EVALUATION OF SPERMATOGENIC ACTIVITY OF SIDDHA DRUG THUTHUVALAI LEGIYAM (TL) IN WISTAR MALE ALBINO...

International Journal of Analytical, Pharmaceutical and Biomedical Sciences 3

Available online at www.ijapbs.com

Volume: 4: Issue-8: August-2015 ISSN:2278-0246

Coden : IJAPBS www.ijapbs.com

EVALUATION OF SPERMATOGENIC ACTIVITY OF SIDDHA DRUG THUTHUVALAI

LEGIYAM (TL) IN WISTAR MALE ALBINO RATS

Parthiban.P1*, Sasireka.R2, Prabakaran.R3, Thillaivanan.S4 1Prof & Head, Department of PG Pothu Maruthuvam, GSMC, Chennai. 2Asst Lecturer, Department of PG Pothu Maruthuvam, GSMC, Chennai.

3PG Scholar, Department of Pothu Maruthuvam, GSMC, Chennai. 4Assistant Medical officer, GPHC, Jamunamarathur, TamilNadu.

* Corresponding Author: [email protected]

ABSTRACT: Infertility is one of the problems of human society. According to the World Health Organization

(WHO), 15-10 percent of couples have experienced some forms of infertility problems which 40% of these

problems are due to male factor. Despite recent advances in the treatment of male infertility, the problem has not

been satisfactorily tackled. This study was aimed for the Spermatogenic activity of Siddha drug Thuthuvalai

Legiyam (TL), in male albino rats. Animals were divided into five groups. Group I serves as control. Group II

serves as Negative control (Ethanol 20%v/v). Group III and IV received suspension of the TL in milk orally at

the dose level of 100mg, 200mg /kg daily for 60 days. Group V serves as standard received Sildenafil citrate (5

mg/kg). The total sexual behavior includes genital grooming, anogenital sniffing, chasing of female rats. The

testis of control group animals showed normal histological texture. The animal in the treated group showed

pronounced effects in terms of sperm count. The positive control intromission latency was less when compared

with TL administered group. The parameter is statistically significant (P<0.001). Post-ejaculatory interval is the

time interval between ejaculation and the first intromission. The parameter shows less in positive control

followed by TL administered group. It was concluded that the trial drug TL increases the spermatogenesis of

adult male Wistar rats. The interventional Siddha drug Thuthuvalai Legiyam can be used more effectively in the

treatment of Oligospermia and male infertility.

Keywords: Siddha, Herbal medicine, Aan maladu, Aphrodisiac, Sildenafil citrate.

INTRODUCTION

Sexual activity has been universally recognized as a vital component of a normal and healthy lifestyle and

general well-being [1]. Infertility is recognized as the inability to conceive after 12 months of sexual practice

without using contraception [2]. According to the World Health Organization (WHO) estimates that 60 to 80

million couples are worldwide currently suffer from infertility [3]. Infertility varies across regions of the world

and is estimated to affect 8 to 12 per cent of couples worldwide [4, 5]. Studies confirm that male sperm counts

are declining, and environmental factors, such as pesticides, exogenous estrogens, and heavy metals may

negatively impact spermatogenesis [6]. Male reproductive health refers to a man’s ability to procreate, and to

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Parthiban & et al IJAPBS ISSN: 2278-0246

International Journal of Analytical, Pharmaceutical and Biomedical Sciences Page 14


bring a living being into existence. Male reproductive health has social and psychological impact on normal life

[7].Male infertility is commonly due to deficiencies in the semen, and semen quality is used as a surrogate

measure of male fecundity [8]. Various factors contribute for sexual dysfunction like psychological disorders,

life style, systemic diseases, etc [9]. Reproductive failure is observed to be a common problem in young

generation due to stress. Sterility and impotence in males is characterized by dramatic change in semniferous

epithelium that leads to decrease in spermatogenesis [10]. Although the use of allopathic medicines have shown

significant improvement in treating sexual disorders, but at the same time there are large number of side

effects[11]. According to several studies, a number of botanical medicines have a positive effect on sperm

parameters [12].The World Health Organization (WHO) in its 31th summit in developed countries has

recommended administration of medicinal herbs, because of their standards, advantages and healthiness [13].

