Indian J. Nat.

Prod, 2021; 35(1):9-15

A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article
http://www.ijnponline.com

Protective Effect of Rasayana Herbs on Lead Acetate-induced

Testicular Toxicity in Wistar Rats
Vikas Sharma, Nagendra S Chauhan, Umesh K Patil, Vinod Kumar Dixit*
Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar, Madhya Pradesh, INDIA.

ABSTRACT
In recent years, the use of the herbs in reducing heavy metal toxicities has increased
worldwide. Rasayana drugs have been reported to have improved reproductive activity. In
this study, we investigated the protective effects of Anacyclus pyrethrum, Spilanthes acmella
and Pedalium murex on lead acetate-induced testicular damage in rats. The sample used 30
male rats divided into 5 groups: negative control (rats were given daily with 1% sodium CMC
solution were given daily with lead acetate in mineral water at 250mg/l (1ml mineral water www.ijnponline.com
poisoned); positive control (rats /day/ rat) orally once in a day for 90 days); and the treatment
group (rats were given the Anacyclus pyrethrum 150 mg/kg, Spilanthes acmella 150 mg/kg DOI : 10.5530/ijnp.2021.1.3
and Pedalium murex 150 mg/kg BW orally once in day for 90 days. After 90 days testicular
tissue and sperm count, motility and viability in the epididymis were measured in rats. Testis
samples were also collected for histopathological studies. Results showed that Lead acetate
decreased the sperm count, motility, viability and altered histopathological testis compared
to the negative control. However, administration of Rasayana drugs significantly improved the
histopathological in testis, increased the sperm count, motility, viability and also significantly
increased the testosterone level in drug treated rats. From the results of this study we
concluded that the Rasayana drugs could be a potent natural product provides a promising
protective effect against lead acetate induced testicular toxicity in rats.
Key words: Vajikaran, Rasayana, Lead acetate, Anacyclus pyrethrum, Spilanthes acmella,
Pedalium murex.

and atrophy in rodents.[4] However, the effects of chemical agents on

INTRODUCTION fertility and development may consider the fact that exposure to some
In recent years, there has been an increasing interest in the contribution materials during critical period of development may affect the adult life
of occupational and environmental exposures to toxic pollutants to and even generations to come. In humans and experimental animals the
declining sperm concentration and human male infertility. There are gastrointestinal absorption of lead after chronic exposure also exerts
epidemiological confirming evidences that exposure to industrial metal a wide number of adverse biological effects during lactation, more so
aerosols may be detrimental to male reproduction system. Heavy metals than in adult life, since neonates absorb 40-50 times more lead than
produce cellular impairments at structural and functional level in male adults. It appears from the evidences that the neonatal period is a critical
reproductive system.[1] The effect of heavy metals, such as lead, mercury, stage in the process of sexual development and maturation in primates.
cadmium, chromium and arsenic on male reproduction system has Interferences with normal brain pituitary gonadal function during
been studied in details in various experimental species. One of the first this period appear to impact adversely on subsequent reproductive
materials to be demonstrated as detrimental to fertility was lead. Lead function. Given the recent evidence that the reproductive potential of
appears in homes in many forms as lead piping, lead-containing solders, the human male has declined rather dramatically over the last 50 years
paints, ceramic glazes, pewter and base metal utensils and fixtures. and that clinical conditions associated with abnormal testicular function
Also, cream powder, lipstick and hair color have lead. Agricultural are on the rise, continued investigation in this area would appear to be
soil contamination may be responsible for lead found in many herbal imperative.[5]
medicines and cigarettes.[2] A special class of Rasayana drugs is known as Vrishya or Vajikarana. They
The role of lead in male sub-fertility factor is of particular current are associated with an improvement of male sexual potency and thereby
interest. In men, especially in professional workers, lead exposure results ensure a supraja, or better progeny. Traditionally, the main aim of using
in infertility and sterility, testicular atrophy, cellular degeneration and Vajikaran was to achieve successful copulation for healthy reproduction,
reductions in seminiferous tubule diameter (STD) and sperm count, along with an improvement in sexual pleasure as an additional benefit.
depending on the dose and time of exposure.[3] In animal models, lead Besides having many specific drugs for enhancing sexual functions, the
exposure consistently decreases male reproductive function at the level most commonly used are Akarkara and Gokharu.[6]
of the hypothalamic-pituitary-testicular axis. The effects of lead on adult Therefore, there is a need to work upon measures that could in some way
rat testis have been widely studied and observations demonstrate that help in restoring the damage that the testis may incur in due course of
lead in particular alters those organs, as evidenced by testicular necrosis time. The prevention of lead induced testicular damage and restoration

