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- Complementary Medicine -
Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells
Paul F. Moundipa1, Nathalie Sara E. Beboyl, Fabien Zelefack2, Silvère Ngouela2, Etienne Tsamo2, Wolf-Bernhard Schill3, Thomas K. Monsees3,4
1Departments of Biochemistry and
2Organic Chemistry, University of Yaounde I, P. O. Box 812 Yaounde, Cameroon
3Centre for Dermatology and Andrology, Justus Liebig University, Gaffkystr.1435385 Giessen, Germany
4Department of Anatomy, University of Technology, Fetscherstr. 74, 01307 Dresden, Germany
Abstract
Aim: To determine the androgenic effects of
Basella alba and Hibiscus macranthus extracts in the rat and the bull,
and to develop a novel in vitro test system using Leydig cells from bull testes.
Methods: The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed
using 125I-radioimmunoassay
(125I-RIA). Rat Leydig cells were obtained by common methods, whereas a novel
technique was used to purify Leydig cells from bull testes.
Results: Bull testes from the slaughter house were a
cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated
by human chorionic gonadotrophin (hCG).
Basella alba extracts significantly enhanced testosterone production in
bull and rat Leydig cells in a concentration-dependent manner.
Hibiscus macranthus showed no androgenic effect but
was shown to inhibit testosterone production at higher concentrations.
Conclusion: Leydig cells purified from bull
testes can be used as an alternative tool in experimental animal research. Certain fractions of
Basella alba extract demonstrated androgenic potential whereas
Hibiscus macranthus extracts did not. (Asian J Androl 2005 Dec; 7:
411每417)
Keywords: bull; rats; Leydig cells; testosterone; Basella alba; Hibiscus macranthus; plant extract
Corresponence to: Prof. Paul F. Moundipa, Laboratory of
Biochemical Toxicology, Department of Biochemistry, University of
Yaounde I, P. O. Box 812 Yaounde, Cameroon.
Tel: +237-780-4010, Fax: +237-222-1320
E-mail: pmoundipa@uycdc.uninet.cm
Received 2004-10-19 Accepted 2005-05-16
DOI: 10.1111/j.1745-7262.2005.00056.x
1 Introduction
Hibiscus macranthus, Basella alba
and many other indigenous plants from Cameroon have been shown to
have an effect on male reproductive function [1每3].
Indeed, the aqueous extract obtained from the mixture
of the leaves of both plants is used in the Western
Province of Cameroon to cure male sexual asthenia and
infertility. Previous works on the aqueous extract
demonstrated their androgenic effect [1, 4]. To better
understand the exact potential of these plants in reproductive
health, we analyzed their mechanism of action in more
detail using extracts of different organic solvents.
However, previous works using rat testis slices failed to
reveal the active principles or fractions (Beboy,
unpublished data, 2001). Thus it is necessary to obtain a new
in vitro test system to analyze the androgenic effect of
the active plant fractions. For this purpose, a novel method
for the purification of Leydig cells from bull testes was
developed. Bull testes are a by-product of the local
slaughter house and are used in this paper as an alternative
material in laboratory research. These Leydig cells as
well as those obtained from rats by common purification
methods were used to test the effect of the methanol
extracts from both plants and the higher purified
fractions from Basella alba on their testosterone production.
2 Materials and methods
2.1 Chemicals
Dubecco*s Modified Eagle*s medium-F12 Ham nutrient mixture (DMEM/Ham F12) (Gibco, Berlin,
Germany), Collagenase Type I, Soybean Trypsin
inhibitor (STI), DNAse, insulin, transferrin, vitamin E,
penicillin and streptomycin were all obtained from Sigma
(Deisenhofen, Germany).
2.2 Preparation of the extracts and fractions
Air-dried leaves of both Hibiscus macranthus
(0.5 kg) and Basella alba (0.5 kg) , obtained from Batoufam
(Western Province, Cameroon) were powdered in a mill.