Natural Products, especially plants, have been used for the treatment of various diseases for thousands of years

[14]. Conventional treatment for infertility usually involves invasive and, expensive procedures. There are many

alternative treatments available that can increase the chance of conception [15]. the availability of the large

number of sexual function improving drugs in the traditional (Ayurveda, Siddha & Unani) System of Medicine is

a unique and distinctive feature of this system [16]. However, many issues related to a lack of scientific evidence

about the efficacy and safety of the drugs remains unresolved [17]. In Siddha system, this ailment is analogues

with ‘Aanmaladu’. Siddha Literatures have described many medicines for the treatment of male infertility. The

interventional Siddha drug Thuthuvalai Legiyam (TL) quoted in the siddha literature ‘Agathiyar paripooranam

400’ has been used for the treatment of Aan maladu (Male infertility). The drug is holding ThathuViruthi

property which is analogues with Spermatogenic activity. Consequently an effort was made to evaluate

Spermatogenic activity of the polyherbal siddha formulation TL in laboratory animals.

MATERIAL METHOD

Source of Drugs

The required raw drugs are procured from a well reputed indigenous drug shop. The raw drugs will be

authenticated by the concerned pharmacognosist at SCRI, Chennai, Tamil Nadu, India.

Ingredients of TL

1. Thuthuvalai poo (Solanum trilobatam) [18],

2. Murungai poo (Moringa olifera) [18] ,

3. Thaneervittan kizhangu (Asparagus racemosus) [18] ,

4. Nannari vaer (Hemidesmus indicus) [18],

5. Nilapannai kizhangu (Curculigo orchioides) [18] ,

6. Nerunjil (Tribulus terrestris) [18],

7. Sarkkarai (Saccharum officinarum) [18] ,

8. Milk,

9. Honey (Apis mellifera) [18]

10. Ghee.





SOP of Trial drug (TL) [19]

All the drugs are powdered. It is mixed with cow’s milk and heated till it gets semisolid consistency. Powdered

sugar is then added to it and mixed. At this time cow’s ghee and honey is added and mixed to get the consistency

of legiyam. The trial drug is stored in clean dry air tight container.

Aim of the Study:

Aim of the study is to evaluate the safety and efficacy of the Siddha drug ‘Thuthuvalai Legiyam’ (TL) In Wistar

Male Albino Rats.

Experimental animals

Adult male Wistar albino rats (150‐200 g) from breeding stock were used for the study. They were housed in

clean and transparent poly propylene cages with three animals in each cage with 12: 12 h light‐dark cycle for a

period of 7days prior to the study. They were fed standard rat pellet feed and water ad libitum. Pelleted feed

supplied by Sai meera foods Pvt Ltd, Bangalore The experimental procedures described were approved by the

Institutional Animal Ethics Committee of K.K college of Pharmacy, Gerugambakkam , Chennai- 122 (Approval

no – KKCP/2013/010/CPCSEA).

Evaluation of Spermatogenic activity:

Grouping of Animals

Healthy and sexually experienced male albino rats (100– 150 gm) that were showing brisk sexual activity were

selected for the study. Sexually experienced male albino rats were divided into 5 groups of 6 animals each;

Animals were divided into five groups. Group I serves as control. Group II serves as Negative control (Ethanol

20%v/v). Group III and IV received suspension of the TL in milk orally at the dose levels of 100mg, 200mg /kg

daily for 60 days. Group V serves as standard received Sildenafil citrate (5 mg/kg), 1 hour prior to the

commencement of the experiment. (Table 1)

Table 1: Grouping of Animals for Experimental

Group Purpose Treatment

Group-I Control Milk (1ml/100gm bw)

Group-II Negative control Ethanol 20%v/v

Group-III Treatment 100mg/kg TL in milk

Group-IV Treatment 200mg/kg TL in milk

Group-V Sildenafil citrate 5 mg/kg

Male rats were housed in temperature controlled 12 hours light/ dark cycle prior to experimental protocols. All

animals were treated in accordance to the Principles of Laboratory Animal Care. All rats were fed a standard diet

and water. Thereafter, the rats were randomly divided into groups (n=6) and experimental (n=6) groups.

