Anti-fertility and Sexual Behavioral Effects of Sub-Acute Administration of Carpolobia Alba (G. Don) Root Extract in Male Wistar Rats: an Immunohistochemical Study

Anti-fertility and Sexual Behavioral Effects of Sub-Acute Administration of Carpolobia Alba (G. Don) Root Extract in Male Wistar Rats: an Immunohistochemical Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-fertility and Sexual Behavioral Effects of Sub-Acute Administration of Carpolobia Alba (G. Don) Root Extract in Male Wistar Rats: an Immunohistochemical Study Gideon Olamilekan Oluwatunase, Olatoye Jubril Otulana, Bamidele Ponle Fakunle, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6953636/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Over 80% of the population in Africa and Asia rely on traditional medicine as their primary form of health care. For centuries, plant-based aphrodisiacs have been used to enhance sexual performance. In sub-Saharan Africa, especially Nigeria, Carpolobia alba (G. Don) is widely used for its perceived sexual stimulatory effects, despite limited scientific evidence on its impact on male fertility. In this study, we investigated the effects of sub-acute administration of methanol root extract of Carpolobia alba on sexual behaviour and testicular immunoexpression of B-Cell lymphoma-2 (Bcl-2 )and Proliferating Cell Nuclear Antigen (PCNA) in adult male Wistar rats. Methods: Twenty adult male rats (weighing 200g-250g) were randomly divided into four groups consisting of ten animals each. Each group was treated daily with distilled water, 100, 200 and 400mg/kg body weight respectively of methanol root extract Carpolobia alba G.Don for 30 days. The animals were anesthetized using Isoflurane via an induction chamber. Mating behavioral assessment was done by direct observation and use of camera, testicular B-Cell lymphoma-2 (Bcl-2 )and Proliferating Cell Nuclear Antigen (PCNA) Immunoexpression was done using immunohistochemical techniques. Results: Our result shows significantly (P < 0.05) increased sexual activity and mating behavior across treated groups. However, testicular sections revealed a weak immunoexpression of Bcl-2, indicating increased apoptotic activity, and strong PCNA expression, indicating increased cell proliferation. Conclusion: Despite its traditional use as an aphrodisiac, Carpolobia alba exhibits potential anti-fertility effects following sub-acute exposure, as evidenced by disrupted testicular cell regulation and structural integrity. These findings raise important concerns about its long-term reproductive safety and need for further investigation. Carpolobia alba sub-acute aphrodisiacs anti-fertility immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The desire to increase sexual arousal, pleasure and potency has existed since the beginning of life. Sexual response in male and female reflects a dynamic balance between excitatory and inhibitory signals of the autonomic nervous system within the external genitalia and throughout the central nervous system (Goldstein, 2022 ). An aphrodisiac is any natural chemical or agent that enhances sexual desire, attractiveness, pleasure, behavior, and performance (Sandroni, 2001 ). It can be derived from animal, plant, spices or mineral (Abdullahi and Tukur 2013 ). Aphrodisiac chemicals are overused and recreationally used for sexual enhancement, even though their primary indication is erectile dysfunction (Christopher and Cindy, 2011 ). The rate of consumption of natural aphrodisiacs especially among young men has increased in recent times. Furthermore, there is limited scientific evidence supporting the efficacy and safety of natural aphrodisiacs which is worrisome as it could lead to abuse of aphrodisiacs (Manortey et al, 2018 ). A common aphrodisiac traditionally used in Nigeria is Carpolobia alba . Carpolobia alba grows as a shrub or small tree which is 3 to 6 metres (9.8 to 19.7 ft) tall. It has querulous branches or short pubescent. Its leaves are membranous or slightly leathery. This type of plant belongs to the milkwort family (Polygalaceae) and is native to Western Tropical and Central Africa's rainforests, forest edges, and savanna parks at elevations lower than 400 meters (1,300 feet). Carpolobia alba is a popular natural recreational aphrodisiac plants commonly used by pygmies of the forest region (Ipassa of Garbon, South-Cameroon; and the Yoruba, Efik, Ibo,and Ijaw peoples of Nigeria, ) and