INTRODUCTION:

Concerns have been raised that man-made and natural compounds may hamper with the endocrine system and thus may affect humans and its progeny. The characterization of various naturals chemicals as compounds with affinity for the androgen receptor (AR) showing androgen or anti-androgen like activity. Male infertility is quite prevalent these days. Male infertility refers to a male's inability to cause pregnancy in a fertile female.¹

Androgen is derived from the Greek words "andros" (man) and "genein" (to produce). Androgen is produced in males by the testes, the two globe-shaped reproductive organs below the penis, and by the adrenal glands, two small hormone-producing organs that each sit atop a kidney. Androgen is also produced in females in the adrenal glands. Overproduction of androgen can generate some male characteristics in women and exaggerate male characteristics in men.

Androgen excess is the most common endocrine disorder in women of reproductive age. Androgens are produced primarily from the adrenal glands and the ovaries. However, peripheral tissues such as fat and skin also play roles in converting weak androgens to more potent ones. Androgen excess can affect different tissues and organs, causing variable clinical features such as acne, hirsutism, virilization, and reproductive dysfunction.²

In humans 40–50% are suffering from infertility. It affects approximately 7% of all men^3,4. Male infertility is occurred due to deficiencies in the semen, and semen quality is used as a surrogate measure of male fertility⁴. Various factors such as physical conditions like torsion, cryptorchidism, trauma, infections like mumps orchitis, epididymitis, naturals factors like excessive heat, radiation, use of different medications like anabolic steroids, alcohol, smoking, presence of diseases or history like thyroid or liver disease (abnormalities of spermatogenesis), diabetic neuropathy (retrograde ejaculation), radical pelvic or retroperitoneal surgery (absent seminal emission secondary to sympathetic nerve injury), or hernia repair (damage to the vas deferens or testicular blood supply), different behavioural variations like sexual habits, frequency and timing of intercourse and lastly hormonal changes, stress, loss of libido and headaches or visual disturbances may cause distinct level of infertility in males. A homeopathy formulation ‘FORCE DROP’ (FD) from NP Dutt and Sons, Kolkata is available in the market, scientific proof is required to establish the androgenic effect of this formulation. Anycompound having the affinity to the androgen receptor may show agonistic or antagonistic activity of androgen³. In the present study, the goal was to investigate the androgenic activity of the FD on male albino Wistar rats. The parameters like dry weights of male androgen dependent organs: Ventral Prostate, Seminal Vesicle, Cowper’s gland, Glans Penis and total body weight of test groups were compared with untreated control.

Although androgens are commonly thought of only as male sex hormones, females also have them, but at lower levels: they function in libido and sexual arousal. Also, androgens are the precursors to oestrogens in both men and women. In addition to their role as natural hormones, androgens are used as medications.⁵

In this preclinical evaluation we are used a homeopathy formulation, FORCE DROP (FD)which is the product of N. P. Dutt and Son, Kolkata. The product is composed of many types of herbal plants. Among of those herbal plants have directly and some have partially androgenic activity, the plants are Origanum vulgare⁶, Turnera diffusa⁷, Tribulus terrestris^8,9, Onosmodium virginianum¹⁰, Dolichosbiflorus¹¹, Crocus sativus¹², Withania somnifera^13,14,15, Salix nigra^16,17, Agnuscastus¹⁸, Avenasativa^,19,20. Other additives are Caramel coloured, Syrup flavoured, Purified Water and Alcohol.

MATERTIALS AND METHOD:

Chemicals:

Testosterone propionate (purity ≥ 97%) was procured from Loba Chemie Pvt. Ltd. India and ‘Forced Drop’ was supplied by N.P Dutt and Sons, Kolkata.

Animal treatment:

Observations were executed according to the OECD guidelines. Forty-one days old male wistar rats were purchased from CPCSEA authorized breeder and supervised under controlled conditions. Adult male wistar rats, weighing 100g, were used in this research. The rats were augmented with a reverse natural light-dark cycle and temperature of 22±2^◦C, a relative humidity of approximately 55%, as practiced in the Animal House of Netaji Subhas Chandra Bose Institute of Pharmacy. All the rats were maintained on standard rat pallets and tap water ad libitum. Five days after their arrival, the animals were castrated. Thereafter they were acclimatised to the laboratory conditions for 1 week until start the treatment. Their condition of health was also monitored during this period. Only healthy animals showing no clinical signs were used for the study. One day before the onset of treatment, castrated rats were assigned to treatment groups (n=6 each) by a randomized procedure in a way that mean body weights were identical in all groups. Six animals were taken in each group. Group 1 (Untreated) received no drug, Group 2 Testosterone Propionate (0.4mg/kg) i.p, Group 3 FD low dose (2.5ml/kg) p.o, Group 4 FD high dose (5ml/kg) p.o given respectively.

