid profile of M. oleifera leaf extract
detected by gas chromatography (ion exchange chro-
matographic method)
Essential Concentration (mg/100 g) Non-essential
Concentration
(mg/100 g)
Alanine 416.67 Arginine <5.00
Histidine 475.27 Aspartic acid 368.14
Hydroxylysine <5.00 Cysteine 133.39
Isoleucine 631.02 Glutamic acid 1422.00
Leucine 910.44 Glycine 131.33
Lysine 652.61 Hydroxyproline <5.00
Methionine 91.32 Proline 283.88
Phenylalanine 2981.00 Serine 1554.00
Threonine 79.72 Tyrosine 546.81
Tryptophan 263.80 Valine 422.97

0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 100 200 300 400 500 600
FR
A
P
a
ct
iv
ity

Ascorbic acid concentration (μmol/L)
Calibration curve of ascorbic acid
Fig. 2 Ferric reducing antioxidant power (FRAP) of
M. oleifera leaf extract using ascorbic acid data to pro-
vide a calibration curve
FRAP activity of M. oleifera is 399 μmol/L AAE/mg extract
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185
(P<0.05, compared to Group II) after a single admin-
istration. Interestingly, rats subjected to the extract at
a dose of 10 mg/kg (Group V) and exposed to im-
mobilization stress showed a significant decrease in
intromission latency, but an increase in the number of
intromissions (P<0.05, compared to Group II) after
the 7-d intervention period. In addition, rats subjected
to a high dose of extract plus stress (Group VII) also
showed increased mounting numbers after the 7-d
intervention period (P<0.05, compared to Group II).
No significant changes in latency or the number of
ejaculations were observed throughout the 7-d study
period.
3.6 PDE-5 activity of M. oleifera leaf extract
PDE-5 activity in the penis of rats given M.
oleifera leaf extract was determined (Fig. 3). The ex-
tract at a dose of 10 mg/kg significantly attenuated the
enhanced PDE-5 activity in the penis of stress-exposed
rats (P<0.05; compared to the control (Group I)). Both




























sildenafil citrate and tianeptine produced significant
reductions in PDE-5 activity in the penis (P<0.01 and
P<0.05, respectively; compared to Group II). Rats in
Group V showed a significant decrease in PDE-5
activity in the penis (P<0.05; compared to Group II).











































Table 2 Effect of hydro-alcoholic extracts of M. oleifera leaves on male sexual behavior of stress-exposed rats at
baseline, after a single dose, and after 7 d of treatment
Group ML (s) MN IL (s) IN EL (s) EF
Baseline
I 15.6±10.7 29.2±5.2 102.5±64.4 23.2±2.9 582.3±114.8 2.3±0.5
II 13.0±3.3 31.0±2.0 292.6±257.0 17.2±4.2 1026.6±318.7 1.6±0.7
III 33.0±12.2 44.3±6.2 155.8±86.2 18.3±3.1 946.5±198.7 1.5±0.3
IV 11.5±4.8 38.2±6.7 45.0±8.3 23.2±1.5 750.8±218.1 1.8±0.4
V 20.5±10.9 34.0±5.1 149.6±65.6 14.5±2.3 707.0±78.3 1.4±0.4
VI 19.7±7.5 32.2±5.2 173.3±86.1 22.4±3.2 1015.1±268.3 2.0±0.7
VII 29.8±13.8 34.7±3.3 68.7±26.5 21.7±3.7 1143.8±163.4 1.3±0.4
Single dose
I 15.7±8.0 39.0±6.3 38.7±8.3 27.6±5.0 613.0±129.9 2.0±0.4
II 29.0±11.2 32.2±3.7 111.4±70.2 26.2±5.2 326.6±196.5 2.2±0.2
III 3.2±0.9* 37.5±5.1 75.3±24.2 20.7±2.9 568.3±139.8 2.7±0.5
IV 6.0±2.3* 28.8±6.4 38.3±8.7 23.7±3.1 502.3±87.1 2.0±0.3
V 28.6±7.3 49.8±6.4* 78.0±21.8 21.2±2.8 447.4±62.9 2.5±0.3
VI 6.4±2.0* 44.8±6.2 58.6±20.1 22.0±4.5 883.2±137.3 1.3±0.3
VII 34.6±11.3 41.0±7.1 46.3±13.8 17.6±4.3 895.2±254.4 1.4±0.4
7-d
I 2.0±0.8 31.0±1.5 15.0±3.8* 29.8±1.9* 421.5±112.5 2.5±0.2
II 15.8±7.7 28.2±4.0 81.8±49.7# 20.3±3.9# 482.5±225.1 2.4±0.4
III 3.3±1.3 33.8±4.9 13.8±3.2* 29.2±1.4* 319.7±137.6 3.0±0.4
IV 18.2±7.3 33.7±7.9 47.3±9.5 26.2±1.2 639.0±55.4 2.3±0.2
V 14.2±7.4 29.7±2.4 12.6±4.4* 32.7±3.0** 397.8±44.5 2.2±0.2
VI 15.2±6.9 44.4±6.6 27.8±10.8 23.6±3.0 672.3±243.9 2.0±0.6
VII 18.2±7.2 46.2±9.2* 39.8±14.2 24.6±4.3 711.8±111.8 1.8±0.2
ML: mount latency; MN: mount number; IL: intromission latency; IN: intromission number; EL: ejaculation latency; EF: ejaculation
frequency. Data are expressed as mean±SEM (n=6). * P<0.05, ** P<0.01, compared with Group II. # P<0.05, compared with Group I

