Structural studies with antimicrobial and antifertility activity of a monofunctional bidentate ligand with its boron(III), palladium(II), and platinum(II) complexes

Article abstract of DOI:10.1080/10426500500330869

The reaction of the sulfur donor Schiff base ligand, 7-nitro-3-(indolin-2- one) hydrazinecarbo-thioamide, with phenyldihydroxyboron in benzene, palladium(II)chloride, and platinum(II) chloride, in ethanol, gave the mononuclear tetracoordinated and hexacoordinated complexes. The Schiff base ligand coordinated to the boron atom in 1:1 and 1:2 molar ratios and to the palladium and platinum metals in only 1:2 molar ratios in the presence of an acidic and basic medium. Tentative structural conclusions are drawn for reaction products based upon elemental analysis, electrical conductance, and spectral (electronic, infrared, ¹H NMR, ¹³C NMR, and ¹¹B NMR) data. The antifertility activity of the ligand and its nonmetal/metal complexes is discussed with a comparative study in an effective manner. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500500330869

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Phosphorus, Sulfur, and Silicon
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Structural Studies with
Antimicrobial and Antifertility
Activity of a Monofunctional
Bidentate Ligand with its
Boron(III), Palladium(II), and
Platinum(II) Complexes
R. V. Singh a & M. K. Biyala a
a Department of Chemistry , University of
Rajasthan , Jaipur, India
Published online: 01 Feb 2007.
To cite this article: R. V. Singh & M. K. Biyala (2006) Structural Studies with
Antimicrobial and Antifertility Activity of a Monofunctional Bidentate Ligand with its
Boron(III), Palladium(II), and Platinum(II) Complexes, Phosphorus, Sulfur, and Silicon
and the Related Elements, 181:6, 1477-1491, DOI: 10.1080/10426500500330869
To link to this article: http://dx.doi.org/10.1080/10426500500330869
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Phosphorus, Sulfur, and Silicon, 181:1477–1491, 2006
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500500330869
Structural Studies with Antimicrobial and Antifertility
Activity of a Monofunctional Bidentate Ligand with its
Boron(III), Palladium(II), and Platinum(II) Complexes
R. V. Singh
M. K. Biyala
Department of Chemistry, University of Rajasthan, Jaipur, India
The reaction of the sulfur donor Schiff base ligand, 7-nitro-3-(indolin-2-
one) hydrazinecarbo-thioamide, with phenyldihydroxyboron in benzene, palla-
dium(II)chloride, and platinum(II) chloride, in ethanol, gave the mononuclear tetra-
coordinated and hexacoordinated complexes. The Schiff base ligand coordinated to
the boron atom in 1:1 and 1:2 molar ratios and to the palladium and platinum met-
als in only 1:2 molar ratios in the presence of an acidic and basic medium. Tentative
structural conclusions are drawn for reaction products based upon elemental anal-
1
13
ysis, electrical conductance, and spectral (electronic, infrared, H NMR, C NMR,
11
and B NMR) data. The antifertility activity of the ligand and its nonmetal/metal
complexes are discussed with a comparative study in an effective manner.
INTRODUCTION
Thiosemicarbazones constitute one of the most important class of bi-
ologically active ligands providing potential binding sites through ni-
trogen and sulphur donor atoms. The preparation of a variety of metal
complexes from these ligands speak for their spectacular progress in co-
ordination and bioinorganic chemistry. Metal complexes, especially of
Pd(II) and Pt(II), with various such heterocyclic compounds have been
extensively studied by several researchers due to their antitumour, an-
tiviral, antibacterial, and antifungal activity.1−4
Platinum complexes with 2-acetyl pyridine thiosemicarbazone were
found to have a completely lethal effect5 on Gram(+) bacteria, while
the same complexes showed no bactericidal effect on Gram(−) bacteria.
Received May 13, 2005; accepted August 1, 2005.
The authors are grateful to CSIR, New Delhi, for financial assistance in the form of
Grant no. 01(1956)/04/EMR-II.
Address correspondence to R. V. Singh, Department of Chemistry, University of
Rajasthan, Jaipur 302004, India. E-mail: singh-rv@uniraj.ernet.in
1477

