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Research Article CODEN: IJPRNK IMPACT FACTOR: 4.278 ISSN: 2277-8713 Singh RV, IJPRBS, 2014; Volume 3(4): 422-438 IJPRBS
Available Online at www.ijprbs.com 422
EFFECT OF SCHIFF BASE LIGAND AND TRANSITION METAL COMPLEXES ON SEX HORMONES, SPERM DYNAMICS AND FERTILITY IN MALE ALBINO RATS
WATTS S1, SHARMA A2, JOSHI SC2, SINGH RV1
1. Department of Chemistry, University of Rajasthan, Jaipur-302004.
2. Department of Zoology, University of Rajasthan, Jaipur-302004. Accepted Date: 22/07/2014; Published Date: 27/08/2014
Abstract: The novel organotitanium(IV) and organozirconium(IV) complexes have been
synthesized by reacting Cp2TiCl2 and Cp2ZrCl2 with sulphur donor ligand (N ′ -[1-2-oxo-2H-
chrome-3yl-ethylidene]-hydrazinecarbodithionic acid benzyl ester) and characterized on the
basis of IR,UV, 1H NMR and 13C NMR spectral studies, elemental analysis and molecular weight
determinations. Zirconium and titanium were estimated by gravimetrical method. The present
research was coined at investigating the effects of metal complexes on the hormones and
sperm profile in male albino rats. The effects of complexes on sex hormones, sperm density,
sperm motility and fertility were observed at dose level of 30 mg/kg.b.wt./day for 45 days in
male albino rats. Findings of the present investigation mention a highly significant decrease in
testosterone, follicle stimulating hormone (FSH) and leutinizing hormone (LH). Decrease in
sperm motility and sperm density was observed after complexes exposure, as compared to
control group. Decrease in the weight of testes, epididymis, vas deferens, and seminal vesicle
were also observed. In conclusion the study shows that ligand (N ′ -[1-2-oxo-2H-chrome-3yl-
ethylidene]-hydrazinecarbodithionic acid benzyl ester) and its metal complexes alter the sex
hormones and fertility in rats.
Keywords: Spectral studies, Organotitanium and organozirconium complexes, Radio immuno assay.
INTERNATIONAL JOURNAL OF
PHARMACEUTICAL RESEARCH AND BIO-SCIENCE
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Corresponding Author: PROF. R. V. SINGH
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Singh RV, Watts S, Sharma A, Joshi SC; IJPRBS, 2014; Volume 3(4): 422-438
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INTRODUCTION
Population growth, a topic most likely insignificant to the common man, but the world’s
population growth and control of that population growth is necessary for our overall
survival. Over population have both major personal and societal impact and it is necessary to
control it on the time. As we know the entire available contraceptive in the market are not safe,
mostly they are steroid in nature and they have more or little hazardous side effects. It includes
adverse effects on sexual function and fertility in adult males and females, as well as
developmental toxicity in the offspring. Attention has now been focused on safe chemicals for
possible contraceptive effect. The chemical control of fertility in the male has received
attention since quite some time and a large number of synthetic compounds have been tested
for their antispermatogenic and antiadrogenic effects1-3. The interest in the construction of
Schiff-base coordination complexes by reacting transition metal ions with bidentate has been
constantly growing over the past years 4-6. Transition metals and their complexes have evolved
great interest due to their biological potential. Organotitanium and organozirconium
compounds of sulphur containing ligands have attracted much attention recently due to their
biological importance. The sulphur containing ligands are well known for their anticarcinogenic,
antibacterial, and antifungal effect. It has been reported that the activity of sulphur-containing
ligands increases on complexation7-9. The aim of present study was to investigate the toxicity
profile on male sex hormones and fertility.
MATERIALS AND METHODS
All reagents were obtained commercially and were purified by standard procedures. All
solvents were of reagent grade. The reactions were carried out under strictly anhydrous
conditions. In the present investigation, ligand and its complexes have been synthesized in our
laboratory.
1. Synthesis of ligand:
1.1 Preparation of the S-benzyldithiocarbazate:-
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A cold solution of KOH (11.4 g) in 90% ethanol (70 mL) was added to hydrazine hydrate (10 g)
with constant stirring. A solution of CS2 was added drop wise with continuous stirring over a
period of one hour and temperature of the reaction mixture was kept below 10 °C. During the
addition, the oily layer so formed was separated and dissolved in cold 40% ethanol (60 mL). The
solution was cooled in a freezing mixture and benzyl- chloride (25 g) was added drop wise while
stirring for two hours. The white solid was separated by filtration, washed with water and dried
in air. The crude product was recrystallized from benzene (M.P.-125° C).