other part of sub-Sahara Africa (Nwidu et al., 2015 ). It is commonly called Cattle stick in English, Egbo Oshunshun in Yoruba, Angalagala in Igbo, Ikpafum in Ibibio (Nwidu et al., 2015 ). The root of Carpolobia alba has long been used to treat male infertility, increase virility, and improves male fertility. It has been reported to have aphrodisiac and androgenic effects. (Manfo et al., 2014 ). The apoptotic pathway in the mitochondria is controlled by proteins from the BCL-2 family, which controls cell survival and death (Czabotar and Garcia-Saez, 2023 ). The BCL-2 is located in the outer membrane of the mitochondria and maintains cell survival by counteracting the effects of the proapoptotic protein BAX, thereby preserving mitochondrial membrane integrity. (Morris et al., 2021 ). BCL-2 plays a crucial role in regulating apoptosis. The expression of BCL2 is tightly controlled and disturbance of BCL2 has been implicated in various disease conditions including cancer (Adelakun et al., 2024 ). Proliferating Cell Nuclear Antigen (PCNA) is a marker for cell proliferation and is involved in DNA synthesis, DNA methylation, chromosome recombination, RNA transcription and nucleic acid metabolism (Zhao et al., 2018 ). Therefore, changes in PCNA expression are used as a marker to evaluate cell proliferation. Spermatogenesis a complex process by which spermatogonia develops into mature sperm cell. It involves continuous cell division and proliferation. Thus, PCNA expression levels in the testis is a standard marker of spermatogenic cell proliferation and can be used also to assess the state of spermatogenic cell proliferation (Farang et al. , 2021). Apoptosis is accompanied by proliferation. Therefore, the positive expression of PCNA indicates increased proliferation of spermatogenic cells in response to apoptotic cell death. A study carried out by Zhao et al. ( 2018 ) reported that positive expression of PCNA in the testes correlates with spermatogenesis dysfunction in fluoride-treated rat. The aim of this study is to investigate the effects of sub-acute administration of of Methanol root Extract of Carpolobia alba (G. Don) on the sexual behavior and the level of expression of BCL-2 and PCNA in the testicular tissue of male Wistar rat. MATERIALS AND METHOD Plant Harvest and Extraction The Carpolobia alba root was obtained from Ayede Ogbese in Akure North Local Government, Ondo state, Nigeria and it was authenticated in the herbarium of Department of Plant Biology and Biotechnology, University of Medical Sciences Ondo state, Nigeria with voucher number UNIMED P.B.T.H No 034. After air drying, the Carpolobia alba root (2.20kg) was pulverized into powder after which Carpolobia alba root was extracted using Soxhlet with pure methanol as the solvent. After filtering the methanol containing the extract, the solvent was vacuum-distilled in a rotary evaporator at 4 o C. To verify that all remaining solvent was gone, the remaining extract was lastly dried for two hours at 30 o C in a vacuum oven. 90.22g (1.69% yields) of the powdery mass were obtained which was stored at room temperature for the study. Distilled water was used to prepare a fresh solution of the extract. ANIMAL EXPERIMENTATION AND DESIGN Twenty male Wistar rats (weighing 200-250g) were procured from the Animal facility of University of Medical Sciences, Ondo State, Nigeria. The rats were acclimatized for two weeks before the experiment begins. The experimental animals were accommodated in standard laboratory conditions. The animals were exposed to light and dark cycle of 12 hours and fed rat show and water ad libitium . Twenty adult Wistar rats were randomized into four groups consisting of five rats each. Group 1 (control) received only distilled water; Group 2,3 and 4 received 100mg/kg, 200mg/kg and 400mg/kg body weight respectively of methanol root extract of Carpolobia alba for 30 days. Assessment of Mating behaviour parameters The primed female rats that exhibited profound receptivity were introduced into the cages containing male rat in ratio 1:1 after which the following mating behavior were observed directly from the cage sides and via camera for 30 minutes (Yakubu and Jimoh, 2014): The following mating behavioural parameters were assessed: Mount frequency (MF) (number of mounts with or without intromission from the time of introduction of the female until ejaculation); Intromission frequency (IF) (number of