Each powder was then subjected to an organic solvent
extraction by maceration under gentle agitation in a glass
vessel for 48 h at room temperature using successively
hexane (500 mL for 5 h, three times), methylene
chloride (500 mL for 5 h, three times) and methanol (500 mL
for 6 h, three times). The methanol extracts of both
Hibiscus macranthus and Basella
alba were concentrated under vacuum for dryness. The greenish viscous residues (12 g for Hibiscus macranthus and 15 g for
Basella alba) were obtained and tested. The extract from
Basella alba was further subjected to flash chromatography
(60 mm × 55 mm column size) on silica gel (230每400
mesh) as a stationary phase. The extract was added in
the form of dry powder to the column. Elution was done
using a CH2Cl2/MeOH system with an increasing polarity
(100 % to 50 % CH2Cl2) as the mobile phase. Eighty-one
fractions of 100 mL each were collected and evaporated
under vacuum at 50 ºC and pooled according to their
retention factor (Rf) on thin layer chromatography (TLC)
using a CH2Cl2/MeOH system as eluent. Twelve main
fractions labeled A to L were obtained. The above
fractions were eluted with the following
CH2Cl2/MeOH system polarities: A (100/0), B and C (95/5), D and E
(90/10), F and G (85/15), H (75/25), I, J and K (50/50) and
L (0/100). All these fractions were further analyzed for
the presence of certain classes of phytochemical
compounds using usual organic phytochemical screening
methods [5] (see Table 1 for details). The hexane and
methylene chloride extracts were not used for the lack
of androgenic activities (Beboy, unpublished data, 2001).
2.3 Purification and culture of Leydig cells from bull
and rat
Adult testes were collected in the local slaughter
house (Giessen, Germany) and were used within 1 h
after isolation. Testes were washed with tap water,
blotted and cleaned with ethanol (70 %). Connective tissues
was removed by dissection and the testis was aseptically
introduced into a sterile hood. The tunicia albuginea was
then removed by dissection using scalpels and scissors,
then a piece of the testis was cut and weighed
(7 g每10 g), washed twice with DMEM/Ham F12 medium
containing gentamicin and cut into thin slices (0.5
mm2). Then it was washed again with the DMEM/Ham F12
medium and quantitatively transferred into 20 mL
of dissociation medium (DMEM/Ham F12 containing
2 mg/mL collagenase type I, 0.2 mg/mL STI and 20 g/mL DNAse)
and incubated for 20 min at 34 ºC under constant shaking.
Thereafter another 20 mL of DMEM/Ham F12 medium was added and the digested mixture was allowed to settle.
The supernatant was carefully collected using a pipette
and put into a sterile tube. The remaining undigested
tissue was subjected to two repeating digestion steps as
described above. All the supernatants were pooled and
centrifuged at 180 × g for 10 min at room temperature.
The pellet, containing the interstitial cells, was washed
three times with DMEM/Ham F12 medium and filtered through a 70 m nylon mesh (Falcon, Becton Dickinson,
NJ, USA).
Bull Leydig cells were further purified using two
different discontinuous percoll gradients by a modification
of the method described by Brun et al. [6]. The first
gradient consisted of 5 %, 30 %, 58 % and 70 % percoll,
four phases. The filtered cell suspension was placed over
the gradient and centrifuged at 1 100 ×g for 20 min at
18 ºC. The second band (cells situated between the 30 %
and the 58 % percoll phases) was collected, washed with
DMEM/Ham F12medium, and layered on a second
gradient (30 % to 60 % percoll). The gradient was
centrifuged as above. Three bands found at 40 %, 45 %
and 50 % were collected, mixed and washed with
DMEM/Ham F12medium. The cells were counted using
a hemocytometer. They were characterized by 3b-hydroxy- steroid
dehydrogenase (3bHSD) histochemistry [7] and hCG
stimulated testosterone production (for 12 h). The purity of
cells was about 92 % and viability >95 % as detected by
trypan blue exclusion.
Leydig cells were also obtained from adult
Sprague每Dawley rats (90 days old) after three enzymatic
dissociations of the testis using collagenase (0.5 mg/mL),
Dnase (20 g/mL) and STI (0.05 mg/mL) in DMEM/Ham F12 at 34 ºC for 10 min. They were further
purified on a percoll gradient by the method described by
Papadopoulos et al.[8].