Correspondence: Prof. Vinod Kumar Dixit, Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar-470003, Madhya Pradesh, INDIA.
Phone No: +91 7582264582; E-mail: [email protected]

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 Sharma, et al.: Protective Effect of Ayurvedic Rasayana Herbs in Reproductive Health

of reproductive functions in patients is an issue that needs scientific epididymis and prostate glands) were dissected out carefully and weighed
attention. The lead for ameliorative substance can be taken from synthesis on a single pan electronic balance. The weights of organs were expressed as
as well as natural sources. The herbs Anacyclus pyrethrum (A. pyrethrum), mg/100 g of body weight.[13]
Spilanthes acmella (S. acmella) and Pedalium murex (P. murex) have
been advocated for improving reproductive functions in male. With this Sperm analysis
aspect in mind the ethanolic extract of the herbs under investigation The motility, viability and number of spermatozoa in the cauda epididymis
were tested for their effectiveness against lead acetate induced damage were assessed in the present investigation. The animals were sacrificed by
on testicular function. It was also envisaged to determine whether the cervical dislocation and the cauda epididymis was transected at the point
herbs could assist in reversing the infertility caused by lead acetate. of origin of the vas deferens at the distal end and placed in a watch glass
In our previous studied A. pyrethrum, S. acmella, P. murex extracts containing 0.5 ml of normal saline (0.9 % NaCl) maintained at 37°C. The
show the maximum effects in dose of 150mg/kg b.w after 28 days tissue was minced carefully with the help of fine forceps and scissors to
treatment.[7-10] This maximum effective dose (150mg/kg) was thus used ensure the extrusion of spermatozoa. The tissue fractions were removed
for further experiments. by using the fine forceps and a needle and the suspension was used for
sperm analysis according to the WHO Laboratory Manual.[14,15]
MATERIALS AND METHODS
Animal stock Sperm motility
The protocol for experimentation was approved by Institutional Animal A drop of sperm suspension was placed on a clear glass slide and then
Ethics Committee of Dr. Hari Singh Gour University, Sagar, India covered with a cover slip. The slide was then examined under the
(Animal Eths Comm/IE/98/Reg No379/01/ab/CPCSEA) and was microscope at 400X and the motility was scored in different fields of
in accordance with international standard on the care and use of view. Spermatozoa showing any degree of movement were considered
experimental animals. Inbred, 30 male rats 10-12 months old and to be motile. All spermatozoa (motile as well as immotile) were counted
weighed 250-300 g was used. The rats were housed at room temperature with the help of a blood cell counter. Sperm motility was calculated using
(24 ± 2°C) on a reversed day-night cycle (dark from 06:00 to 18:00) and the following formula:
relative humidity of 50–55%. They were fed with a standard pellet diet
Motility (%) = number of motile spermatozoa / total number of spermatozoa
and water ad libitum.
(motile + immotile) ×100
Preparation of extracts
Sperm viability
The flowers of the Spilanthes acmella and fruits of Pedalium murex plant
were collected from the area in vicinity of the campus and were dried at Sperm viablity was assessed by using a supra-vital staining technique
