INTRODUCTION
Natural products are powerful biochemical tools; which serve as Path Finders
for molecular biology and chemistry and in the investigation of cellular function
(Lixin and Arnold, 2005). The family Bombacaceae is best
known for showy flowers and woody or thin-shelled pods filled with small seeds
and silky or cotton like fiber. The durian, Durio zibenthinus L., is
one member that differs radically in having large seeds surrounded by fleshy
arils.
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| Fig. 1: |
Durio zibenthinus tree |
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| Fig. 2: |
Durio zibenthinus fruit |
Apart from variants of the word durian in native dialects, there are few other
vernacular names, though the notorious odor has given rise to the unflattering
terms, civet cat tree (Fig. 1) and civet fruit (Fig.
2) in India. Nevertheless the durian is the most important native fruit
of South-Eastern Asia and neighboring islands. The fruits are ovoid or ovoid-oblong
to nearly round, 18 lbs (8 kg) in weight and some fruits split into 5 segments,
others do not split, but all fall to the ground when mature (Morton,
1987).
Earlier study reported that β-galactosidase were isolated from durian
(Tanboly, 2001), forty-three sulphur-containing constituents
were found in a pentane extract of the durian fruit (Naf
and Velluz, 1998), 63 constituents were identified, comprising 30 esters,
16 sulphur containing compounds, 5 ketones, 8 alcohols and 4 miscellaneous compounds
(Wong and Tie, 2006) and ethyl 2-methylbutanoate was
found to have the highest odor impact among the non-sulfurous odorants in durian
(Weenen et al., 1996).
The term infertility is used to describe a couple who has been unable to conceive
naturally after two years of unprotected intercourse (Miller
et al., 2007). Traditionally the fruits of Durio zibenthinus
are being used by people all over the world for their fertility enhancing activity.
Hence an attempt to evaluate the plant for its influence on copulatory behavior,
sperm count and sperm motility in the male species of Swiss mice is being considered
as worthwhile investigative undertaking.
MATERIALS AND METHODS
Collection and Identification of Plant
The plant fruits were collected in the month of August 2008 from the State
Horticulture Farm, Burliar, The Niligiris, Tamil Nadu, India. The plant
was authenticated by comparing it with authentic specimen at the Botanical survey
of India, Coimbatore, Tamil Nadu, India.
Extraction of the Fruits of Durio zibenthinus Linn.
The fresh fruits of Durio zibenthinus Linn. which were made free
from mud and other impurities and dried in shade. The dried fruit pulp was then
powdered and subjected to successive hot extraction using non polar to polar
solvents. The solvents used for successive extracts were petroleum ether, chloroform,
ethyl acetate and aqueous alcohol.
To 380 g of dried fruit pulp powder, 1.5 L of petroleum ether was added and
extracted at 60°C for 24 h in a Soxhlet apparatus. After 24 h the petroleum
ether fraction was filtered through a Whattman filter paper and the marc was
again successively extracted using chloroform, ethyl acetate and aqueous alcohol
respectively by the same procedure. All the extracts were then concentrated
and dried under reduced pressure with a controlled temperature (40-55°C)
using the rotary evaporator (Kokate et al., 1997).
Isolation of Phytoconstituents of Durio zibenthinus Linn. Extracts
The petroleum ether extract was subjected to isolation of phytoconstituents
by counter current method using various solvents like acetone, chloroform, methanol
and hexane.
In vivo Screening Studies of the Petroleum Ether Extract and Isolated
Compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane)
The sexually active male Swiss mice (25-35 g) were grouped separately and
divided into 5 groups, each group consisting of 6 animals. Group I received
solvent control (0.3% CMC), group II received petroleum ether extract 200 mg
kg-1 b.wt. in 0.3% CMC, group III received petroleum ether extract
400 mg kg-1 b.wt. in 0.3% CMC, group IV received isolated compound
(3-β-hydroxy-21-Normethyl-19-vinylidenylursane) 20 mg kg-1 b.wt.
in 0.3% CMC and group V received isolated compound (3 β-hydroxy-21-Normethyl-19-vinylidenylursane)
40 mg kg-1 b.wt. in 0.3% CMC, daily, orally using oral catheter.
Sexual behavior was observed in a dim light at day time in specially designed
cages having glass on all sides and measuring 50x30x30 cm (Fig.
3). The male experimental mice were transferred to the cage and the female
mice in oestrous phase were introduced with males.