1478
R. V. Singh and M. K. Biyala
Similarly, boron and its compounds occupy a unique position in the
development of inorganic chemistry because of the complexity of the
structure and a subtle type of bonding, as boron compounds exhibit
structures ranging from a trigonal planar to a tetrahedral to open or
close polyhedra. Many boron complexes having B N bonds have been
synthesized due to their use in the industry and agriculture.6 On the
other hand, multifarious roles of transition metals in biochemistry7
suggest that considerable potential exists for the development of new
chemistry with these metals in a ligand system specifically designed to
serve these roles. Keeping all these aspects into consideration, we here-
in reported the synthesis, characterization, and biological activity of a
sulfur donor ligand, and its boron, palladium, and platinum complexes.
RESULTS AND DISCUSSION
The elemental analysis and spectral data are consistent with formu-
lations of compounds as [PhB(OH)(L)], [PhB(L)2], [M(LH)2]Cl2, and
[M(L)2]. Reactions of phenyldihydroxyboron with the ligand (LH) have
been carried out in 1:1 and 1:2 molar ratios in dry benzene, and the
reactions of palladium chloride and platinum chloride with the ligand
(LH) have been carried out in 1:2 molar ratios in ethanol.
The addition products, [M(LH)2]Cl2, were synthesized in the pres-
ence of a few drops of concentrated HCl. However, the substituted prod-
ucts, [M(L)2], were obtained when the reaction was carried out in the
presence of aqueous amonium hydroxide.
1:1
[PhB(OH)2] + LH ———- −→ [PhB(OH)(L)] + H2O
Dry Benzene
1:2
[PhB(OH)2] + 2LH ———- −→ [PhB(L)2] + 2H2O
Dry Benzene
1:2
[MCl2] + 2LH
——−→ [M(L)2]Cl2
Ethanol
1:2
[MCl2] + 2LH + 2NH4OH ——−→ [M(L)2] + 2NH4Cl + 2H2O
Ethanol
where M = Pd(II) and Pt(II) and LH = Ligand molecule.
These reactions proceeded easily; reactions of boron could be com-
pleted within 12–24 h of refluxing in benzene, and the reactions of pal-
ladium and platinum could be completed within 3–6 h in an ethanol
medium. All complexes are soluble in DMF, DMSO, and chloroform.
The complexes are monomers as revealed by their molecular weight
determinations.
Low molar conductance values (8–12 ohm−1 cm2 mol−1) of these
boron derivatives in DMF at 10−3 M concentrations show them to be