room temperature (25-35°C). The plant Spilanthes acmella (L.) Murr. and based on the principle that cells with damaged plasma membrane take
Pedalium murex Linn. was identified and authenticated at Department of up the stain, while viable ones do not. All glass wares as well as the eosin–
Botany, Dr. H. S. Gour University Sagar (M.P.) The roots of Anacyclus nigrosin stain were maintained at 37°C. A drop of sperm suspension and
pyrethrum DC. Were procured from the market and authenticated by a drop of eosin–nigrosin stain (1% eosin + 5% nigrosin, 1:1) were placed
Agharkar Research Institute, Pune (Authentication no. Auth. 07-86). on a clear glass slide and mixed thoroughly with the help of a fine glass
Next, the plant materials were reduced to powder and passed through rod. A portion of the mixture was transferred to a second slide and a thin
a sieve (60 mesh), fed in a soxhlet extractor and extracted with ethanol film was prepared. The slide was then examined under the microscope
(95%) till complete exhaustion. The extract was collected and dried (400X) and about one hundred spermatozoa (viable and dead) were
under vacuum by using a rota vapor (Heidolph, Germany). counted from different fields of the slide. Spermatozoa appearing pinkish
A. pyrethrum ethanolic extract was dark brown colored and semisolid in (stained) were considered to be dead, while those appearing colorless
consistency (yield 7.2%w/w). Ethanolic extract of S. acmella was greenish (unstained) were counted as viable.
brown and semisolid with a yield of 6.1% w/w. P. murex ethanolic extract Sperm viability was calculated in percent by using the following formula:
was brown colored, semisolid with a characteristic odor (yield 5.2% Viability (%) = number of viable spermatozoa / total number of spermatozoa
w/w). Before oral administration, all extracts was suspended in 1%
(viable + dead) ×100.
sodium CMC.
Sperm count
Treatment
A hemocytometer with improved Neubauer ruling was employed for
The male rats 10-12 months old and weighed 250-300 g was used for
counting the spermatozoa. A 20-fold dilution was made by mixing the
present study. The animals were divided into six groups of six rats each.
Group I (Control) animals served as the control and received only sperm suspension with normal saline (0.9 % NaCl). The preparation
vehicle 1 ml of 1% sodium CMC solution. Group II (LA) Groups III was then thoroughly mixed and one drop was added to both sides of the
(LA + AP 150), IV (LA + SP 150) and V (LA + PM 150) were given hemocytometer. The number of spermatozoa was counted in the four
ethanolic extracts of A. pyrethrum, S. acmella and P. murex respectively, corner squares of the hemocytometer under a microscope at 400X. If
with dose of 150mg/kg b.w. for 90 days. The present study protocol was spermatozoa crossed the lines of the grid, only those at the top and right-
pursued.[11,12] hand sides of the squares were counted. Spermatozoa on both sides of
the hemocytometer were counted and the average number was recorded.
Functional studies Number of spermatozoa per cauda epididymidis was expressed as
Effect on body weight and weight of sexual organs follows:
After 28 days of treatment as described above, all the treated and control Sperm number = averaged no. of spermatozoa counted (N) ×
animals were weighed and the body weights were recorded. Animals were multiplication factor (10’000) × dilution factor (20) = N × 10’000 × 20 =
sacrificed by decapitation and the sexual organs (testis, seminal vesicles, N × 0.2 × 106 spermatozoa.