1.2 Preparation of the 3-acetyl coumarin S -benzyldithiocarbazate
Ligand was prepared by the condensation of 3-acetyl coumarin with S-benzyldithiocarbazate in
1:1 molar ratio and refluxed on a water bath for five-six hours. Alcohol was used as the solvent.
The solution then concentrated under reduced pressure. On cooling overnight, crystals
separated out which were further purified by washing with ethanol and finally recrystallized
with acetone (Figure. 1).
HN
NH2
S
S
O O
O
+
Ethanol
- H2OO O
C N
CH3
NH
S
S
Ligand
O O
C N
CH3
N
S
HS
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Figure.1 Structure of the Ligand
1.3 Synthesis of the complexes
For the preparation of the complexes, Cp2TiCl2 and Cp2ZrCl2 were mixed with the corresponding
ligand in 1:1:1 ratio in dry THF using triethylamine (Et3N) as hydrogen chloride acceptor. The
solution was refluxed for a period of 8–10 hours. The precipitate of triethylamine hydrogen
chloride formed during the course of the reaction was removed by filtration, and the filtrate
was dried under reduced pressure. The resulting product was repeatedly washed with n-hexane
and then finally dried under vacuum. The purity was further checked by thin layer
chromatography with silica gel-G using DMSO as a solvent. The synthetic details and elemental
analyses of the resulting complexes are listed in Table I.
The ligand and complexes synthesized are soluble in DMF and DMSO. Molecular weights were
determined by the Rast Camphor method. Nitrogen was estimated by the Kjeldahl’s method
and sulphur was estimated by the Messenger’s method. Carbon and hydrogen analyses of the
ligand and its metal complexes were carried out at CDRI, Lucknow. Infrared spectra of the
ligand and its metal complexes were recorded with the help of Nicolet Megna FTIR-550
spectrophotometer on KBr pellets. 1H NMR spectra was recorded on a JEOL-AL-300 FTNMR
spectrometer in DMSO-d6. using TMS as the internal standard.
2. Spectral Studies
The molecular weights of the complexes were determined by the Rast Camphor method which
indicated their monomeric nature. These complexes have low conductance values (10-15 ohm-1
cm2 mol-1) in anhydrous DMF, which reveal their non-electrolytic nature.
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2.1 Electronic Spectra
The electronic spectra of the ligand and its metal complexes were recorded in dry methanol.
The electronic spectra of the ligand exhibits two bands in the regions, 280nm and 295nm. The
bands in this regions are assignable to -* transitions within the benzene and coumarin rings.
A band around 365nm due to the >C=N chromophore shows considerable hypsochromic shift in
the spectra of the metal complexes which may be attributed to the coordination of the
azomethine nitrogen to the metal atom.
2.2 IR Spectra
In the IR spectra of ligands and their metal complexes the band at 3250 cm-1 due to -NH
stretching vibrations of the ligand disappears due to the complexation and a band at 1625-1590
cm-1 is observed due to the (C=N) vibrations. The appearance of strong and medium intensity
bands in the regions 395-380 cm-1, 520-500 cm-1 and 375-366 cm-1 be assigned to (TiN),
(Ti-O) and (Ti-S) vibrations, respectively and thus confirms the formation of complexes. In 1:1
molar metal complexes band at 370-325 cm-1 due to (M-Cl) vibrations is also observed. In
addition, the presence of cyclopentadienyl groups in these complexes is indicated by the
absorption bands at ca 3000-2990 cm-1 for (C-H), 1430-1420 cm-1 for (C-C), 1020-1010 cm-1
for (C-H) and ~ 810-800 cm-1 for (C-H) out of plane deformation.