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| Fig. 3: |
Male mice showing mounting behavior |
The first 15 min were considered as acclimatization period (Ageel
et al., 1994). The activities of male mice in each group were recorded
individually for 60 min after 30 min of the drug administration on 1st, 7th
and 14th day treatments. The parameters viz. mounting (Subramoniam
et al., 1997), intromission (Suresh-Kumar et
al., 2000), sperm count and sperm motility were observed.
Sperm Count
One milliliter of diluting fluid (sodium bicarbonate 5 g and formalin neutral
1 mL in 100 mL of distilled water) was measured into a clean test tube. To this
0.1 mL of seminal fluid collected from epididymis using forced extraction was
added, which yields a 1:10 dilution. Neubauer’s chamber (0.0025 mm2,
Tiefe depth profounder 0.100 mm, Superior Marienfield Germany) was filled with
the diluted seminal fluid. The chamber was left on the bench for 2 min. This
allows the immobilized sperms to settle down. Then the numbers of sperms in
the four corner squares covering 4 mm2 were counted under the high
power objective (40x) (Mukherjee, 1997). Sperm count was
calculated as per the formula:
Sperm count (mL-1) = Sperm count x10x1000/4x 0.1
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Sperm Motility
Single Cauda of the epididymis was punctured with a 21 gauge hypodermic
needle. Fluid content from the epididymal lumen was collected. A small drop
of fluid was placed in pre-warmed microscopic slide, approximately at body temperature.
The drop was covered with a cover slip by the WET MOUNTING TECHNIQUE. The cover
slip was rimmed with vaseline to avoid drying. The slide was examined under
the high power objective (40X) with reduced illumination. Several fields were
scanned until a total of at least 200 sperms have been observed. The percentage
of sperm showing actual progressive motion was calculated. Sperm motility was
expressed in terms of the percentage of sperms which were active (Mukherjee,
1997).
RESULTS
Isolation of Phytoconstituents of Durio zibenthinus Linn. Extracts
The phytoconstituents present in the petroleum ether extract of Durio
zibenthinus Linn. were isolated by counter current extraction and were partially
characterized by 1H NMR, 13C NMR and Mass are shown in
the Fig. 4-6. Compound I (Fig.
4 and 5, the 1H NMR spectrum displayed signals
for two tertiary methyl groups at δ 0.68 (s, 3H) and 1.01 (s, 3H) and three
secondary methyl groups at δ 0.93 (d, 3H), 0.81 (d, 3H) and 0.83 (d, 3H)
and primary methyl group at δ 0.85 (m, 3H). The broad singlet at δ
5.35 was attributed to H-6 and the hydroxyl methane proton signal at δ
3.55 was attributed to H-3 proton based on biogenetic considerations. From Fig.
7 the molecular ion peak was found to be 415.00, the above data and knowledge
of known sterols the compound I was found to be β-Sitosterol).
Compound II (Fig. 10), the 1H NMR spectrum showed
signals for six C-methyl singlet’s at δ 0.76, 0.78, 0.88, 0.94, 0.98
and 1.01, an allylic methyl group at δ 1.68, Fig. 11
showed signals for a vinylidene grouping exhibited by a pair of doublet signals
at δ 4.56 and 4.68 each integrating for one proton and a hydroxy methine
group at δ 3.19 (dd) which may be placed between a tetrasubstituted sp3
carbon atom and a methylene grouping. From a biogenetic point of view,
the C-3 hydroxy group was assigned equatorial β-orientation, it is common
feature in Triterpenoids.
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| Fig. 4: |
1H NMR of cmpd I isolated compound I |
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| Fig. 5: |
1H NMR of isolated compound I |
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| Fig. 6: |
13C NMR of isolated compound I |
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| Fig. 7: |
Mass of isolated compound I |
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| Fig. 8: |
1H NMR of isolated compound II |
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| Fig. 9: |
1H NMR of isolated compound II |
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| Fig. 10: |
1H NMR of isolated compound II |
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| Fig. 11: |
1H NMR of isolated compound II |
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| Fig. 12: |
13C NMR of isolated compound II |
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| Fig. 13: |
13C NMR of isolated compound II |
The presence of C-3 hydroxy group was supported by C13 NMR (Fig.
12 and 13) signal at δ 79.00. The signals at δ
109.32 and 150.97 are due to unsaturated carbon atoms between C20
and C30. From Fig. 8 and 9 the
signals at δ 2.38 is due to H-13β and at δ 1.9 is due to H-19.