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 Sharma, et al.: Protective Effect of Ayurvedic Rasayana Herbs in Reproductive Health

Histological studies the method described for the kit. Serum concentrations of hormones
After 28 days of treatments to animals of all respective groups, testis were determined in triplicate samples.[17]
of animals from each group was dissected out. The testis was fixed in
Statistical analysis
Bouin’s fluid for histological studies. Twenty-four hours after fixation,
Results are expressed as mean ± standard error mean (SEM). The groups
the tissues were dehydrated in an ascending series of alcohol, treated
were compared by ANOVA, followed by Dunnet’s test. All the statistical
with xylene and embedded in paraffin wax. 6-mm-thick sections were
analysis was carried out using Instate version, 2.1 software. Etholog 2.1
cut by a rotary microtome (York Scientific Industries PVT. LTD, India) was used for computation of the behavioral parameters.
and stained with Hematoxylin and Eosin (H&E). Histological sections
of the testis were viewed in microscope and images were captured with RESULTS
a digital camera.
For determination of the percentage of affected seminiferous tubules, all Effect on body weight and weight of reproductive
the tubules in a randomly selected section of the testis from three rats of organs
each group were counted.[16] The seminiferous tubules were considered A significant decrease in body weights of lead acetate group animals was
affected if they showed any of the following details: regenerated observed upon completion of experimentation on days 91. Similarly, the
appearance of germ cells; condition of germinal epithelium; presence testicular weight was also reduced significantly in lead acetate intoxicated
of spermatids of different stages of spermatogenic cycle in the same group animals. There was a significant recovery in body and testicular
tubule; diameter of the seminiferous tubule; presence of spermatogonia; weights of animals in extract treated animals. The percent reduction
presence of primary and secondary spermatocytes; differentiation of in body weight of lead acetate intoxicated group animals was 10.6%
spermatids; lumen of the seminiferous tubule; sertoli cell cytoplasm; while there was only 3.5% reduction of body weight in A. pyrethrum
presence of stages of spermatogenesis; formation of clones of germ cells, treated group, in S. acmella extract treated group the reduction was
structure and distribution of Leydig cells. only 4.4% and in P. murex treated group it was 4.07%. The result clearly
demonstrated the effectiveness of extracts in preserving the loss of body
Serum total testosterone measurement weight in following order (Table 1 and Figure 1).
On the 28th day, blood was collected to measure serum testosterone level. A) pyrethrum> P. murex > S. acmella
Blood samples were spun at 2500 g for 10 min in a table top centrifuge. The In case of testicular weights, a significant reduction was observed in lead
serum samples obtained were analyzed to determine the concentration acetate group animals. Treatment with extract of all the herbs was highly
of testosterone. Serum concentration of total testosterone was measured effective in ameliorating the damages caused by exposure to lead acetate.
by using a double antibody ELISA kit (Eiagen Testosterone kit, Italy). There was a remarkable reduction in testicular weight (15.2%) in lead
The protocol used for the testosterone determination was according to acetate intoxicated group animals. On treatment with A. pyrethrum, S.

Table 1: Effect of ethanolic extract of A. pyrethrum, S. acmella and P. murex on body and sexual organ weights in lead acetate intoxicated male rats.

Weight of Weight of seminal

Body weight Weight of testis Weight of epididymis
prostate vesicles
Group and of animals (g) (mg /100 g b.w) (mg /100 g b.w.)
(mg / 100 g b.w.) (mg /100 g b.w.)
treatment

0 day After 28 day After 28 day treatment

Control 160.5±10.11 176.1±5.17** 710.1±12.1** 162.3±9.2** 592.6±5.1** 715.3±21.2**

LA 157.1±8.7 140.4±9.21 602.1±9.7 106.1±7.3 501.3±11.1 613.1±15.1

LA+ AP150 152.5±9.5 147.1±7.0** 685.2±10.5 ** 142.1±5.5** 581.9±14.2** 695.4±12.8**

LA+ SA150 161.1±7.9 154±8.05** 675.1±12.2** 132.3±6.4** 576.1±10.06** 673±11.49**

LA + PM150 156.9±10.2 150.5±8.2 ** 681.6±7.3 ** 138.7±3.7** 580.8±8.3** 684.7±10.17**

Weight of testes, prostate, seminal vesicles is expressed as mg/100g body weight.

All values are expressed as mean ±S.E.M, n=6; P*<0.05 and P**<0.01 Considered significant as compared to control. Control: No drug;
LA: Lead acetate diluted in mineral water at 250mg/l (1ml mineral water poisoned/day/ rat) for 90 days.
LA+AP 150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
A. pyrethrum, p.o., for 90 days.;
LA+SA150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
S. acmella in 1% sodium CMC solution, p.o., for 90 days;
LA+PM 150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
P. murex in 1% sodium CMC solution, p.o., for 90 days