In ligand one strong band located at 1050 cm-1 in the ligand was attributed to ν (C = S) moiety,
which in the case of complexes shifted to lower frequency due to formation of ν (C- S) . These
data on comparison with the spectrum of the ligand suggested that the azomethine nitrogen
and thiolic sulphur atom of the ligand are involved in coordination with the metal ion. A
doublet at ∼ 2950 and ∼ 2900 cm-1 is assigned to symmetric and asymmetric vibrations of S-
CH2-C6H5 grouping. In organozirconium complexes the coordination of the azomethine nitrogen
and bonding of the ketonic oxygen/thiolic sulphur to the zirconium atom is supported by the
appearance of three bands in the regions, 387-378, 480-473 and 372-368 cm-1 in the complexes
which may be assigned to (ZrN), (Zr-O) and (Zr-S) vibrations, respectively
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2.3 1H NMR Spectra
The 1H NMR spectra of the free ligand and its metal complexes were recorded in DMSO-d6. The
signal due to the -NH proton of the ligand δ 10.20 ppm disappears in the complexes and this
confirms the deprotonation and complexation through this functional group. The spectra of the
ligand shows multiplets in the region δ 6.38-7.50 ppm assignable to aromatic protons which
remain almost at the same position in the free ligand as well as its respective complexes. The
presence of -C5H5 group in all the complexes is suggested by the signal at 6.10-6.19 ppm.
The 1H NMR spectra of the ligand and its metal complexes have been recorded in Table 2
In the 13C NMR spectra, the considerable shifts in the position of carbon atoms adjacent to the
azomethine nitrogen 163.16 ppm and carbons attached to the - thiolic sulphur 180.30 ppm
support the proposed coordination in the complexes. The 13C NMR spectra of the ligand and its
metal complexes have been recorded in Table 3.
On The Basis of above Spectral studies, the structures of metal complexes considered as
follows:
N
S S
N
M
Cp
Cp
1:1 1:2
M = Ti or Zr
Figure.2 Structure of the Complexes
S
Cp
Cp
M
Cl
N
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3. Animal model used:
Male albino rats (Rattus norvegicus) weighing 150-200 gms were used in this study and they are
housed in a separate room cages, under controlled conditions of temperature (22±3°C),
humidity (50± 5%) and light (12 hrs light: 12 hrs dark cycle), fed with standard pellet diet
(Ashirwad Industrial Ltd., Punjab, India) and water ad libitum.
3.1 Ligand and metal complexes administration
For this study, the rats were divided into groups containing 5 animals each. Ligand and Metal
complexes (1:2) were administered 30mg/kg.b.wt./day in the groups for 45 days to the rats.
Only olive oil was given to the control group. At the end of the experimentation rats were
sacrificed under light ether anesthesia for testicular and accessory sex organ weight analysis,
sperm dynamics and hormonal studies.
3.2 Hormonal measurements and sperm dynamics hormonal analysis:-
Radioimmunoassay of testosterone, leutinizing hormone (LH), and follicle stimulating hormone
(FSH) were performed10.
3.3 Sperm motility
The epididymis was removed immediately after anesthesia and known weight of cauda
epididymis was gently teased in a specific volume of physiological saline (0.9% NaCl) to release
the spermatozoa from the tubules. The sperm suspension was examined within five minutes
after their isolation from epididymis. The results were determined by counting both motile and
immotile sperms in at least ten separate and randomly selected fields. The results were finally
expressed as percent motility11.
3.4 Sperm density
Total number of sperms were counted using haemocytometer after further diluting the sperm
suspension from cauda epididymis and testis. The sperm density was calculated in million per
mL as per dilution11.
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Statistical Analysis: The data were analyzed statistically by using ANNOVA test and the
significance of differences was set at P < 0.05 and P < 0.01.
RESULTS
Analysis of male reproductive hormones and sperm dynamics is an important tool to detect
male reproductive toxicity occurred by chemicals. All the results are as following:
1. Body and Organ weight determination (Table 4)
The results presented in Table 4 clearly revealed that the weights of testes of the treated rats
decreased significantly in comparison to the control group, even though non significant
reductions were observed in the weight of Vas Deferens, Seminal Vesicle and Epididymis and a
normal decrease in the body weight was found in both the treated as well as control groups.
2. Sperm Motility (Table 5)
(i) Cauda Epididymis
There was a significant (P ≤ 0.05) reduction in case of ligand and highly significant reduction (P ≤
0.01) was observed in the motility of sperm in the rats treated with the starting materials and
their complexes when treated rats were compared with the control.
(ii) Sperm Density (Table 5)
Testes
Significant and highly significant reductions in testicular sperm density were observed
respectively when rats were fed with the ligand, starting material and their complexes.
(iii) Cauda Epididymis
The density of sperm in cauda epididymis was reduced highly significantly (P ≤ 0.01) after the
treatment with the ligand, starting material and their complexes.