Antimicrobial and Antifertility Activity
1479
TABLE I Physical Properties and Analytical Data of LH and Its
Complexes
Mol. wt.
found
(calcd.)
Elemental analysis (%) found (calcd.)
Color,
State
M.p.,
(◦C)
Compound
LH
M
—
C
H
N
S
Dim yellow,
solid
236
40.34
2.72
26.48
12.22
278.36
(40.75) (2.65) (26.40) (12.08) (265.24)
48.22 3.17 18.39 8.39 382.75
[PhB(OH)(L)] Brown, solid 252
[PhB(L)2] Cock, solid 257
[Pd(LH)2]Cl2 Brown, solid 267
2.84
(2.92) (48.80) (3.27) (18.97) (8.68) (369.15)
1.34 47.04 2.84 23.14 10.24 628.78
(1.75) (46.76) (2.77) (22.72) (10.40) (616.38)
15.34 30.65 2.17 19.56 9.14 719.68
(15.03) (30.54) (1.99) (19.78) (9.05) (707.81)
16.56 34.18 1.93 21.84 10.03 641.55
(16.76) (34.05) (1.90) (22.06) (10.09) (634.89)
24.65 27.34 1.96 17.36 8.13 782.98
(24.49) (27.14) (1.77) (17.58) (8.05) (796.47)
26.82 29.96 1.78 19.43 8.96 738.68
(26.96) (29.58) (1.67) (19.35) (8.86) (723.55)
[Pd(L)2]
[Pt(LH)2]Cl2 Grey, solid
Grey, solid
264
252
[Pt(L)2]
Dark brown, 248
solid
nonelectrolytes, and molar conductance values of 10−3 M solutions of
[M(L)2] types of complexes lie in the range of 10–15 ohm−1 cm2 mol−1
in dry DMF, indicating their nonelectrolytic behavior. However, the
[M(LH)2]Cl2 complexes are 1:2 electrolytes with conductance values
of 200–220 ohm−1 cm2 mol−1. Analytical data of the ligand and its com-
plexes are given in Table I.
Infrared spectra (4000–200 cm−1) were recorded with the help of
a Nicolet Magena FT-IR 550 spectrophotometer in KBr pellets. NMR
(1H, 13C, and 11B) spectra were recorded in CDCl3 (1H) or DMSO-d6 (13C,
11B) solutions on a JEOL-FX-90Q spectrometer. Molecular weights were
determined by the Rast camphor method. Nitrogen was determined
by the Kjeldal’s method, and sulfur was estimated by the Messanger’s
method. Boron was determined by the methyl borate procedure,8 and
palladium and platinum were estimated gravimetrically.
Infrared Spectra
IR spectra of the free ligand reveals a band at ca 3280 cm−1 assigned to
NH vibrations of the functional group, which disappears in the corre-
sponding [M(L)2] type of complexes, indicating possible deprotonation
of the functional group upon complexation. The band at 1615 cm−1 in
LH, due to ν(C N), is shifted to a higher wave number (ν10–15 cm−1
)
in the [PhB(OH)L], [M(LH)2]Cl2, and [M(L)2] complexes, suggesting

1480
R. V. Singh and M. K. Biyala
coordination through the azomethine nitrogen atom to the central atom,
but in the case of the [PhB(L)2] type of complex, it shifted to a lower
wave number at 1605 cm−1 and a higher wave number at 1630 cm−1
,
suggesting the uncoordinated and coordinated (C N) groups, respec-
tively, to the boron atom. The band at 1020 cm−1, due to ν(C S), is
shifted toward lower frequencies in complexes, indicating the coordina-
tion of sulfur to the central atom and the formation of B/Pd/Pt–S bonds.
In addition, new weak to strong intensity bands are observed in the far
IR spectra of the complexes. Bands in the regions of 830–836 cm−1
,
1520–1524 cm−1, and 1230–1238 cm−1 in boron complexes are due to
ν(B − S), ν(B ← N), and ν(Ph−B) frequency,8 respectively, and 364–369,
440–445, and 308–314 cm−1 can be assigned to ν(M-S), ν(M ← N), and
ν(M-Cl) binds9 in the spectra of the [M(LH)2]Cl2 complexes.
The IR spectra of free ligands display two sharp bands at ca 3446
and 3325 cm−1 assignable to asymmetric and symmetric NH2 group
modes, respectively, which remain at almost the same positions in
the complexes, suggesting that the amino group is not involved in
chelation.
1H NMR Spectra
1H NMR spectra of the free ligand and its compelxes were recorded
in DMSO-d6. The free ligand shows a sharp singlet for the –NH pro-
ton at δ11.24 ppm, which disappears from the spectra of [PhB(OH)(L)],
[PhB(L)2], and [M(L)2], suggesting that this proton has been lost via the
thioenolization of the (>C S) group and the coordination of the sulfur
atom to the central atom. This signal however, suffers a downfield shift
in the complexes [M(LH)2]Cl2 (δ11.52–11.58 ppm) as a consequence of
their deshielding due to the coordination of the ligand to the metal
atom. The ligand shows multiplets in the region of δ6.70–8.30 ppm
attributable to aromatic protons, which appear almost in the same po-
sition as in the respective complexes. The NH2 group gives a singlet of
δ3.36–3.44 ppm in the free ligand as well as in the complexes. It shows
that the −NH2 group is not taking part in the complexes (Table II).
13C NMR Spectra
13C NMR spectral data also support the authenticity of the proposed
structures. Considerable shifts in positions of carbon atoms adjacent
to the azomethine nitrogen (δ157.28–163.76 ppm) and thiolic sulfur
(δ169.42–171.85 ppm) support the proposed coordination in the com-
plexes.