Leydig cells were used for experiments just after
purification and cultivated in DMEM/Ham F12 medium
containing 10 g/mL insulin, 5 g/mL transferrin and
10 g/mL vitamin E, 100 U/mL penicillin and 100 pg/mL
streptomycin in 96 well plates (50 ×
103 cells in 0.2 mL).
2.4 Effects of extracts and fractions on Leydig cells
Methanol extract was dissolved in
Dimethylsulfoxide (DMSO) and added to cells in culture
(0.01 g每100.00 g extract/mL final concentration in culture medium). The
control well contained DMSO to a level not exceeding
0.5 %. After 12 h, medium was collected from each well and
testosterone assayed by
125I-radioimmunoassay(125IRIA) (DSL, Sinsheim, Germany) (detection limit:
0.006 ng/mL, Intra assay variation 5 %). Testosterone production by
fractions obtained from Basella alba methanol extract
were additionally tested after 4 h of incubation time.
Extracts mixed to culture medium without Leydig
cells were also assayed for the presence of testosterone.
The RIA count obtained were even higher than the zero
concentration of the standard. This means the compounds
present in the extract do not cross react with the
testosterone assay system.
2.5 Statistical analysis
All data obtained were analyzed using MedCalc
computer program (MedCal Software, Mariakerke, Belgium).
They were analyzed for normal distribution using the
Kolmogorov每Smirnow test, before any further analysis
either by oneway ANOVA or Kruskal每Wallis test. When
ANOVA was significant, t-test was used for pair
comparison between means.
3 Results
3.1 Bull Leydig cells: purity and testosterone production
From 11 different purifications, the number of Leydig
cells collected was (2.4 ± 1.0) × 106/g of testis, with a
viability of 95 %每98 % (Figure 1). Testosterone
production in these cells was significantly stimulated by human
chorionic gonadotrophin (Figure 2). When the cells were
cultured without changing the medium, testosterone was
produced continuously reaching a plateau after 5 days
(Figure 3). When the medium was changed every day,
we obtained a rapid decline of testosterone production
within 2每3 days (data not shown).
3.2 Effect of Basella alba or Hibiscus macranthus
methanol extract on testosterone production in rat Leydig cells
Methanol extracts of Basella alba caused a
significant (P < 0.001, ANOVA) concentration-dependent
increase in testosterone production after 12 h exposure to
rat Leydig cells. We observed 190 % and 55 % increased
testosterone levels, respectively, compared with the
control and hCG-stimulated control containing DMSO for a
concentration of 10 g/mL Basella alba extract
(Table 1). When DMSO was not added in the hCG control, a
2.5-fold stimulation of testosterone production was observed.
In contrast, extracts from Hibiscus macranthus
increased the level of testosterone slightly at 10 g/mL, but
decreased testosterone production significantly by 60 %
compared with the control at 100 g/mL (P < 0.05, paired
t-test).
3.3 Effect of different fractions from Basella alba on
testosterone production of Leydig cells from bull
The methanol extract of Basella alba was further
separated by silica gel column chromatography. When
bull Leydig cells were incubated with various fractions
(100 g/mL each) for 12 h, significantly (P < 0.05,
paired t-test) enhanced testosterone levels compared with the
control were obtained in culture supernatants containing
fractions B, J§, K, K* and L and hCG control not
containing DMSO (Figure 4). When bull Leydig cells were
incubated with different concentrations (10 g/mL and
100 g/mL) of these fractions for 4 h, testosterone was
only detectable in incubations containing hCG without
DMSO and fraction B (Table 2).
3.4 Testosterone production of rat Leydig cells in presence
of various concentrations of Basella alba fraction B
Fraction B seems to contain most of the active
principle of Basella alba. Therefore, we also analyzed the
effect of various concentrations of Fraction B on rat
Leydig cell culture. After 4 h of stimulation, a
concentration-dependent increase (r = 0.89) of testosterone
production was observed (Table 3). Significant higher
hormone levels compared with the control were achieved
after stimulation with 10 g/mL or higher of fraction
B (P = 0.02, Kruskal-Wallis test).