Testing for androgenicity:

The Homeo formulation FD was investigated for its androgenic property by Hershberger Bioassay as per OCED guidelines³. Twenty-four male albino wistar rats, aged 41 days, weighing around 100gm were selected. On 46^th day all the animals were orchiectomized and left for 7 days to acclimatize to the laboratory condition. Then all the rats were divided into 4 groups with 6 rats in each Group. Group 1: Untreated control Group 2 standard - Testosterone treatment (0.4mg/kg) i.p Group 3 FD low dose (2.5ml/kg) p.o, Group 4 FD high dose (5ml/kg) p.o given. All the animals were received treatment continuously for 10 days as per their treatment schedule. On 10th post treatment day all the animals were weighed, sacrificed and androgen dependent organs like Ventral Prostate (VP), Seminal Vesicles (SV), Cowper’s gland (CG), Glans Penis (GP) isolated and dry weights of all these organs were observed²¹.

Statistical analysis:^22,23

Results ware expressed as mean+S.E.M. The differences between experimental groups ware compared by one way Analysis of Variance (ANOVA) followed by Dunnett’s testand were considered statistically significant when P<0.05.

RESULTS AND DISCUSSION:

In this experiment FD was evaluated for its androgenic property by taking the standard Testosterone Propionate and found unique result. FD had shown significant (p<0.001) androgenic property while compared with the untreated control in dose dependant manner. When dry weights of the androgen dependent organs were calculated and compared, the dry weight of ventral prostate, seminal vesicle, cowper’s gland, glans penis was 6.33, 1.33, 3.83, 8.66 in untreated group whereas Testosterone and FD (5ml/kg) has shown significant increase in the dry organ weight i.e in testosterone treated group 16.66, 4.16, 12, 29.33 respectively and FD high dose has shown 13.66, 3.16, 9.5, 12.83. In the other hand FD (2.5/kg) has also shown increase in the weight of organs 11.33, 2.33, 7.66, 18.16 respectively. From the above findings it can be concluded Force Drop has shown significant increase in weight of the organs but in dose dependent manner. In most of the cases FD (5ml/kg) had shown results which resembles with standard testosterone readings.

Table 1: Dry weights of Androgen Sensitive Tissue of various groups.

Treatment

Ventral Prostate

Seminal Vesicle

Cowper’s Gland

Glans Penis

Untreated

6.33±

0.21

1.33±

0.21

3.83±

0.30

8.66±

0.33

Testosterone

16.66±

0.21***

4.16±

0.16***

12±

0.36***

29.33±

0.494***

FD (2.5ml/

kg)

11.33±

0.21***

2.33±

0.21*

7.66±

0.33***

18.16±

0.30***

(5ml/kg)

13.66±

0.21***

3.16±

0.16***

9.5±

0.22***

25.83±

0.40***

All the values are mean + SEM, n=6, p*<0.05, p***<0.001 compared with untreated control.

Figure 1: Dry weights of androgen dependent organs of various groups.

Testosterone is an anabolic hormone, it causes increase in body mass, primary and secondary sex organs in male body^24,25,26,27. Any drug that possesses androgen like activity must involve in growth same as testosterone. As far as the androgenic parameters are concerned, percentage increase in body mass in testosterone, FD (2.5ml/kg), FD (5ml/kg) groups had shown 158, 123, 145% respectively. Testosterone and FD (5ml/kg) groups had shown significant (p<0.05) increase in body mass when compared with untreated (121%).

Table-2: Total body weight increase of various groups after treatments

Treatment	Body weight on 0^th day	Body weight on 10^th day	% increase in body weight
Untreated	55.5+0.562	67.16+0.307	121
Testosterone	54.33+0.760	86.33+0.666***	158.89
FD (2.5ml/kg)	53.66+0.614	69.33+0.421*	123.61
FD(5ml/kg)	54+1.065	78.5+0.428***	145.37

All the values are mean + SEM, n=6, p*<0.05, p***<0.001 compared with untreated control.