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 Sharma, et al.: Protective Effect of Ayurvedic Rasayana Herbs in Reproductive Health

acmella and P. murex extract reduction in testicular weight noted was Effect on serum testosterone level
3.5, 4.9 and 4.0%, respectively as compared to lead acetate intoxicated A significant reduction occurred in the serum level of testosterone in
group animals. The weight of epididymis, seminal vesicles and prostate rats exposed to lead as compared to control rats (Table 2 and Figure 5).
in all extract treated groups is either better or comparable with lead The testosterone level of lead acetate intoxicated group was found to be
acetate intoxicated group animals (Table 1 and Figure 2). 1.04±0.15 (ng/ml). The results indicate that serum testosterone level
was restored in extract treated animals. The level of testosterone was
Effect on sperm parameter observed 2.27±0.17, 2.15±0.211 and 2.09±0.16 ng/ml in A. pyrethrum, S.
acmella and P. murex treated animals, respectively.
The effect of lead acetate exposure on total epididymal sperm count, sperm
motility and sperm viability is shown in Table 2 and Figure 3. There was a Effect on testes histomorpholgy
significant reduction in sperm count in the testicular section of lead acetate The sections of testes from control rats showed the normal structure of
intoxicated group animal as compared to extract treated animals. The the testis. The seminiferous tubules appeared rounded or oval in their
observation in lead acetate group animals was 3.31×106/ml. In contrast the outlines and lined by germinal epithelium showing two types of cells;
sperm count in A. pyrethrum, S. acmella and P. murex extract treated group germ cells and Sertoli cells (Photograph 1a). Sertoli cells were detected
animals was 5.58×106, 5.21×106 and 5.65×106/ml, respectively (Figure 4). in between spermatogenic cells as pyramidal cells with pale basal
oval or triangular nuclei and prominent nucleoli. The spermatogenic
A significant decline in the levels of sperm motility and viability was also
cells were seen in regularly arranged rows with different stages of
observed in rats exposed to lead for 90 days. Rats treated with lead acetate
spermatogenesis. They were arranged from the basal compartment to
plus ethanolic extracts of A. pyrethrum, S. acmella and P. murex showed the lumen of the tubules starting from spermatogonia (type A and B),
higher percentage for sperm motility and viability than the lead acetate primary spermatocytes, rounded and elongated spermatids till mature
intoxicated group animal. spermatozoa in the lumen. The seminiferous tubules were surrounded

Figure 1: Effect of ethanolic extract of A. pyrethrum, S. acmella and P. Figure 3: Effect of administration of ethanolic extract of A. pyrethrum,
murex on body weight in lead acetate intoxicated male rats. S. acmella and P. murex on male rat’s sperm motility and viability in lead
acetate intoxicated male rats

Figure 2: Effect of ethanolic extract of A. pyrethrum, S. acmella and P. Figure 4: Effect of administration of ethanolic extract of A. pyrethrum, S.
murex on sexual organ weights in lead acetate intoxicated male rats. acmella and P. murex on sperm count in lead acetate intoxicated male rats

12 Indian J. Nat. Prod., Vol 35, Issue 1, Jan-Mar, 2021

 Sharma, et al.: Protective Effect of Ayurvedic Rasayana Herbs in Reproductive Health