The entire animal including the control were treated with 20% v/v of ethanol for a period of 35 days (5 weeks).

All the animals were subjected to the treatment for 60 days. During the treatment of the animals, the behavioral

observations were recorded for each animal. The male animals were introduced into the cage containing females

pretreated with estradiol and checked for estrous cycle. The behavioral observations like rearing, licking,





anogenital smelling, non-genital grooming, genital grooming and exploration were observed and recorded.

Finally on the 61 day, the animals were sacrificed and testis excited and sperm collected for counting [20].

Sexual Behavior

The experiment was carried out in a specially designed box measuring 50×30×30 cm with a dim yellow light.

One male rat marked by picric acid was kept initially in the box and two female rats which were prepared as

explained above were introduced into the box. After a 15 minutes acclimatization period, the following sexual

behavior such as Mount frequency (MF), Intromission frequency (IF), Mount latency (ML), Intromission latency

(IL), Ejaculation latency (EL), Post-ejaculatory interval (PEI) and Total sexual behavior (TSB) includes genital

grooming, anogenital sniffing, chasing of female rats was observed for one hour till ejaculation of semen in any

one of the female[21].

Sperm count Analysis (no of sperm x 106)

After treatment, the sperm count was carried out by using Haemocytometer. Haemocytometer is generally used

for RBC as well as WBC count. It is provided with the pipettes for the dilution of the blood samples and

Neaubaur’s slide with special type of ruling. 1The counting is done in the ruled squares on the slide. The

epididymis was removed and placed in a pre-chilled petri-plate. 2 ml. of 0.9% saline was added to it and the

cauda epididymis was gently minced with the help of sharp razor. This sample was used for the sperm count. The

sample was pipetted out with the help of pipette provided in the Haemocytometer. A clean and dry cover slip

was kept on the Neaubaur’s ruling. The ruling was loaded with the sample by touching the tip of the pipette to

the slide [22].

RESULT

The trial drug Thuthuvalai Legiyam has been in use by the traditional healers in the southern regions of India as

a means of treating sexual inadequacy and stimulating sexual drive even without choice to the scientific authority

of the claim. Hence this study was carried out to validate scientifically this tribal claim.

Table 2 shows that the mean activity of licking and Anogenital smelling of male experimental rats towards

female. The trial drug was exerted the maximum licking activity (19.83±1.922) towards female at the dose level

of 200mg/kg of BW, which is comparable with the standard drug Sildenafil (21.67±1.498). The trial drug was

showed only the minimal Anogenital smelling activity (16.33±1.358) towards female at the dose level of

200mg/kg of BW, which is not comparable with the standard (20.67±0.6146).

Table 2: Effect of Thuthuvalai Legiyam on Male Wistar rats – Licking, Anogenital smelling towards

female rats

Group Treatment Mean activity towards female

Licking Anogenital smelling

Control Milk 11.50±1.500 9.167±0.6540

Ethanol Ethanol 20%v/v 4.66±1.257* 5.500±1.245*

Trial drug 100mg/kg 17.50±2.187** 16.33±1.358***

Trial drug 200mg/kg 19.83±1.922** 16.33±1.358***

Sildenafil citrate 5 mg/kg 21.67±1.498** 20.67±0.6146***





Table 3 shows that the mean activity of Exploration and Rearing of male experimental rats towards environment.