Antimicrobial and Antifertility Activity
1481
TABLE II 1H NMR Spectral Data of the Ligand and Its
Complexes
1H NMR (δ, ppm)
Compound
NH2
NH(1)
NH(2)
OH Aromatic protons
LH
3.36
3.42
3.44
3.38
3.40
3.39
3.42
11.24
—
—
11.52
—
11.58
—
11.92
11.94
11.95
11.93
11.95
11.94
11.96
4.20
4.52
—
—
—
6.70–8.26
6.76–8.30
6.75–8.28
6.72–8.20
6.75–8.28
6.73–8.24
6.76–8.28
[PhB(OH)(L)]
[PhB(L)2]
[Pd(LH)2]Cl2
[Pd(L)2]
[Pt(LH)2]Cl2
[Pt(L)2]
—
—
11B NMR Spectra
11B NMR spectra of boron complexes have signals in DMSO-d6 in the re-
gion of δ2.5–5.8 ppm, which unequivocally suggests a tetracoordinated
environment around the boron atom and the presence of a B ← N coor-
dinate bond. The driving force for the formation of this coordinate bond
is the ability of a trivalent PhB(OH)2 to accept a pair of electrons from
a suitable donor atom.
X-Ray Spectra
The X-ray diffraction spectrum of one powdered sample has been
recorded in order to further substantiate the lattice of the complexes.
˚
The observed interplanar spacing values d(A) have been measured from
the differactogram of the complex PhB(L)OH, and the Miller Indices
h, k, and l have been assigned to each “d” value. The data suggest an
“orthorhombic” lattice to this derivative having unit cell dimensions;
˚
˚
˚
a = 9.9777A, b = 6.7131A, c = 7.4079A; the Miller Indices h, k, and l are
recorded in Table III and α = β = γ = 90◦ (Table III).
On the basis of the previously discussed spectral studies, suitable
structures have been suggested for new complexes (Figure 1).
Biocidal Screening
Antifungal and antibacterial activities of the ligand and its complexes
have been evaluated. Results reveal that the activity increases on com-
plexation. Newly synthesized complexes have indeed been found to be
more active in inhibiting the growth of fungi and bacteria than the lig-
and itself. The greater toxicity of metal/non-metal complexes than the
ligand can be explained on the basis of the chelation theory. Chelation
reduces the polarity of the metal ion mainly because of the partial