Fig. 3 Effect of hydro-ethanolic extracts of M. oleifera
leaves on phosphodiesterase type 5 (PDE-5) activity in the
penis of stress-exposed rats after 7 d of treatment
Data are expressed as mean±SEM (n=6). * P<0.05, ** P<0.01,
compared with Group II. # P<0.05, compared with Group I
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186
3.7 Effect of M. oleifera leaf extract on testos-
terone and corticosterone levels
Fig. 4 shows that the male rats receiving
sildenafil citrate and M. oleifera leaf extract at doses
of 10 and 250 mg/kg and exposed to immobilization
stress for 7 d showed elevated levels of serum tes-
tosterone (P<0.001, P<0.05, and P<0.01, respectively;
compared to Group II). We also evaluated the effect
of the extract on the serum corticosterone level
(Fig. 5). Rats subjected to the extract treatment at
doses of 10, 50, and 250 mg/kg and exposed to im-
mobilization stress for 7 d showed elevated serum
corticosterone levels (P<0.01, P<0.001, and P<0.05,
respectively; compared to Group II).

































3.8 Effect of M. oleifera leaf extract on testis
histology
The histological morphology of the testis was
evaluated and the results are shown in Fig. 6. In the
vehicle plus stress treated group (Group II), the
seminiferous epithelium was disorganized and fewer
interstitial cells of Leydig were observed. Rats sub-
jected to stress and sildenafil, or stress plus tianeptine,
or stress plus M. oleifera extract at doses used in this
study showed a more organized seminiferous epithe-
lium than those in Group II. In addition, rats in all
these groups appeared to have more interstitial cells
of Leydig and more spermatozoa in the lumen of the
seminiferous tubules than those in Group II.































0
2
4
6
8
10
12
14
16
18
20
S
er
um
le
ve
l o
f t
es
to
st
er
on
e

(n
g/
m
l)
Group
***
* **
###
***
###
###
###
###
###
I II III IV V VI VII
Fig. 4 Effect of hydro-ethanolic extracts of M. oleifera
leaves on serum testosterone levels of stress-exposed rats
after 7 d of treatment
Data are expressed as mean±SEM (n=6). * P<0.05, ** P<0.01,
*** P<0.001, compared with Group II. ### P<0.001, compared
with Group I
Fig. 6 Haematoxylin and eosin (H&E) stained frozen sections of rat testis showing the histomorphology of seminiferous
tubules, Sertoli cells (SC), spermatogonia (SG), primary spermatocytes (PS), spermatids (SP), sperm, and Leydig cells
(LC) in the following treatment groups: (a) naive control; (b) vehicle plus stress; (c) sildenafil citrate plus stress;
(d) Tianeptine plus stress; (e)–(g) M. oleifera at doses of 10, 50, and 250 mg/kg plus stress, respectively

Fig. 5 Effect of hydro-ethanolic extracts of M. oleifera
leaves on serum corticosterone levels of stress-exposed
rats after 7 d of treatment
Data are expressed as mean±SEM (n=6). * P<0.05, ** P<0.01,
*** P<0.001, compared with Group II. # P<0.05, ## P<0.01,
compared with Group I
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187
4 Discussion