The trial drug was showed the maximum Exploration activity (22.17±1.797) towards environment at the dose

level of 200mg/kg of BW, which is more comparable with the standard drug Sildenafil (24.33±3.765). The trial

drug was showed the maximal Rearing activity (18.33±0.9545) towards environment at the dose level of

200mg/kg of BW, which is more than that of the standard (12.33±1.745).

Table 4 shows that the mean activity of non genital grooming and genital grooming of male experimental rats

towards self. The trial drug was exerted the significant non genital grooming activity (16.17±1.138) towards self

at the dose level of 200mg/kg of BW, which is more than that of the standard drug Sildenafil (14.67±1.585). The

trial drug was showed only the minimal genital grooming activity (27.50±3.041) towards self at the dose level of

200mg/kg of BW, which is less significant with the standard (31.17±4.262).

Table 4: Exploration towards non genital grooming and genital grooming

Table 5 shows that the sperm count of the Male Wistar rats at the end of the study. Administration of the trial

drug Thuthuvalai Legiyam at the dose of 100 and 200 mg/kg, significantly increased the sperm concentration

(sperm count) (45.17±2.4) in testes and epididymis as compared to control group. Similarly the standard group

animal which received Sildenafil, also showed significant increase in the sperm concentration (sperm count)

(50.37±1.75) as compared with the control group.

Table 3. Effect of Thuthuvalai Legiyam on Male Wistar rats – Exploration, Rearing towards

environment

Group Treatment

Mean activity towards environment

Exploration Rearing

Control Milk 12.17±1.887 26.17±2.197

Ethanol Ethanol 20%v/v 11.67±4.13 27.67±1.145ns

Trial drug 100mg/kg 17.33±1.498 25.00±2.295ns

Trial drug 200mg/kg 22.17±1.797* 18.33±0.9545*


Group Treatment Mean activity towards self

Non genital grooming Genital grooming

Control Milk 21.50±1.057 9.500±0.6708

Ethanol Ethanol 20%v/v 23.67±3.001* 22.33±2.67*

Trial drug 100mg/kg 19.50±2.078* 27.50±3.041*

Trial drug 200mg/kg 16.17±1.138* 27.50±3.041**






Table 5: Effect of Thuthuvalai Legiyam on Sperm count.

Treatment Groups Dose mg/kg Sperm count (sperm x 106)

Control Vehicle 36.67±3.04

Ethanol Ethanol 20%v/v 17.83±3.96

Trial drug 100mg/kg 40.97±1.49

Trial drug 200mg/kg 45.17±2.43*

Sildenafil citrate 5 mg/kg 50.37±1.75**

[Values are expressed as mean ± SEM; n=6; ns p>0.05, *p<0.05, **p<0.01]

Histopathological Analysis

At the end of 60th day testis were isolated for histopathological examination and fixed in 10 % formal saline (10

parts of formaldehyde and 30 parts of normal saline). Tissues were processed and embedded in paraffin wax.

Sections were cut at 5 micron thickness and stained with Haematoxylin and Eosin. Light microscopic

examination of the sections was then carried out and micrographs produced using Vanox-T Olympus

photographing microscope. The histopathological examinations were reviewed by the pathologist.

DISCUSSION

Male fertility requires the production by the testis of large numbers of normal spermatozoa through a complex

process known as spermatogenesis. This process can be subdivided into three major steps: (i) the multiplication

of spermatogonia by the process of mitosis; (ii) meiosis, which reduces the chromosome number from diploid to

haploid and commences with the entry of type B spermatogonia into the prophase of the first meiotic division.