1482
R. V. Singh and M. K. Biyala
TABLE III X-Ray Powder Diffraction Data of
[PhB(OH)(L)]
˚
Peak
2θ (deg.)
d-spacing (A)
h
k
l
1
2
3
4
5
6
7
8
9
14.6022
21.8468
30.3523
33.0726
34.5589
36.3355
40.2568
52.3622
58.1818
72.98264
86.67176
7.4564
5.0379
3.6619
3.3483
3.2317
3.0801
2.7933
2.1829
1.9228
1.62264
1.40729
0
0
0
0
0
1
2
1
3
3
5
0
1
0
2
1
1
2
3
0
0
2
1
1
2
0
2
2
0
0
3
4
3
10
11
sharing of its positive charge with the donor groups and possible π-
electron-delocalization over the whole chelate ring. This increases the
lipophilic character of the metal complexes, which subsequently favors
its permeation through the lipid layers of the organism cell membrane
and normal cell process being impaired. The results are discussed and
reported in Tables IV and V.
Antifertility Activity
Weight Response
There were no significant differences in body weights. However, the
weights of testes, the epididymis, the ventral prostate, and the seminal
vesicle were decreased significantly (P ≤ 0.01 to P ≤ 0.001) after the
treatment with the ligand and its boron, palladium, and platinum com-
plexes (Table VI).
TABLE IV Fungicidal Screening Data of the Ligand and
Its Metal Complexes
Inhibition after 96 h (%) (Conc. in ppm)
Macrophomina phaseolina
Fusarium oxysporum
Compound
50
100
200
50
100
200
LH
30
40
42
49
47
52
56
38
50
54
54
52
58
60
46
59
62
62
61
65
68
29
41
42
48
46
52
55
38
51
55
53
54
58
60
45
60
62
62
63
64
68
[PhB(OH)(L)]
[PhB(L)2]
[Pd(LH)2]Cl2
[Pd(L)2]
[Pt(LH)2]Cl2
[Pt(L)2]

Antimicrobial and Antifertility Activity
1483
FIGURE 1 Structures of complexes where M = Pd (II) and Pt (II).
Sperm Dynamics and Fertility
The sperm motility in the cauda epididymis and sperm density in
testes and the cauda epididymis were decreased significantly after
treatment with the ligand and its various complexes (Table VII).

1484
R. V. Singh and M. K. Biyala
TABLE V Antibacterial Screening Data of the Ligand
and Its Complexes
Diameter of Inhibition zone (mm) (Conc. in ppm)
Bacillus subtilis
Salmonella species
Compound
500
1000
500
1000
LH
7
9
10
10
9
10
11
12
12
11
14
16
6
9
10
9
9
11
12
9
[PhB(OH)(L)]
[PhB(L)2]
[Pd(LH)2]Cl2
[Pd(L)2]
[Pt(LH)2]Cl2
[Pt(L)2]
11
13
14
13
13
15
11
13
Biochemical Findings
The ligand and its boron, palladium, and platinum complexes bring
about a significant decrease in the testicular, total protein, and total
cholesterol contents. A significant increase was observed in testicular
glycogen, cholesterol, acid, and alkaline phosphatase activity after the
treatment with the various compounds (Table VIII).
The administration of the ligand (LH) and its boron, palladium, and
platinum complexes bring about a reduction in the weights of testes
and sex accessories. The weight of testes are largely dependent on
the mass of differentiated spermatogenic cells, and the reduction in
weight may be due to the decreased number of germ cells and elon-
gated spermatids.10 The observed reduction in the weight of accessory
sex organs may be due to reduced bioavailability and the estrogenic
and/or antiandrogenic activities of the compounds.11 Low causal epi-
didymal sperm density may be due to the alteration in the androgen
metabolism. The physiological and biochemical integrity of the epi-
didymis is dependent on androgens.12 The 50–95% negative fertility
test may be attributed to the lack of forward progression and reduc-
tion in the density of spermatozoa and altered biochemical milieu of
the cauda e´pididymis.13 The treatment with the ligand (LH) and its
boron, palladium, and platinum complexes also changes the testicu-
lar biochemical parameters. The reduction in testicular sialic acid con-
tents may be due to the absence of spermatozoa or reduced androgen
production.14,15 The increased level of testicular cholesterol contents
is attributed to the decreased androgen concentration, which resulted
in impaired spermatogenesis.16 An increase in the testicular glycogen
of the ligand and its complexes treated animals might be associated