3.5 Phytochemical composition of various fractions
The results of the phytochemical test were shown
on Table 4. Terpenoids and courmarins were the major
classes of compounds found in both Hibiscus macranthus
and Basella alba fractions. Some limonoids were also
present in Basella alba. On TLC plates of the active
fraction B two spots with Rf values of 0.43 and 0.57,
respec-tively, were observed (data not shown; elution
solvent: methylene chloride: methanol [97:3 v/v]).
4 Discussion
In vitro cell culture is a useful tool to analyze various
functions of Leydig cells. This technique was used to
explore the direct effects of hormones, paracrine
factors or xenobiotics on Leydig cells [6, 9每14]. However,
these cell preparations were of various purity according
to the type of biochemical study investigated. In most
cases Leydig cells were purified from laboratory animals
such as the rat and mouse. This fact is often criticized
by animal protection associations. The purification of
Leydig cells from bull testes 每 waste products from the
slaughter house 每 is a contribution to the development of
an alternative animal test method in laboratory research.
Purification of Leydig cells from bulls have not yet
been described in the scientific literatures. But it is known
that Leydig cells are colored in blue by 3bHSD
histochemistry and upon centrifugation on a percoll gradient,
they are usually found between gradient concentrations
35 % and 60 %, which corresponds to buoyant densities
of 1.067每1.075 [15]. All these characteristics were
exploited advantageously in a procedure described herein
which combines two percoll gradients. The cells
purified from the bull testes produced testosterone and were
stimulated by hCG. When they were cultured with the
medium changed every day, they had a rapid decline of
testosterone production. This may be because of our
culture conditions were not optimal for bull Leydig cells.
Changing the medium may have affected them more rapidly. However, when the cells were cultured without
changing the medium, testosterone was produced
continuously reaching a plateau after 5每6 days. This could
mean that in our culture conditions, these Leydig cells
are functional up till day 5. Our purified Leydig cells were
used in the following experiments without changing the
medium. When methanol extracts were added to rat Leydig cell cultures, it appeared clearly that,
Basella alba was the plant responsible for the induction of
testosterone production, since the production of this hormone in
the medium increased with increasing concentrations of
its extract. In contrast, Hibiscus macranthus
methanol extract had no androgenic effect but inhibited
testosterone production at a concentration of 100 g/mL. The
leaves of this plant are also used in a different herbal
medication together with other three different plants, for
fertility regulation in women. It has been recently shown
that this herbal medication has estrogenic properties [16,
17].
Based on the above results, only fractions from
Basella alba were further analyzed for their androgenic
activities. In purified bull Leydig cells, many of the
fractions tested (mainly B, J§, K, K* and L) induced
testosterone production after 12 h exposition. Usually, for
studies on the effects of various compounds on Leydig
cell function, these cells are incubated for 3每5 h [6, 14,
18, 19]. Thus, Leydig cells were also incubated for 4 h
with the above named fractions in order to compare the
response to that obtained after a 12-hour incubation
period. Here, testosterone was found only in incubations
stimulated by fraction B. In other incubations, including
the controls, testosterone levels were below the
detection limit of the RIA (0.006 ng/mL). Generally, the levels
of testosterone produced by the purified Leydig cells from
bull in this study was lower compared with those of rats.
That might be the reason why testosterone was not
detected in the control. Moreover, fraction B of
Basella alba caused a concentration-dependent increase in
testosterone production in rats Leydig cells culture, thus
confirming the results obtained with bull Leydig cells. This
suggested fraction B as the active one. As shown by
TLC analysis, fraction B is a mixture of two fluorescent
products with Rf values of 0.43 and 0.57 (on
CH2Cl2/methanol 97: 3 v/v solvent system), respectively.
Phytochemical tests demonstrated the presence of terpenoid
or steroid compounds in these fractions. Some steroid
compounds such as cholesterol or dehydroepiandroste-
rone are known as a precursor of the biosynthesis of
testosterone. Further studies will probably help to
elucidate the structure of compounds responsible for
androgenic activity.
Acknowledgment
This work was supported by grants from the
International Foundation for Science (PFM, F/22862) and
the DFG (TKM, 445 KAM-18/2/03). We are grateful to
Mr Tchouanguep C谷lestin for providing the plant material.
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