These cells, now called primary spermatocytes, divide to form secondary spermatocytes, and then divide again to

form round spermatids; (iii) the successful transformation of the round spermatid into the complex structure of

the spermatozoon, this phase is being called spermiogenesis. Each of these steps represents a key element in the

Spermatogenic process. Defects which occur in any of them can result in the failure of the entire process and

lead to the production of defective spermatozoa and reduction or absence of sperm production. It is therefore

essential that our understanding of these processes is expanded to provide information concerning the regulatory

mechanisms [23]. The sperm count was significantly (P<0.01) higher in trial drug (Thuthuvalai Legiyam) groups

than the control in a dose related manner.In the Thuthuvalai Legiyam treated groups, there was a significant

excess in the number of primary and secondary spermatocytes and round spermatids. The administration of

Thuthuvalai Legiyam for 60 days to male rats resulted in remarkable increase in the sexual vigor of the male rats,

as evidenced by the different parameters studied. The results of mating behaviour test show the trial drug

Thuthuvalai Legiyam at the dose of 100 & 200 mg/kg body weight significantly increased the Mounting

Frequency (MF). The significant increase in Ejaculation latency (EL) suggests that the all experimental extracts

and standard drug prolonged the duration of coitus, which is an indicator of increase in sexual motivation.

Administration of Thuthuvalai Legiyam at the dose of 100 & 200 mg/kg significantly increased the sperm count

(P<0.05) in testes and epididymis as compared to control group. Similarly the standard group animal also

showed significant increase in the sperm count (P<0.01) as compared with the control group (Table 5). The

present results clearly indicate that administration of Thuthuvalai Legiyam has positive effect on

spermatogenesis in rats. The total sexual behavior also increase which includes genital grooming, Anogenital

sniffing, chasing of female rats etc. The testis of control group animals showed normal histological texture. The

animal in the trial drug treated group showed pronounced effects in terms of sperm count.





CONCLUSION

The results obtained in this study strongly confirms the positive beneficial effects on male reproductive system

by Increased number of spermatozoa in semniferous tubules and which is evident by increase in Spermatogenic

elements as compared to control. The present study exposed that the trial drug Thuthuvalai Legiyam (TL)

possess significant Spermatogenic activity at the dose levels of 100 and 200 mg/kg of body weight. The findings

of this study comparatively supported the traditional claims of the trial drug TL as Spermatogenic.

Figure 1: Effect of Thuthuvalai Legiyam on Male Wistar rats – Licking towards Female.





Figure 2: Effect of Thuthuvalai Legiyam on Male Wistar rats- Anogenital smelling

towards female

anogenital smelling (towards female)

control ethanol 100mg/kg 200mg/kg standard0

5

10

15

20

25

Groups

no o

f tim

es

Figure 3: Effect of Thuthuvalai Legiyam on Male Wistar rats – Exploration towards environment

Exploration (towards environment)

control ethanol100mg/kg200mg/kgstandard0

10

20

30

Groups

no o

f tim

es





Figure 4: Effect of Thuthuvalai Legiyam on Male Wistar rats –Rearing towards environment

rearing (towards environment)

control ethanol 100mg 200mg std0

10

20

30

40

Groups

no

of

tim

es

Figure 5: Effect of Thuthuvalai Legiyam on Male Wistar rats- Exploration towards non genital grooming

(towards self)

non genital grooming (towards self)


10

20

30

Groups

no

of

tim

es





Figure 6: Effect of Thuthuvalai Legiyam on Male Wistar rats- Exploration towards genital grooming

(towards self)

Genital grooming (towards self)


10

20

30

40

Groups

no

of

tim

es

Figure 7: Effect of Thuthuvalai Legiyam on Sperm count

Evaluation of the siddha drug Thuthuvalai legiyam for its toxicological

and Spermatogenic activity in Wistar Albino rats

(Sperm count no of sperm X 106)

control ETHANOL 100mg 200mg std0

20

40

60

Groups

no

of

spe

rm X

10

6





PHOTOMICROGRAPHS OF TESTES TISSUE SECTIONS (Histopathology) OF RATS:

Control Ethanol

100Mg/Kg TL 200 Mg/Kg TL

Standard





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EVALUATION OF SPERMATOGENIC ACTIVITY OF SIDDHA DRUG THUTHUVALAI LEGIYAM (TL) IN WISTAR MALE ALBINO...

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