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(iv) Fertility test (Table 5)
Mating exposure test showed 100% positive fertility in control rats whereas 71% negative
results in group II and 53% ,92%, 56% and 90% negative results in groups III,IV,V and VI
respectively.
3. Radio-Immuno Assays (RIA) (Table.6).
Leutinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) decreased significantly in
group II and highly significantly in groups III to VI whereas Serum testosterone level decreased
highly significantly in all groups..
DISCUSSION
The present study revealed that administration of ligand (N ′ -[1-2-oxo-2H-chrome-3yl-
ethylidene]-hydrazinecarbodithionic acid benzyl ester) and metal complexes at dose level of
30 mg/kg/b.wt./day for 45 days to male rats resulted in antifertility.
The weight of testes and accessory sex organs were decreased The epididymis, seminal vesicle
and Vas Deferens are all androgen dependent organs, relying on testosterone for their normal
growth and function12 . A reduction in their weights may reflect a decreased bioavailability and
production of androgens, which caused Leydig cell disintegration13 .
Sperm motility is considered one of the most important parameters evaluating the sperm
fertilizing ability12. The motility of sperm in cauda epididymis indicates less ability of sperm to
interact with the oocyte plasma membrane15. Decreased sperm density in the epididymis is an
indicator of reduced spermatogenesis as a result of the antispermatogenic nature of any
agent16. Low caudal epididymal sperm density may be due to alteration in androgen
metabolism17-18. The physiological and biochemical integrity of epididymis are dependent on
androgens. The negative fertility may be attributed to lack of forward progression and
reduction in density of spermatozoa and altered biochemical milieu of cauda epididymis19-20.
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Researchers have demonstrated that physiologic concentrations of testosterone, LH and FSH
play an important role in spermatogenesis21, so a significant decrease of these hormones in our
study could decrease the number and function of somatic and germinal cells of testis. The
reduction in the serum testosterone, FSH and LH clearly demonstrate the inhibitory effects on
the secretion of pituitary gonadotrophins and in turn on the testosterone biosynthesis in the
testis of rats22.
CONCLUSION
The oral administration of ligand, starting materials and their metal complexes induced
reproductive toxicity in male albino rats. Toxic effects of complexes were more pronounced
than ligand and starting materials and the results also revealed that the starting materials of
titanium and zirconium(Cp2TiCl2 and Cp2ZrCl2)are more effective than the ligand. Thus a
conclusion may be drawn that ligand, starting materials and their complexes may be a
potential source for the development of an antifertility drug for males because of their
antispermatogenic nature and some antifertility effects on reproductive organs.
ACKNOWLEDGEMENT
The authors are thankful to Council of Scientific and Industrial Research, New Delhi, and UGC,
New Delhi for financial assistance.
Table 1. Analytical data and physical properties of the ligand and its metal complexes.
Compound Colour Melting Point(0C)
Found (Calcd.)(%) Mol.Wt Found (Calcd.)
C H N S Ti/Zr L1H Reddish
orange 155 60. 91
(61.93) 4.19 (4.38)
6.56 (7.60)
16.54 (17.40)
- 366.85 (368.47)
Cp2TiCl Brown 215oC 59.21 (60.16)
3.42 (4.00)
3.52 (4.84)
10.25 (11.08)
7.85 (8.27)
576.35 (578.96)
Cp2Ti (L1)2 Brown 250oC 62.89 (63.28)
3.01 (4.20)
5.27 (6.15)
13.20 (14.08)
3.69 (5.25)
827.38 (910.98)
Cp2ZrCl(L1) Brown 185 oC 54.25 3.10 3.24 9.28 12.87 620.98