1485

1486
R. V. Singh and M. K. Biyala
TABLE VII Effects of the Ligand (LH) and Its Boron, Palladium, and
Platinum Complexes on Body and Reproductive Organ Weights on
Male Rats
Sperm density million/cm3
Sperm motility Cauda
epididymis (%)
Cauda
Group Treatment
Testes
epididymis Fertility test (%)
A
B
C
D
E
F
Control
LH
85.0 4.6
65.0 4.5a
50.0 3.8a
42.0 3.5b
41.0 2.9b
35.0 3.1b
34.1 4.1b
27.0 2.1b
5.1 0.60
4.1 6.30a
3.1 0.20a
2.2 0.10b
2.9 0.25a
1.9 0.10b
2.1 0.15b
1.4 0.10b
57.5 3.6
47.3 1.5a
35.8 1.8a
30.0 1.6b
31.0 1.7b
28.0 2.1b
25.0 2.5b
19.0 1.1b
100% positive
50% negative
55% negative
60% negative
65% negative
80% negative
90% negative
95% negative
[PhB(OH)(L)]
[PhB(L)2]
[Pd(LH)2]Cl2
[Pd(L)2]
[Pt(LH)2]Cl2
[Pt(L)2]
G
H
Group B was compared with Group A.
Groups C, D, E, F, G, and H were compared with Group A.
Mean SEM of five animals; a = P ≤ 0.01; b = P ≤ 0.001.
with the poor utilization of glycogen due to a decrease in phosphary-
lase activity.17 Furthermore, an increase in testicular acid and alka-
line phosphatase activities indicate an impairment of the functional
integrity of testes.18 From these results, it can be conducted that the
addition of boron, palladium, or platinum moiety to the ligand enhances
its activity, and the compounds of platinum are more effective in regu-
lating the fertility in male rats.
EXPERIMENTAL
All syntheses and manipulations were carried out under strict exclusion
of moisture using an apparatus made up of standard interchangeable
quick-fit joints. The [PhB(OH)2], PdCl2, PtCl2, and o-nitroaniline were
purchased from L obachemie and used as such. 7-nitroisatin was pre-
pared in the laboratory. All preparations were carried out under acidic
or basic conditions. All solvents were dried and distilled before use.
Preparation of the Ligands
The Synthesis of 7-nitroisatin
7-nitroisatin can be synthesized conveniently by Sandmeyer
isonitroso-acetanilide synthesis. o-nitroaniline-HCl, chloralhydrate-
Na2SO4, and hydroxylamine hydrochloride are dissolved in water,
and the solution is heated for a few minutes. The solution is shaken

1487

1488
R. V. Singh and M. K. Biyala
vigorously and cooled whereby crystals of isonitrosoacetanilide are ob-
tained. The isonitrosoacetanilide is filtered and dried.
The dried isonitrosoacetanilide is then cyclized in conc. H2SO4 at
60–80◦C to yield 7-nitroisatin. The crude isatin is then recrystallized
from glacial acetic acid or purified by dissolving in aq. NaOH and re-
precipitating with HCl.
The Synthesis of LH of 7-nitroisatin
The ligand LH was prepared by the condensation of 7-nitroisatin
with hydrazinecarbothioamide in the presence of sodium acetate in a
1:1 molar ratio, in absolute alcohol. The reaction mixture was refluxed
over a water bath for 3–4 h and allowed to stand over night (Scheme 1).
The product was recrystallized from the ethanol and dried in vacuo. Its
physico-chemical properties and analytical data are given in Table I.
SCHEME 1
The Preparation of [PhB(OH)(L)] and [PhB(L)2] Complexes
Phenylboronic acid was dissolved in dry benzene in a 100-mL RB flask,
and to this the requisite amounts (1:1 or 1:2 molar ratios) of the lig-
and were added. The resulting mixture was refluxed for 12–24 h. The
progress of the reaction was monitored azeotropically by the liberation
of water. The solid product was dried in vacuo. It was then washed sev-
eral times with dry cyclohexane and again dried in vacuo for 3–4 h. It
is interesting to note that if the same reactions were carried out under
microwave irridation, reactions completed within 5 min. This shows
that the microwave technology is more useful than the conventional
method.
The Preparation of [M(LH)2]Cl2 Complexes
These complexes were prepared by dissolving MCl2(0.01 mol) in ethanol
and then adding an ethanolic solution of a the ligand (0.02 mol) to this
solution in 1:2 molar ratios. The reaction mixture was heated under
reflux for about 1 h in the presence of a few drops of concentrated HCl.