Figure 5: Effect of administration of ethanolic extract of A. pyrethrum,

S. acmella and P. murex on serum testosterone level in lead acetate
intoxicated male rats.

by a thin connective tissue layer with myoid cells. The other control
subgroups showed similar histological characters.
Examination of sections obtained from testes of lead intoxicated rats
revealed seminiferous tubules with vacuolations in the spermatogenic Photograph 1: Histopathological observations of the effect of different
epithelium mostly separating primary spermatocytes from spermatogonia ethanolic extracts in lead acetate induced testis toxicity in male rats. L
indicates Lumen of seminiferous tubules, LC indicates the Leydig cells
and surrounding nuclei of Sertoli cells (Photograph 1 b). Testes of few
and arrows of SG, SC and ST indicates spermatogonia, Spermatocytes and
animals showed some thin walled seminiferous tubules with wide lumen Spermatids. (a) Control (b) LA (c) LA+AP150 (d) LA+SA 150 (e) LA+PM150
and vacuolations in the basal part of the spermatogenic epithelium with
decreased number of germ cells. Apoptotic bodies were found within
the basal part of the spermatogenic epithelium. Focal thickening of the (O- 2) or hydrogen peroxide (H2O2). Enhanced generation of ROS can
connective tissue bounding the tubules was noticed in some tubules. overwhelm cells’ intrinsic antioxidant defenses and result in a condition
Some spermatogonia appeared shrunken with pyknotic nuclei. known as ‘‘oxidative stress’’. Cells under oxidative stress display various
Animals that received lead together with ethanolic extract of A. dysfunctions due to lesions caused by ROS to lipids, proteins and DNA.
pyrethrum, S. acmella and P. murex showed wide areas of testicular tissue Consequently, it is suggested that metal-induced oxidative stress in
similar to the examined control sections. However, focal areas of basal cells can be partially responsible for the toxic effects of heavy metals.[26]
vacuolations in the germinal epithelium were noticed only in limited In recent research literature the effect of antioxidant supplementation
tubules (Photograph 1 c, d and e). followed heavy metals exposure was reported. They suggest that
It is noteworthy that all the three herbal extracts are helpful in preventing antioxidants may play an important role in abating some health hazards
the testicular damage caused by lead acetate and preserve the testicular of heavy metals in connection with an interaction of physiological free
integrity. The most effective treatment in the experiment was with A. radicals (health effects). So, multiple mechanisms may be responsible for
pyrethrum extract, the others following closely. ROS production in toxic metal exposure. Among them, duction of ROS
and damage to cell’s antioxidant defense systems are well known for all
DISCUSSION redox-active and inactive elements.[27]
It was demonstrated that intratesticular testosterone levels are decreased From the present results, it is obvious that treating heavy metals-
in rats treated with lead acetate;[18] therefore we suggest that decreased intoxicated rats with all selected ethanolic extracts significantly protected
spermatogenesis were caused by a reduction in intra-testicular the testis structures and functions as compared to the lead intoxicated
testosterone levels. Supporting this hypothesis, other researchers group. Ethanolic extracts of selected herbs allows free radicals to
concluded that lead alters sperm function by altering the hormonal abstract a hydrogen atom from the antioxidant molecule rather than
control of spermatogenesis rather than by direct toxic action on from polyunsaturated fatty acids, thus breaking the chain of free radical
spermatozoa.[19] Ethanolic extracts of selected herbs was able to bring reactions, the resulting antioxidant radicals being a relatively unreactive
back serum testosterone levels suggesting that herbs may affect sperm species.[28,29]
count through a mechanism related to testosterone levels. Therefore ethanolic extracts of all selected herbs are sufficient to protect
Many studies indicate that heavy metals act as catalysts in the oxidative the organism from toxic agent and free radical damage. It has been
reactions of biological macromolecules therefore the toxicities with reported that reactive oxygen species, generated in vitro, can cause DNA
these metals might be due to oxidative tissue damage;[20-22] Cuypers et al. fragmentation in human sperm, but that this could be prevented by
1999;[23] Leonard et al. 2004;[24] Flora et al. 2008).[25] Redox-active metals, preincubation with antioxidants.
such as iron (Fe), Cu and chromium (Cr), undergo redox cycling whereas Previous phytochemical investigations have shown the presence of
redox-inactive metals, such as Pb, Cd, Hg and others deplete cells’ major phenolic compounds such as flavonoids, flavonol and quercetin in
antioxidants, particularly thiol-containing antioxidants and enzymes. selected herbs. In fact, all selected herbs have been demonstrated to
Either redox-active or redox-inactive metals may cause an increase in have antioxidant properties in vitro and in vivo. For instance, lead
production of ROS such as hydroxyl radical (HO-), superoxide radical acetate administration causes an increase in oxidative status and it has

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 Sharma, et al.: Protective Effect of Ayurvedic Rasayana Herbs in Reproductive Health

Table 2: Effect of administration of ethanolic extract of A. pyrethrum, S. acmella and P. murex on male rats sperm motility, viability and number of
spermatozoa in cauda epididymidis ,Fructose content in seminal vesicle and serum Testosterone level in lead acetate intoxicated male rats.

