Androgenic and Spermatogenic Potential of Justicia Carnea on Testosterone-Propionate Induced Benign Prostatic Hyperplasia in Male Wistar Rats

no UPH/CEREMAD/REC/MM116/037 by the University of Port Harcourt ethical Committee, University of Port Harcourt, Choba, Rivers State, Nigeria. Experimental Design BPH was induced in rats through daily subcutaneous injection (S.C) of testosterone-propionate (10mg/kg b.wt), adopting the method of [18, 19] with modification. Rats were randomized into six groups; Group 1 received feed and water only (normal control), Group 2 received TP injection only for durations of 11 days. Group 3 (Positive control) was induced with 10mg/kg b.wt TP for 11 days and treated with 5mg/kg b.wt Finasteride for 56 days . Group 4 (Low dose extract) received 500mg/kg b.wt of the plant extract for 56 days. Group 5 (medium dose extract) received 1500mg/kg b.wt of the plant extract for 56 days. Finally, Group 6 (high dose extract) received 2500mg/kg b.wt of the plant extract for 56 days. The animal grouping and treatment protocol is summarized in Table 1. Table 1. Experimental design Group Procedure 1 Food and water only 2 Induced with TP for 11 days and not treated 3 Induced with TP for 11 days and treated with 5mg/kg b.wt finasteride 4 Induced with TP for 11 days and treated with 500mg/kg AEJC for 56 days 5 Induced with TP for 11 days and treated with 1500mg/kg AEJC for 56 days 6 Induced with TP for 11days and treated with 2500mg/kg AEJC for 56 days Determination of Prostatic Index The body weight (BW) and prostate weight (PW) of each animal were recorded at the terminal sacrifice of the study. Following this, group mean body weights were calculated. For each individual study group animal, the prostatic index (PI) was calculated by dividing the prostate weight by the animal’s body weight i.e., Weight of prostate (in grams) Prostatic Index (PI) = Body Weight of Rat (in grams) Determination of Hormonal Parameters The rats underwent fasting 24 hours prior to sacrifice. Blood samples were carefully collected in plain bottles, spun for 15mins at 3000 rpm to obtain serum. The serum TEST, FSH and LH levels were estimated using Elabscience kit (Elabscience Biotechnology Co., Ltd., China) following manufacturer’s instructions. Semen Analysis The rat seminal fliud was extracted from the epididymis into a clean microscope slide, a few drops of normal saline was added and this was used for evaluation of sperm quality (ie sperm count, sperm motility and sperm morphology). The concentration of spermatozoa was determined using the improved Neubauer Improved Counting Chamber (Deep 1/10 mm, LABART, Germany). Statistical Analysis Data collected were analysed using one-way analysis of variance followed by Tukey’s multiple comparison. The significance of the data was assessed using a p-value of less than 0.05 and reported as Mean ± Standard Error of Mean . 3. Results Qualitative Phytochemical Analysis of the Leaves of Justicia carnea Qualitative Phytochemical Screening of the leaves is shown in Table 2. The phytochemicals present include alkaloids, flavonoids, saponins, phenols, steroids, tannins and terpenoids. Table 2: Qualitative Phytochemical Composition of the Leaves of J. carnea Phytochemical Status Alkaloids + Flavonoids ++ Phenols + Steroids + Tannins + Terpenoids + Therapeutic Effect of Justicia carnea Aqueous Extract on Hormonal Profile Figures 1- 3 show the effect of treatment with various doses of the aqueous extract of Justicia carnea on testosterone-propionate induced rats as presented. There was an increase in TEST level from 4.08 ± 0.04ng/ml in the normal control (group 1) to 8.72ng/ml for group 2 (negative control, which received TP only). At low, medium and high doses, the levels of TEST were observed to be significantly (p < 0.05) lowered when compared to group 2. The levels of LH and FSH for group 2 were significantly (p < 0.05) reduced when compared to the normal control (group 1). From 3.46 ± 0.04mμ/ml to 0.49 ± 0.12mμ/ml for LH. And for FSH, the values decreased from 9.90 ± 0.10mμ/ml (for group 1) to 2.70 ± 0.04mμ/ml (for group 2). Group 4, 5 and 6 which received low, medium and high doses of the plant extract had values which compared favourably with the normal control (group 1). Effect of Aqueous Extract of J.carnea on Semen Parameters Following exposure to testosterone propionate, there was a significant (p < 0.05) decrease in the total sperm count, sperm motility, activeness and viability of the negative control (group 2) when compared to the normal control (group 1). However, treatment with 500mg/kg b.wt, 1500mg/kg b.wt and 2500mg/kg b.wt (group 4, 5 and 6 respectively) significantly abated the pathological conditions by restoring the sertoli and leydig cells of the testes and the stroma cells of the prostate (figures 6- 10). This is demonstrated by the levels of LH and FSH for groups 4, 5 and 6 which compared favourably to the normal control, group 1. Effect of Aqueous Extract of J.carnea on Prostate Index Relative prostate weight is normally used to evaluate the growth of BPH. The rats in the TP- induced BPH group recorded a significantly (p <0.05) increased weight of prostate (1.65 ± 0.05g) when compared to the normal control (0.89 ± 0.01g). Similarly, the prostate index for the negative control, group 2 (8.0±0.12x10−3) was significantly (p < 0.05) elevated when compared to the normal control (4.2±0.20x10−3). The standard drugs treated groups and the extract treated groups had values which compared favourably to the normal control (group 1) as presented in figure 5. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 2 4 6 8 10 Testosterone (ng/ml) Groups Concentration ✱✱✱✱ ✱✱✱✱ ✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱✱✱✱✱ ✱✱✱✱ ✱ Fig 1. Graph showing the testosterone concentrations of animal model induced with TP and treated with AEJC at different concentrations. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 2 4 6 8 LH (ng/ml) Groups Concentration ✱✱✱✱ ✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱ ✱✱✱✱ ✱✱✱✱ Fig 2. Graph showing the luteinizing hormone of animal model induced with TP and treated with AEJC at different concentrations. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 5 10 15 FSH (ng/ml) Groups Concentration ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ Fig 3. Graph showing the follicle stimulating hormone of animal model induced with TP and treated with AEJC at different concentrations. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 1 2 3 4 Prostate Wt (g) Groups Concentration Fig 4. Graph showing prostatic weight of animal model induced with TP and treated with AEJC at different concentrations. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0.000 0.005 0.010 0.015 0.020 Prostate Index Groups Concentration Fig 5. Graph showing prostatic index of animal model induced with TP and treated with AEJC at different concentrations. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 50 100 150 Motility (%) Groups Concentration ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱ ✱✱✱✱ ✱✱✱✱ Fig 6. Showing the effect of AEJC on semen motility of animal model induced with TP. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 20 40 60 80 100 Active (%) Groups Concentration ✱✱✱✱ ✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱ ✱✱✱ ✱✱✱✱ Fig 7. Showing the effect of AEJC on the active cells of animal model induced with TP. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 5 10 15 Sluggish (%) Groups Concentration ✱✱ ✱✱✱✱ ✱✱ ✱ ✱✱✱ ✱ Fig 8. Showing the effect of AEJC on the sluggish cells of animal model induced with TP. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 50 100 150 Dead Cell (%) Groups Concentration ✱✱✱✱ ✱✱✱✱ ✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱ ✱ ✱✱✱✱ ✱✱ Fig 9. Showing the effect of AEJC on dead cells of animal model induced with TP. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. N-Control -ve Control 5mg/kg Ref Drug 500mg/kg 1500mg/kg 2500mg/kg 0 10 20 30 TSC (x 106/ml) Groups Concentration ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ Fig 10. Showing the effect of AEJC on the total sperm count of animal model induced with TP. Values are mean ± SEM. All comparison with superscripts is significant at (p≤0.05). Four superscripts (****) indicated adjusted p-value less than 10-4, three superscripts (***) indicated adjusted p-value is 10-4, two superscripts (**) indicated adjusted p-value 10-3 and one superscripts (*) indicated adjusted p-value 10-2. 4. Discussion Male fertility requires the cooperation of the different organs of the male urogenital system each carrying out its assigned function. Through their interactions, the testes (which contain germ cells, Sertoli cells and Leydig cells), the epididymis and the male accessory glands (prostate, seminal vesicles and bulbourethral glands) simultaneously contribute to the production of the human seminal fluid. [5, 20] in their review of the role of the prostate in male fertility, health and disease reported that male fertility is controlled by a Zn2+ dependent short circuit of the Krebs cycle within the prostate epithelial cells. Homeostasis and pathophysiological status of the prostate epithelium depends on the ultra-cellular androgen- dependant accumulation of Zn2+, citrate and Kallikreins and the semenogelin-enriched seminal vesicle secretion. The proliferative disorders of the prostate gland will negatively impart male fertility function. The burden of clinical BPH still remains a significant health concern especially among elderly men worldwide [21]. This investigation sought to evaluate the anti-proliferative effects of Justicia carnea aqueous extracts on the development of BPH using a TP-induced model in rats. The rats with TP-induced BPH displayed hormonal dysregulation and elevated prostate weights. The semen parameters also were negatively imparted with TP-treatment. The total sperm count , viable sperm cells and motility of the sperm cells were also low in the TP- only treated groups. This study however reveals that J. carnea aqueous extract ameliorated the hormonal dysregulation, lowered the induced increment in prostate weight and improved the sperm quality parameters. Testosterone is involved in the pathogenesis of BPH and plays an important role in the development of male reproductive organs [6]. The serum concentration of testosterone may vary with age [22]. Testosterone is converted to dihydrotestosterone in the prostate, hair follicles and testes via the enzymatic action of 5 α-reductase. DHT has a greater affinity towards androgen receptors when compared to that of testosterone and other adrenal androgens [23]. Elevated levels of testosterone in the induced groups have direct correlation with increased level of DHT and hence the prostatic enlargement noticed in the current study. The reduction in the testosterone concentration for groups treated with the plant extract suggests the plant extract had the potential to ameliorate prostatic proliferation. This can be posited as having followed the same mechanism as the α -blocker agents such as finasteride. Increased levels of LH and FSH is an indication of the plant extract potential to restore and improve sexual function and hence fertility [24]. Increased luteinizing hormone will contribute to the increased production of testosterone by the interstitial cells besides the other mechanism for its production via increased cholesterol synthesis [25]. With increased FSH, spermatogenesis will be enhanced by the sertoli cells leading to the increased production of viable and motile spermatozoa. Several research reports have proven that an increase in the prostate is a vital indicator of the development of BPH [19, 26]. The findings of our study agree with these reports. Significant enlargement in the size of the prostate gland was observed in testosterone propionate only. An increase in the size of the prostate results in urethral canal constriction, resulting in partial or complete urinary canal obstruction. According to [27], increased relative prostate weight is used as one of the significant markers indicating the development of BPH. BPH