Antimicrobial and Antifertility Activity
1489
On cooling, complexes separated out, which were filtered and washed
several times with alcohol and dried in vacuo.
The Preparation of [M(L)2] Complexes
The ethanol solution of MCl2 (0.01 mol) was mixed with an ethanolic
solution of a ligand (0.02 mol) in 1:2 molar ratios. Aqueous NH4OH was
added dropwise to the reaction mixture until it was weakly alkaline
(pH ca. 8.0). The mixture was then stirred on a magnetic stirrer for 2–
3 h, and the resulting product was recovered by filtration, washed with
ethanol, and dried in vacuo. Physical properties and analytical data of
these complexes are listed in Table I.
BIOCIDAL ACTIVITY
Antimicrobial Activity
The antifungal activity was evaluated against Macrophomina phase-
olina and Fusarium oxysporum by the agar plate technique. Solutions
of compounds in different concentrations (50, 100, and 200 ppm) in DMF
were used. The linear growth of the fungus was recorded by measuring
the diameter of the colony after 96 h, and the percentage inhibition was
calculated as 102 (dc − dT)/dc, where dc and dT were the diameters of
the fungus colony in the control and test plates, respectively.
The antibacterial activity was screened by the paper-disc plate
method. The nutrient agar medium (Peptone, beef extract, NaCl, and
agar-agar) and 5-mm diameter paper discs (Whatman No. 1) were used.
Compounds were dissolved in DMF in 500 and 1000 ppm concentra-
tions. Filter paper discs were soaked in different solutions of com-
pounds, dried, and then placed in petri dishes previously seeded with
the test organism (Bacillus subtilis and salmonella species). Plates were
incubated for 24–30 h at 28 2◦C, and the inhibition zone around each
disc was measured.
Antifertility Activity
Male rats obtained from ICMR, New Delhi were used. Animals were
housed in steel cages and maintained under standard conditions (14 h
light/10 h dark cycle; 25 3◦C, 35–50% humidity); water and food were
given ad libitum. Proven fertile male rats were taken and divided into 8
groups of 6 each. Group A served as a vehicle (olive oil) treated control.
Groups B, C, D, E, F, G, and H were respective to ligand, boron (1:1
and 1:2), palladium (1:2 add. and 1:2 sub.), and platinum (1:2 add. and

1490
R. V. Singh and M. K. Biyala
1:2 sub.), respectively, for the same period and dose. On the 61st day,
these animals were autopsied, and testes, the epididymis, the seminal,
the vesicle, and the ventral prostate were removed; fat and connective
tissue was cleared off and kept at −20◦C until they were assayed for
total protein, sialic acid, cholesterol, frustose, and glycogen, by standard
laboratory techniques.
Fertility Test
The mating exposure test of all animals was performed. They were
cohabited with progesterone females in the ratio of 1:3. The vaginal
plug and presence of sperm in the vaginal smear was checked for posi-
tive mating. Females were separated, and resultant pregnancies were
noted when dams gave birth. Fertility was calculated in control as well
as treated groups. Animals were weighed and autopsized under light
ether anesthesia. The sperm motility in cauda epididymis and den-
sity of testis and cauda epididymis suspended sperm were calculated.19
The weight of testes, the epididymis, the ventral prostate, and the sem-
inal vesicle were recorded, testes were frozen for the measurements
of glycogen, total protein, total cholesterol, sialic acid, acid, and alka-
line phosphatase, by standard laboratory techniques. The difference
between control and treated groups were evaluated statistically using
a student’s “t” test. Data were expressed as mean 5 EM. The signifi-
cance was set at P ≤ 0.01 and P ≤ 0.001.
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