The current study has revealed that M. oleifera
leaf extract has antioxidant and MAO-B suppression
effects. Phenylalanine was the main amino acid found
in the extract. In vivo data showed that a single con-
sumption of medium or low doses of extract increased
libido. When the treatment was prolonged to 7 d, the
low dose of extract enhanced penile erection capacity
by decreasing intromission latency and increasing
intromission number, decreased both PDE-5 activity
and corticosterone levels, increased the numbers of
interstitial cells of Leydig and spermatozoa, and in-
creased testosterone levels. The high dose extract also
increased the numbers of mountings, interstitial cells
of Leydig and spermatozoa, increased testosterone
levels, and reduced corticosterone levels.
Male sexual behavior is governed by a complex
interaction between different systems in the brain that
process sensory inputs, regulate reward and motiva-
tion, and integrate hormonal signals (Hull et al.,
2004). The regulation of sexual behavior can occur
both at the brain and at peripheral sites. It has been
reported that testosterone plays an important role
in priming brain neural circuits for sexual behavior.
The priming effect is achieved partly by the modifi-
cation of regulating enzymes, receptors, or other
proteins which affect neurotransmitter function.
Testosterone can increase local dopamine synthesis
and metabolism of dopamine (Purves-Tyson et al.,
2012), which in turn exerts an influence on various
aspects of male sexual function including sexual mo-
tivation, copulatory proficiency, and genital reflexes
(Hull et al., 2004).
Accumulated evidence has shown that dopamine
plays a crucial role in male sexual behavior via the
enhanced release of oxytocin from the paraventricular
nucleus (PVN) of the hypothalamus, which in turn
increases the release of nitric oxide from the carver-
nosal nerve. Nitric oxide penetrates the cytoplasm
of smooth muscle cells and interacts with guanylyl
cyclase, catalyzing conformational changes which
in turn induce the conversion of guanosine
5-triphosphate (GTP) to 3-5-cyclic guanosine
monophosphate. cGMP in turn phosphorylates sev-
eral proteins, resulting in decreased intracellular cal-
cium levels causing the relaxation of arterial and
trabecular smooth muscle, resulting in arterial dilata-
tion, venous constriction, and the rigidity of penile
erection. The inactivation of cGMP via PDE-5 de-
creases the dilation of arterial vessels and the con-
striction of venous blood vessels and increases penile
tumescence (Andersson, 2001).
In this study, stress exposure induced a reduction
in the number of interstitial cells of Leydig and in
serum testosterone levels. These changes may possi-
bly have been due to enhanced oxidative stress (Rai et
al., 2004; Bitgul et al., 2013). M. oleifera extract
attenuated the reduction in the interstitial cells of
Leydig and serum testosterone levels induced by
stress exposure. Based on the results of this study, we
suggest that the antioxidant effect of the extract alle-
viates oxidative stress-related cell toxicity, resulting
in increased numbers of interstitial cells of Leydig
and serum testosterone levels. It has been reported
that the elevation of glucocorticoid levels induced by
stress can also inhibit the synthesis of testosterone in
interstitial cells of Leydig (Gao et al., 1996).
Therefore, the mitigating effect of M. oleifera extract
on the reduction in testosterone induced by stress may
occur partly via decreased corticosterone levels.
It has been shown that M. oleifera extract also
suppresses MAO-B. Due to the crucial role of dopa-
mine in male sexual behavior mentioned earlier, it is
possible that suppression of MAO-B activity may
enhance the available dopamine, which in turn in-
creases oxytocin release from the PVN and induces
the release of nitric oxide from the carvernosal nerve,
triggering the elevation of cGMP in the penis. The
elevation of cGMP induced by M. oleifera extract and
the suppression effect of the extract on PDE-5 may
enhance the cGMP-dependent vasodilation effect of
nitric oxide, leading to enhanced penile blood flow
and erection. Since the high dose of M. oleifera ex-
tract failed to show a PDE-5 suppression effect, no
significant change in the intromission phase was ob-
served in stress-exposed rats that received a high dose
of extract. In addition, it has been reported that the
elevation of testosterone can regulate PDE-5 function
(Aversa et al., 2009) and modify dopamine inactiva-
tion via MAO-B, leading to increased penile erection
(Sanna et al., 2011). Therefore, the enhanced
intromission phase observed in this study may have
been related to MAO-B suppression activity, the
ability to increase the testosterone level or the PDE-5
suppression effect.
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188
In this study, an increased number of spermatozoa
was also observed in stress-exposed rats subjected to
M. oleifera leaf extract treatment. It has been reported
that spermatozoa are vulnerable to oxidative stress
because of the high concentration of polyunsaturated
fatty acids involved in the regulation of sperm matu-
ration, spermatogenesis, capacitation, acrosome re-
action, and eventually membrane fusion, and their low
antioxidant capacity. An attack of oxidative stress may
lead to structural damage and decreased viability.
Therefore, the increased spermatozoa density induced
by the M. oleifera leaf extract may possibly be due to
the antioxidant effect of the extract.
Our data also suggest that the enhanced dopa-
minergic function induced by the M. oleifera leaf
extract may occur not only via the MAO-B suppres-
sion effect but also via the high concentration of
phenylalanine in the plant extract. Since phenylala-
nine serves as a precursor for tyrosine and dopamine
synthesis, the enhanced dopamine function observed
in this study may also occur partly via the increased
dopamine precursor supplement. Moreover, the M.
oleifera leaf extract used in this study also contained
abundant phenolics and flavonoids. Therefore, the
antioxidant effect of the extract may have been due to
these ingredients.
This study is the first to demonstrate the possible
benefit of M. oleifera active amino acids on dopa-
minergic and sexual function besides the possible role
of phenolics and flavonoids in the extract, and the
biochemical and histological changes related to stress
and sexual function. However, the determination of
the effect of the plant extract on serum testosterone
and PDE-5 activity in the penis at only a single time
point and the lack of nitric oxide and penile vascular
blood flow measurement, were limitations of this
study. The effect of the plant extract on serum tes-
tosterone before and during sexual performance
should be determined to confirm the modulating role
of the extract on the sexual enhancement effect of
testosterone. The effect of the extract on PDE-5 ac-
tivity in testis both before and during the sexual in-
tromission phase should also be determined together
with the levels of nitric oxide, cGMP, and penile
blood flow, to confirm that the suppression of PDE-5
induced by the plant extract can enhance the
cGMP-dependent vasodilation effects of nitric oxide,
penile blood flow, and penile erection.
Therefore, further investigations of the effect of
M. oleifera leaf extract on the alterations of testos-
terone and dopamine levels before and during sexual
performance, together with evaluation of the cGMP-
dependent vasodilation effects of nitric oxide, penile
blood flow and penile erection, are required to
improve understanding of the precise underlying
mechanisms of the sexual enhancing effect of M.
oleifera leaf extracts.