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(55.97) (3.72) (4.50) (10.31) (14.66) (622.32) Cp2Zr(L1)2 Brown 210 oC 58.63
(60.41) 3.27 (4.01)
4.10 (5.87)
12.58 (13.44)
8.35 (9.56)
952.14 (954.34)
Table 2. 1H NMR Spectral data of the ligand and its corresponding metal complexes
Compound 1 H NMR spectral data (cm−1)
-NH (bs) Aromatic
protons(m)
-S-CH2(s) Cp-Ti/Cp-Zr
L1H 10.20 (s) 6.38-7.50 4.20 -
Cp2TiCl(L1) - 6.28-7.45 4.20 6.10
Cp2Ti(L1)2 - 6.30-7.48 4.19 6.12
Cp2ZrCl(L1) - 6.30-7.47 4.19 6.15
Cp2Zr(L1)2 - 6.34-7.49 4.18 6.19
Table 3. 13C NMR Spectral data of the ligand and its corresponding metal complexes
Compound
13 C NMR spectral data (cm−1)
-HC=N -C=S/-C-S Aromatic protons
C1
C4
C2
C6
C3
C5
C7
C9
C8
C10
L1H 163.16 180.30 --
134.80
160.75
125.30
111.46
127.21
128.16
154.12
118.66
132.76
Cp2TiCl(L1) 164.78 175.23 -
134.50
159.48
125.12
110.95
126.35
127.83
153.90
118.32
132.15
Cp2Ti(L1)2 164.12 176.95 -
133.90
159.12
124.90
110.62
126.10
127.70
153.10
118.20
132.12
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Cp2ZrCl(L1) 166.78 170.25 -
134.60
159.45
125.12
111.25
126.65
127.93
153.94
118.30
132.30
Cp2Zr(L1)2 166.10 170.89 -
134.10
159.40
124.85
110.62
126.10
127.70
153.90
118.20
132.35
TABLE: 4
Body and organ weight measurements of ligand (L1H), starting materials and complexes treated male rats
(Mean ± SEM of 5 animals)
Group I compared with all groups
ns = Non significant
* = Significant (P< 0.05)
** = Highly significant (P< 0.01)
Group
Treatment
(30 mg/kg
b.wt./day)
Body weight Testes Vas Deferens Seminal Vesicle Epididymis
Initial Final
G mg/100g body wt.
I Vehicle
treated
(Control)
208.11±12.78 180.20±16.43 999.60±2.71 139.30±1.98 621.40±17.31 366.80±10.41
II Ligand
(L1H)
206.25±19.40ns 164.24±13.54ns 882.13±1.46ns 134.28±1.14ns 577.65±11.24ns 341.74±12.32ns
III Cp2TiCl2 192.21±11.87ns 160.81±13.37ns 791.64±1.65* 130.19±0.74ns 570.14±11.3ns 331.00±12.2ns
IV Cp2Ti(L1)2 182.15±12.80ns 145.29±13.13ns 724.35±1.95** 124.20±1.02ns 563.27±10.9ns 318.69 ±13.8ns
VI Cp2ZrCl2 196.10±11.97ns 162.40±10.07ns 819.41±2.51* 132.19±0.86ns 572.25±11.1ns 335.64±13.5ns
VII Cp2Zr(L1)2 187.32±14.14ns 165.54±11.76ns 7 92.39±2.47** 126.45±1.73ns 567.31±11.3ns 321.9±13.2ns
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TABLE 5
Sperm motility and fertility test of ligand (L1H), starting materials and complexes treated male rats
(Mean ± SEM of 5 animals)
Group I compared with all groups
ns = Non significant
* = Significant (P< 0.05)
** =Highly significant (P< 0.01)
Group Treatment
(30 mg/kg
b.wt./day)
Sperm
motility
(%)
Sperm density (million/ml) Fertility
(%)
Testes Cauda
epididymis
I
Vehicle treated
(Control)
73.60±4.34 6.03±0.50 43.20±0.85 100%(+ve)
II Ligand (L1H) 53.31±4.77* 3.95±0.80* 31.98±1.08** 71%( -ve)
III Cp2TiCl2 49.35±5.15** 3.32±0.53* 29.58±1.09** 53%( -ve )
IV Cp2Ti(L1)2 41.87±4.28** 2.30±0.70** 26.87±0.86** 92%( -ve )
V Cp2ZrCl2 48.27±4.96** 3.56±0.71* 30.37±1.02** 56% ( -ve )
VI Cp2Zr(L1)2 43.69±5.87** 2.41±0.62** 27.93±1.08** 90% ( -ve)
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TABLE 6
Serum harmonal analysis of ligand (L1H), starting materials and complexes treated male rats
(Mean ± SEM of 5 animals)
Group I compared with all groups
ns = Non significant
* = Significant (P< 0.05)
** =Highly significant (P< 0.01)
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b.wt./day)
Testosterone
ng/ml
Luteinizing
Hormone (LH)
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Follicle Stimulating
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IV Cp2Ti(L1)2 0.93±0.02**,c 0.66±0.05**,b 0.35±.04**,b
V Cp2ZrCl2 1.21±0.17**,a 0.94±0.16**,a 0.49±0.06**,a
VI Cp2Zr(L1)2 1.08±0.06**,b 0.74±0.09**,b 0.37±0.06**,b
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