Figure 2: Percent increase in body weight of 4 treatment groups of rats after androgenic study

CONCLUSION:

The Homeo formulation, FD was investigated for its androgenic activity by Hershberger bioassay model. Two doses of FD (2.5ml/kg and 5ml/kg) were studied with untreated control and TP as standard. Both the doses of FD shown significant effect compared to the untreated control and standard TP. In this present investigation FD has shown significant androgenic activity by measuring different parameters like the dry weight of androgen sensitive tissues i.e ventral prostate, seminal vesicle, cowper’s gland, glans penis. At the same time the percentage increase in body mass in testosterone, FD (2.5ml/kg), FD (5ml/kg) groups had shown 158, 123, 145 % respectively. Testosterone and FD (5ml/kg) groups had shown significant (p<0.05) increase in body mass when compared with untreated (121%).

At the end we can conclude Force Drop a marketed formulation by N.P Dutt and Sons has shown significant androgenic activity comparable with standard Testosterone and having enough potency to withstand in the market.

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors would like to thank N.P. Dutt and Sons providing the marketed valuable product ‘Force Drop’ to conduct my project work and their encouragement and moreover for their timely support and guidance till the completion of our project work.We wish to offer my sincere thanks to our principal of Netaji Subhas Chandra Bose Institute of Pharmacy, Dr. Arnab Samanta, for providing all necessary valuable help and support. And we also thank full to our lab assistants, they helped us lots throughout our research work.

REFERENCES:

1. Freyberger A, Ellinger-Ziegelbauer H, Krötlinger F.et al. Evaluation of the rodent Hershberger bioassay: testing of coded chemicals and supplementary molecular-biological and biochemical investigations. Toxicology. 2007 Sep 24;239(1-2):77-88. doi:10.1016/j.tox.2007.06.093

2. Lizneva D, Gavrilova-Jordan L, Walker W, et al. Androgen excess: investigations and management. Best Practice and Research Clinical Obstetrics and Gynaecology. 2016 Nov; 1(37): 98-118. doi: 10.1016/j.bpobgyn.2016.05.003

3. Soto AM, Calabro JM, Prechtl NV, Yau AY, Orlando EF, Daxenberger A, Kolok AS, GuilletteJr LJ, Le Bizec B, Lange IG, Sonnenschein C, et al. Androgenic and estrogenic activity in water bodies receiving cattle feedlot effluent in Eastern Nebraska, USA. Environmental health perspectives. 2004 Mar; 112(3):346. doi: 10.1289/ehp.6590

4. Moon HJ, et al OECD validation of phase 3 Hershberger assay in Korea using surgically castrated male rats with coded chemicals. Journal of Applied Toxicology. 2009 May 1; 29(4):350-355.doi: 10.1002/jat.1418

5. Carlson, Neil. Physiology of Behavior. Reproductive Behavior. 11th edition. Pearson.2012.

6. Kazemi P, et al. Androgenic Effect of Origanum vulgarel L.spp viride extract on Hormone Level of Pituitary - gonadal Axis in Mature Male Vistar Rats. J Arak Uni Med Sci. 2012; 14 (6) :89-96

7. Estrada-Reyes R, et al. Turnera diffusa Wild (Turneraceae) recovers sexual behavior in sexually exhausted males. Journal of Ethnopharmacology. 2009Jun25; 123(3): 423-9. doi:10.1016/j.jep.2009.03.032

8. Kumar DJ, Sonia K, Madhan R, Selvakumar K, PT K. Antiyeast, antioxidant and anticancer activity of Tribulus terrestris Linn and Bougainvillea spectabilis Linn. Research Journal of Pharmacy and Technology. 2011;4(9):1483-1489.

9. Abdulqawi LN, Quadri SA. Evaluation of Antibacterial and Antioxidant activities of Tribulus terrestris L. Fruits. Research Journal of Pharmacy and Technology. 2021;14(1):331-336.doi: 10.5958/0974-360X.2021.00061.5

10. Sex medicines list in homeopathy for firm erection, longer duration - Page 2 of 2 [Internet]. Homeopathy Remedies Online. 2022 [cited 11 March 2022]. Available from: https://homeomart.net/2016/06/13/sex-medicines-list-homeopathy-for-firm-erection-longer-duration/2/

11. MD(Ayu) D. Horse Gram Uses, Recipes, Dose, Side Effects [Internet]. Easy Ayurveda. 2022 [cited 11 September 2022]. Available from: https://www.easyayurveda.com/2016/02/29/horse-gram-uses-recipes-side-effects/#uses

12. Khayatnouri M, et al. The effect of saffron orally administration on spermatogenesis index in rat. Adv Environ Biol. 2011 Jun 1; 5(7):1514-21.