From Fig. 14 the molecular ion peak was found to be 440.85, the above data suggested the compound II was (3-β-hydroxy-21-Normethyl-19-vinylidenylursane).
In vivo Screening Studies of the Petroleum Ether Extract and Isolated
Compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane)
The parameters viz., mounting (Fig. 3), intromission,
sperm count and sperm motility were observed and results are shown in the Table
1-5.
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| Fig. 14: |
Mass of the isolated compound II |
| Table 1: |
Control |
 |
| Route of administration: Oral, Values are Mean±SEM
of 6 animals, Statistical significance: d = ns = p>0.05, c = p<0.05,
b = p<0.01, a = p<0.001 as compared to the solvent control group,
Bonferroni compare selected pairs of columns |
| Table 2: |
Petroleum ether extract 200 mg kg-1
b.wt. in 0.3% CMC |
 |
| Route of administration: Oral, Values are Mean±SEM
of 6 animals, Statistical significance: d = ns = p>0.05, c = p<0.05,
b = p<0.01, a = p<0.001 as compared to the solvent control group,
Bonferroni compare selected pairs of columns |
| Table 3: |
Petroleum ether extract 400 mg kg-1
b.wt. in 0.3% CMC |
 |
| Route of administration: Oral, Values are Mean±SEM
of 6 animals, Statistical significance: d = ns = p>0.05, c = p<0.05,
b = p<0.01, a = p<0.001 as compared to the solvent control group,
Bonferroni compare selected pairs of columns |
| Table 4: |
Isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane)
20 mg kg-1 b.wt. in 0.3% CMC |
 |
| Route of administration: Oral, Values are Mean±SEM
of 6 animals, Statistical significance: d = ns = p>0.05, c = p<0.05,
b = p<0.01, a = p<0.001 as compared to the solvent control group,
Bonferroni compare selected pairs of columns |
| Table 5: |
Isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane)
40 mg kg-1 b.wt. in 0.3% CMC |
 |
| Route of administration: Oral, Values are Mean±SEM
of 6 animals, Statistical significance: d = ns = p>0.05, c = p<0.05,
b = p<0.01, a = p<0.001 as compared to the solvent control group,
Bonferroni compare selected pairs of columns |
DISCUSSION
On the first day of the treatment all the treated groups showed increased copulatory
sexual behavior in all the experimental animals as revealed by the results.
The prolonged treatment of all the treated groups was highly effective to increase
the sexual libidity as compared to the solvent control. This indicates that
the aphrodisiac activity has been shown by the tested petroleum ether extract
and isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane) at
all the tested dose levels. The order of potency for the petroleum ether extract
and isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane) were:
Finally, two compounds were isolated. The petroleum ether extract and isolated
compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane) were screened
for aphrodisiac activity. The petroleum ether extract at 400 mg kg-1
b.wt. doses showed better aphrodisiac activity than all other treated doses.
In support to the above work, phytochemical investigations have shown the presence
of glycoside, saponins, flavanoids and sterols in Durian fruit. It is likely
that these steroidal constituent increase the steroidogenesis and elevate androgen
levels which results in observed effect (Chauhan and Dixit,
2008). So, the petroleum ether extract showed better activity due to presences
of sterols.
Generally sexual behaviours are enhanced by elevated testosterone levels. Drug
induced changes in neurotransmitter levels or their action in the cells could
also change sexual behavior. In this connection it should be remembered that
on ethnomedical practices this herb is also considered as a nervous stimulant
(Chopra et al., 1956). Investigations are in progress
to explore the possible mechanism of action.
CONCLUSION
There can be no doubt that most herbs rely for their effects on a variety constituents
and the idea of synergy within and between them. So this plant may also contain
other constituents which also possess fertility enhancing activity. Hence, future
study on this plant should be in isolation of other constituents which may show
fertility enhancing activity. Once a molecule has been isolated which possess
activity then its derivatives could be synthesized and QSAR studies can be conducted
which gives optimized result for activity (Wolf, 1997).
ACKNOWLEDGMENT
We are thankful to the management of J.S.S. College of Pharmacy, Ooty, Tamil Nadu, India for sponsoring this project and Sir C.R. Reddy College of Pharmaceutical Sciences, Eluru, Andhra Pradesh, India for providing other facilities.
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