Serum
Sperm number
Group and treatment Motility (%) Viability (%) Testosterone level
(×106 /ml)
(ng/ml)
Control 71.12±4.03** 72.12±5.81** 6.28±0.15** 1.88±0.12**

LA 49.22±3.10 48.12±3.71 3.31±0.11 1.04±0.15

LA + AP150 66.31±4.72** 63.20±4.90** 5.58±0.21** 2.27±0.17*

LA + SA150 60.14±2.40** 59.60±3.41** 5.21±0.19** 2.15±0.11**

LA + PM150 70.10±3.18** 68.18±2.87** 5.65±0.31** 2.09±0.16**

All values are expressed as mean ±S.E.M, n=6; P*<0.05 and P**<0.01 Considered significant as compared to control.
Control: No drug;
LA: Lead acetate diluted in mineral water at 250mg/l (1ml mineral water poisoned/day/ rat) for 90 days.
LA+AP 150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
A. pyrethrum, p.o., for 90 days.;
LA+SA150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
S. acmella in 1% sodium CMC solution, p.o., for 90 days;
LA+PM 150: Lead acetate diluted in mineral water 250mg/l (1ml mineral water poisoned/day/ rat) + 150 mg/kg body weight of ethanolic extract of
P. murex in 1% sodium CMC solution, p.o., for 90 days

been shown in male rats to reduce spermatogenesis and epididymal 5. Dorostghoal M, Dezfoolian A, Sorooshnia F. Effects of maternal lead acetate
exposure during lactation on postnatal development of testis in offspring wistar
sperm count; Ethanolic extracts reversed this effect. Thus, ethanolic rats Iran J Basic Med Sci. 2011;14(2):122-31.
extract of selected herbs could play a role in regulating sperm number 6. Chauhan NS, Sharma V, Dixit VK, Thakur M. A Review on Plants Used for Im-
by maintaining the balance between oxidant and antioxidant status. In provement of Sexual Performance and Virility. Bio Med Research International.
2014;1-19.
addition, it has been demonstrated that these constituents have potent
7. Sharma V, Thakur M, Chauhan NS, Dixit VK. Effect of petroleum ether extract of
scavenging activity. Again, other investigators found a high total phenolic Anacyclus pyrethrum DC on sexual behaviour in male rats. J Chin Integr Med.
content an ethanolic extracts, supporting the hypothesis that the effect 2010;8(8):767-73.
observed on sperm production might be related to these compounds and 8. Sharma V, Boonen J, Chauhan NS, Thakur M, DeSpiegeleer B, Dixit VK. Spilan-
thes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects
their antioxidant activity. on sexual behavior in male rats. Phytomedicine. 2011;18(13):1161-9.
Vajikarana therapy improves the function of the reproductive organs and 9. Sharma V, Thakur M, Dixit VK. A comparative study of ethanolic extracts of
vitalizes reproductive tissues, increasing sperm count and strengthening Pedalium murex Linn. fruits and sildenafil citrate on sexual behaviors and serum
testosterone level inmale rats during and after treatment. Journal of Ethnophar-
their motility and viability. While some Vajikarana herbs work as macology. 2012;143(1):201-6.
aphrodisiacs, they also engender reproductive strength. Vajikarana not 10. Sharma V, Boonen J, Spiegeleer BD, Dixit VK. Androgenic and spermatogenic
only enhances the quality and longevity of individual life, but also treats activity of alkylamide-rich ethanol solution extract of Anacyclus pyrethrum DC.
Phytotherapy Research. 2013;27(1):99-106.
infertility by nourishing the whole body as well as the reproductive
11. Rubio J, Riqueros MI, Gasco M, Yucra S, Miranda S, Gonzales GF. Lepidium
tissues and sexual fluids. meyenii (Maca) reversed the lead acetate induced-damage on reproductive
function in male rats. Food Chem Toxicol. 2006;44(7):1114-22.
ACKNOWLEDGEMENT 12. Allouchea L, Hamadouche M, Touabti A. Chronic effects of low lead levels on
sperm quality, gonadotropins and testosterone in albino rats. Exp Toxicol Pathol.
Funding by the All India Council, New Delhi, India for technical education 2009;61(5):503-10.
in the form of National Doctoral Fellowship is duly acknowledged. 13. Thakur M, Dixit VK. Effect of Chlorophytum borivilianum on androgenic and
sexual behavior of male rats. Ind Drugs. 2006;43(4):300-6.