5 Conclusions

The use of Moringa oleifera leaf extract may
provide a cheap and simple approach for improving
sexual function under stressful conditions. Therefore,
the extract appears to be a potential aphrodisiac, par-
ticularly for people with stressful daily lifestyles.
However, more research is needed concerning the
possible underlying mechanisms of action of M.
oleifera leaf extracts, such as the determination of
testosterone levels before and during sexual perfor-
mance, and the evaluation of the cGMP-dependent
vasodilation effects of nitric oxide, penile blood flow,
and penile erection.

Acknowledgements
We would like to express our sincere thanks to Assoc.
Prof. Panee SIRISA-ARD (Faculty of Pharmacy, Chiangmai
University, Thailand) for her authentication.

Compliance with ethics guidelines
Thawatchai PRABSATTROO, Jintanaporn WATTAN-
ATHORN, Sitthichai IAMSAARD, Pichet SOMSAPT, Opass
SRITRAGOOL, Wipawee THUKHUMMEE, and Supaporn
MUCHIMAPURA declare that they have no conflict of interest.
All institutional and national guidelines for the care and
use of laboratory animals were followed.

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中文概要

题 目：辣木提取物对应激大鼠性能力具有增强作用
目 的：探讨辣木叶提取物对改善应激条件下雄性性功能
障碍的作用。
创新点：根据氧化应激和多巴胺对雄性性功能的影响，推
测具有抗氧化和单胺调节功能的辣木叶能改善应
激条件下的雄性性功能障碍。
方 法：本文以雄性 Wistar 大鼠作为实验对象，通过对影
响雄性性功能的各种因素进行测量及统计学分析，
包括抗氧化性、B 型单胺氧化酶（MAO-B）和
5 型磷酸二酯酶（PDE-5）活性、血清睾酮和皮质
甾酮水平、睾丸内组织形态学变化。
结 论：研究结果表明，辣木叶提取物作为一种抗氧化剂
具有抑制 MAO-B 和 PDE-5 活性的作用，同时能
降低血清皮质酮水平，提高血清睾酮水平，增加
睾丸间质细胞和精子的数量。睾丸间质细胞和精
子的数量的增加可能是由于辣木叶提取物的抗氧
化作用，插入阶段性功能增强可能是由于MAO-B
和 PDE-5 活性的抑制和睾酮水平的增加。因此，
辣木可以作为一种潜在的壮阳药。
关键词：辣木；性功能；应激