13. Kour P, Tiwari A. Medicinal Values of Secondary metabolites of Withania somnifera. Research journal of Pharmacy and Technology. 2018; 11(7): 3167-70.doi: 10.5958/0974-360X.2018.00582.6

14. Nasimi Doost Azgomi R, et al. Effects of Withania somnifera on reproductive system: a systematic review of the available evidence. BioMed Research International. 2018 Jan 24; 2018:1-17. doi:10.1155/2018/4076430

15. Vasavan SS, Sivanesan S, Jagadesan V. Antiperoxidative effect of Withania somnifera on lipid peroxidation and antioxidant capacity in the serum of nandrolone decanoate treated rats. Research Journal of Pharmacy and Technology. 2021 Feb 1; 14(2): 1065-8. doi:10.5958/0974-360X.2021.00191.8.

16. SBL Salix Nigra Mother Tincture Q: Find SBL Salix Nigra Mother Tincture Q Information Online | Lybrate [Internet]. Lybrate. 2022 [cited 11 September 2022]. Available from: https://www.lybrate.com/otc/sbl-salix-nigra-mother-tincture-q

17. Chevallier A. Encyclopedia of Herbal Medicine. Dorling Kindersley.2000

18. Saul S. Effects of vitex agnes castus on hormonal imbalances in Polycystic Ovary Syndrome. Int J Basic Clin Pharmacol. 2017; 6(8): 2015-55.doi: 10.18203/2319-2003.ijbcp20173295.

19. Saka VP, et al. Effect of avena sativa (Oats) on spermatogenesis and reproductive health. Journal of Endocrinology and Reproduction. 2016; 20(2): 118-25.doi:10.18311/jer/2016/15471

20. Rashid M, et al. Consumption of pure oats by individuals with celiac disease: a position statement by the Canadian Celiac Association Can J Gastroenterol. 2007 Oct; 21(10): 649–651 .doi:10.1155/2007/34059

21. Owens W, et al. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses. Environmental health perspectives. 2003 Sep; 111(12): 1559–1567. doi: 10.1289/ehp.5949

22. Saha K, Sen T, Majumdar M, Das H, Baisya O. Evaluation of Hydro-alcoholic extract of Corchorus capsularis seeds for its Anti-ulcer activity on Albino Wistar Rat. Research Journal of Pharmacy and Technology. 2022; 15(12): 5751-6. doi: 10.52711/0974-360X.2022.00970

23. Majumdar M, Samanta A, Roy A. Study of wound healing activity of different formulations of Nigella sativa seed extract. Research Journal of Pharmacy and Technology. 2016; 9(12): 2097-105.doi: 10.5958/0974-360X.2016.00427.3

24. Reddy YV, Reddy PS, Shivalingam MR. Protective effects of testosterone on cisplatin induced impairment of spermatogenesis and steroidogenesis in rats. Research Journal of Pharmacy and Technology. 2010;3(2):535-9.

25. Benmebrouk L, Kaddour A, Mohammedi L, Achouri A. Chemical detection of certain greenhouse gases by the LIDAR Technique.Asian Journal of Research in Chemistry. 2021; 14(3): 195-2. doi: 10.52711/0974-4150.2021.00035

26. Ibrahim MK, Tikamdas R, Kamal M, Nouh RA, Sayed M. Testosterone Undecanoate effects on behavior and Cognitive Functions in male swiss Albino mice exposed to Chronic Social Defeat. Research Journal of Pharmacy and Technology. 2020; 13(12): 6041-9.doi:10.5958/0974-360X.2020.01053.7

27. Revathi R, Julius A. A biological effect of sex hormone binding globulin and testosterone in polycystic ovary syndrome (PCOS) obese women. Research Journal of Pharmacy and Technology. 2017; 10(7): 2143-5.doi: 10.5958/0974-360X.2017.00377.8

Received on 03.04.2023 Modified on 22.07.2023

Research J. Pharm. and Tech 2024; 17(5):1991-1994.

DOI: 10.52711/0974-360X.2024.00315