CONFLICT OF INTEREST 14. World Health Organization. WHO Laboratory Manual for the Examination of
Human Semen and Semen–Cervical Mucus Interaction. Cambridge University
Press: Cambridge.1999.
The authors declare no conflict of interest.
15. Sethi S, Chaturvedi CM. Temporal synergism of neurotransmitters (serotonin and
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GRAPHICAL ABSTRACT SUMMARY

In recent years, the use of the herbs in reducing heavy metal toxicities has in-
creased worldwide. Rasayana drugs have been reported to have improved repro-
ductive activity. The male rats were given daily with lead acetate and Anacyclus
pyrethrum, Spilanthes acmella and Pedalium murex BW orally once in day for 90
days. Results showed that Lead acetate decreased the sperm count, motility,
viability, and altered histopathological testis compared to the negative control.
On the other hand, administration of Rasayana drugs significantly improved the
histopathological in testis, increased the sperm count, motility, viability, and also
significantly increased the testosterone level in drug treated rats. From the re-
sults of this study we concluded that the studied Rasayana drugs are potent
natural products in providing a promising protective effect against lead acetate
induced testicular toxicity in rats.

ABOUT AUTHORS

Dr.Vikas Sharma has more than 14 years of teaching and research experience. He has made significant contributions
in the area of medicinal plant research. He has been working on evidence-based validation of medicinal plants
and pharmacological screening of herbal drugs. Dr. Sharma works on marker and biomarker analysis of herbs for
quality evaluation. He has also made significant contributions to development of chemical profiling of extracts and
formulation through TLC, HPTLC and LC-MS. He is supervising scientific research of the post-graduation and the
doctoral level. He has written several researches and review paper in reputed journals and has contributed several
chapters in books on pharmaceutical and indigenous drugs. He is a member of various professional and academic
bodies.
Dr. Nagendra Singh Chauhan has around 15 years of research experience. He is presently working as Senior
Scientific Officer Grade-II and Government Analyst at Drugs Testing Laboratory Avam Anusandhan Kendra ,Raipur,
Chhattisgarh, India. He has written more than 65 articles published in national and international journals, 24
book chapters and edited four books in publishers like Academic Press, CRC and Willey. He is a member of
various professional and academic bodies like Society of Pharmacognosy India, International Natural Product
Sciences Taskforce (INPST) society, SILAE: Società Italo-Latinoamericana di Etnomedicina (The Scientific Network
on Ethnomedicine, Italy), Institutional Human ethical committee, Association of Pharmaceutical Teachers of India
(APTI).
Dr. Umesh K. Patil is presently working as Professor of Pharmacognosy at Department of Pharmaceutical
Sciences, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar. His research interest includes Herbal
Drug Technology, Ethnopharmacology and Natural Products. He has more than 22 years of experience in teaching
and research. He is recipient of 7 prestigious awards given in the field of HMPs and remained BOYSCAST fellow
of DST, Govt of India.

Dr. Vinod Kumar Dixit has worked in academic and administrative capacities for more than 30 years and retired
from active services as Professor of Pharmacognosy at Department of Pharmaceutical Sciences, Dr. Harisingh Gour
Vishwavidyalaya (A Central University), Sagar. He has more than 40 years of experience in teaching and research.
His research interest includes Standardization of Herbal Products, Herbal Drug Technology, Ethnopharmacology
and Natural Product Research. He has served as General Secretary, Vice president, President of Indian Society of
Pharmacognosy (ISP) and currently contributing to the society as one of the National Advisors of ISP.

History: Submission Date: 07-12-2020; Review Completed: 08-01-2021; Accepted Date: 31-01-2021
Cite this article: Sharma V, Chauhan NS, Patil UK, Dixit VK. Protective Effect of Rasayana Herbs on Lead Acetate-induced
Testicular Toxicity in Wistar Rats. Indian J Nat Prod. 2021;35(1):9-15.

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