is characterized by epithelial and stromal hyperplasia of the prostate, which results in an increase in prostate weight. When the prostate dilates, it results in the constriction of urethral canal, causing partial or complete obstruction. Treatment of BPH will therefore correspond with the reduction in prostate weight. Several studies have evaluated the effect of various natural products in the treatment of BPH by investigating the volume/size of the prostate. Treatment with the aqueous extract of Justicia carnea in the current study at the both the low, medium and high doses showed a significant reduction in the prostate weight of BPH-induced rats. The deduction is that there was a positive impact on the BPH condition and brought about a restoration of normal physiological function. The high death rate of the sperms cells observed in the groups administered with testosterone propionate only could be attributed to the inflammatory response elicited by administration of TP. This also corresponded with the observed reduction in total sperm death recorded. Other works have stated that factors which damage the testes and imparts semen parameters could hamper fertility [28]. In addition, the previous report and several others [29] have associated sperm count, sperm motility and morphology with spermatogenic and androgenic properties of plant extracts. The extract treatment groups in our investigation, showed an improved sperm count, sperm motility and morphology in comparison with the negative control group. The ameliorative properties recorded our observations in this study may be attributed to the phytochemical constitution present in the plant which could have significantly improved the spermatogenic and androgenic activities. 5. Conclusion The results of this investigative study revealed that aqueous extract of Justicia carnea leaves had a curative effect on the prostate and testis alteration caused by testosterone propionate- induced benign prostatic hyperplasia by restoring and bringing a balance to hormonal control. A restoration of normal spermatogenic parameters also gives credence to the possible androgenic and spermatogenic potential of the plant extract. References [1] Eruotor, H. O., Ezendiokwere, E. O., & Oghenemavwe, E. L. (2025). Impact of Testosterone Propionate on Semen Quality and Endocrine Profile in Male Wistar Rats. Asian Journal of Research in Medical and Pharmaceutical Sciences, 14 (2):93-104. [2] Kefer, J. C., A. Agarwal & Sabanegh. E. (2019). Role of antioxidants in the treatment of male infertility. International journal of Urology, 16 (5):449–457. [3] Agarwal, A. (2023). Significance of oxidative stress and sperm chromatin damage in male infertility. Male fertility and lipid metabolism, 13: 157–183, 2003. [4] Agarwal, A & Saleh, R.A. (2021). Oxidative stress and DNA damage in human sperm: The cleeland clinic story. [5] Verze, T., Cai, P & Lorenzetti, M. (2016). The role of the prostate in male fertility, health and disease. Nature Reviews Urology, 13(7):379–386. [6] Xin, M. J. Kwon, J. W. Lee, K. S. Kim, H. Chen, M. G. Campos, V., & Cao, F. (2019). Gamma irradiated rhodiola sachalinensis extract ameliorates testosterone-induced benign prostatic hyperplasia by downregulating 5-alpha reductase and restoring testosterone in rats. Molecules, 24(21):3981. [7] Onyegeme-Okerenta, B.M, Anacletus, F.C., & Omeje, H.C. (2023). Annona muricata Leaf Extract Modulates Chloramphenicol-Induced Lymphoma in Female Wistar Rats. Tropical Journal of Natural Product Research, 7(5):3042-3049. [8] Adnan, D.S. Jairajpuri, M. Chaddha, M.S. Khan, D.K. Yadav, E.A., & Mohammad, T (2022) Discovering tuberosin and villosol as potent and selective inhibitors of AKT1 for therapeutic targeting of oral squamous cell carcinoma. Journal of Personalised Medicine, 12 (7): 1087. [9] Tong, Y & Zhou. Y.R. (2020). Review of the roles and interaction of androgen and inflammation in benign prostatic hyperplasia. Mediators of Inflammation, 20(1):7958316 . [10] P. S. Cooke, M. K. Nanjappa, C. Ko, G. S. Prins, and R. A. Hess (2017). Estrogens in male physiology. Physiological reviews, 97(3):995–1043, 2017. [11] Egharevba, H.O., Ibrahim, J.A., Kassam, C.D. and Kunle, O.F. (2015) Integrating traditional medicine practice into the formal health care delivery system in the new millennium–the nigerian approach: a review. Int J Life Sci, 4(2):120–8. [12] B. L. Rice. Mysore and Coorg. A Gazetteer Compiled for the Government of India. BoD–Books on Demand, 2024. [13] Shah, M.D., Yong, Y.S. and Iqbal, M (2014). Phytochemical investigation and free radical scavenging activities of essential oil, methanol extract and methanol fractions of nephrolepis biserrata. International Journal of Pharmaceutical and Pharmacological Science, 6(9):269–277. [14] Malati,S.N., Rao, G.N and Kumar, P.R. (2022). study on antimicrobial activity of nephrolepis biserrata from eastern ghats of india, visakhapatnam district, andhra pradesh. Journal of Pharmacy and Biological Sciences, 17(4):1–6. [15] Oyawaluja, B.O., Oyawaluja, A.A., Akinyimika, D.E., Odukoya, O.A., & Coker, A. H. (2024). Antioxidant profiling, phytochemical investigation and pharmacognostic evaluation of nephrolepis biserrata (sw.) schott (nephrolepidaceae). Tropical Journal of Phytochemistry and Pharmaceutical Sciences, 3(2):208–215. [16] M. Duenas-Lopez. Nephrolepis biserrata (giant sword fern), 2022. URL http://www.cabi.org/ISC/datasheet/36 [17] J. B. Harborne. Phytochemical methods. Chapman and Hall Publications, London, 2nd edition, 1984. [18] G. E. Trease & M. C. Evans. Textbook of Pharmacognosy. Bailliere, Tindall, London, 13th edition, 1989. [19] Li, J., Tian, Y., Guo, S., Gu, H., Yuan, Q. & Xie. X. (2018). Testosterone-induced benign prostatic hyperplasia rat and dog as facile models to assess drugs targeting lower urinary tract symptoms. PloS one, 13(1):e0191469. [20] Huang, Y., Chen, Y., Zhou, X., Wu, X., Hu, E & Jiang, E. (2017). Inhibition effects of chlorogenic acid on benign prostatic hyperplasia in mice. European Journal of Pharmacology, 809:191–195. [21] Gilany, K., Minai-Tehrani, E. Savadi-Shiraz, H. Rezadoost, B & Lakpour, N. (2018). Exploring the human seminal plasma proteome: an unexplored gold mine of biomarker for male infertility and male reproduction disorder. Journal of reproduction infertility, 16(2):61. [22] Yu, Z.J., Yan, L.J., Xu, F.H., Chao, H.C., Deng, L.H., Xu, X.D & Zeng, T. (2020). Efficacy and side effects of drugs commonly used for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia. Frontiers in Pharmacology, 11:658. [23] Izumi, K., Mizokami, A., Lin, W.J., Lai, K.P & Chang, C. (2019). Androgen receptor roles in the development of benign prostate hyperplasia. The American journal of pathology, 182(6):1942–1949. [24] Hwangbo, H., Kwon, D.H., Choi, E.O., Kim, Y.M., Ahn, K.I., Ji,S.I & Choi, Y.H. (2018). Corni fructus attenuates testosterone induced benign prostatic hyperplasia by suppressing 5-reductase and androgen receptor expression in rats. Nutrition Research and Practice, 12(5):378–386, 2018. [25] Shah, J. (2002). Erectile dysfunction through the ages. BJU international, 90(4):20 [26] Kayode, O.T., & Yakubu, M.T (2017). Parquetina nigrescens leaves: chemical profile and influence on the physical and biochemical indices of sexual activity of male wistar rats. Journal of integrative medicine, 15(1):64–76. [27] Akanni, O.O., Owumi, S.E., Olowofela, O.G., Adeyanju, A.A., Abiola, O.J & Adaramoye, O. A. (2020). Protocatechuic acid ameliorates testosterone-induced benign prostatic hyperplasia through the regulation of inflammation and oxidative stress in castrated rats. Journal of Biochemical and Molecular Toxicology, 34(8):e22502. [28] Babu, S. V., Veeresh, A. A., Patil, B. & Warke, Y.B. (2010). Lauric acid and myristic acid prevent testosterone induced prostatic hyperplasia in rats. European journal of pharmacology, 626(2-3):262–265. [29] Emmanuel, O.Y., Obasi, K.K., & Lawal, I. (2018). Spermatogenic and spermatotoxic effects of telfairia occidentalis aqueous leaves extract in adult male wistar rats (rattus novergicus). Toxicology Reports, 5:954–958.