vaginal penetrations during the observation period); Ejaculatory frequency (EF) (number of ejaculatory recorded during the time of observation); Mount latency (ML) (time interval between the introduction of the female and the first mount by the male); Ejaculation latency (EL) (time interval between the first intromission and ejaculatory); Intromission latency (IL) (time interval between the introduction of the female and the first intromission by the male). The assessment was done around 1:30pm local time to avoid interference of with the natural increase in libido in male rat in the morning (Kenmogne et al ., 2016). IMMUNOHISTOCHEMISTRY ASSAY Paraffin embedded sections where deparaffinized with xylene, and rehydrated through descending grades of ethanol (100%, 95%, 70 % ethanol) and taken to water. Heat-mediated antigen retrieval was performed using a citrate-based antigen unmasking solution, pH 6.0 (Vector ® , Burlingame, CA, USA; #H3300) in a steamer for 30 mins. Thereafter, it was Wash sections in PBS for 2 minutes. Endogenous peroxidase blocking in 0.3 % hydrogen peroxide solution in PBS for 10 minutes and Wash sections in PBS for 2 minutes. Sections were then incubated at room temperature for 2 hours in primary rabbit antibodies diluted in a universal antibody diluent and blocking reagent, UltraCruz® Blocking Reagent (Santa Cruz, USA). The sections were subsequently incubated with specific primary antibodies against PCNA (Cell Signalling, USA; #13110) and Bcl-2 (Novus Biologicals, USA; NB100-56098) overnight at 4 °C in a humidified chamber. Section was washed in PBS for 5 minutes, incubated in ImmPRESS™ HRP Anti-Rabbit IgG (Peroxidase) Polymer Reagent, made in horse for 30 mins, Wash in PBS for 5 minutes twice. Colour is developed with DAB Peroxidase (HRP) Substrate Kit (Vector ® Labs, USA). Sections were rinsed well in tap water. Sections were counter-stained in hematoxylin and observed under a microscope. Quantification of Immunoexpression The expressions for BCL-2 and PCNA in the testicular tissue were quantified using Image J software version 1.54p. The data obtained were statistically analyzed. Statistical Analysis The analysis was done with GraphPad Prism software version 10.1. Both descriptive and inferential statistics (one-way ANOVA) were done on all obtained data. Turkey's multiple comparison was used to test for statistical significant difference between control and experimental groups. The data were represented as Standard Error of Mean and the level of differences were considered significant at p<0.05. Ethical Consideration Ethical approval was obtained from Olabisi Onabanjo University’s research Ethic Committee with reference number: OOU/SCIENG/EC/0009/240924. RESULT Assessment of mating behavior The mounting (MF), Intromission (IF), and Ejaculation (EF) frequencies were significantly increased (P< 0.05) in the experimental groups when compared to the control (fig 1,2 and 3). This means that animals that received the extract had more sexual motivation, penile thrusting and sexual climax than the control group. The mounting latency (fig 4) and intromission latency (fig 5) was significantly reduced (P< 0.05) in the experimental group when compared to control. This mean the animals that received the extract took lesser time to make their first mount and penile thrust after the introduction of a receptive female rat. On the contrary, the ejaculation latency (fig 6) was significantly increased in the experimental group when compared to the control. This implies that animals that receive the extract took longer time before ejaculation during mating. IHC Assay of BCL-2 Expression in the Testicular Tissue IHC Assay of PCNA Expression in the Testicular Tissue DISCUSSION In this study the effects of sub-acute administration of Methanol root Extract of Carpolobia alba (G. Don) on mating behavior and testicular PCNA and BCL-2 immunoexpression in male Wistar rat was investigated. The result obtained from the mating behavioral assessments shows that there is significant increase in mounting frequency (MF), intromission frequency (IF) and ejaculation frequency (EF) in all the experimental groups (100,200 and 400mg/kg groups) when compared to control. An increase in MF, IF, and EF reflects enhanced sexual motivation. This implies that Carpolobia alba increases sexual motivation of the male rats to make with the female rat. This result agrees with study reported by (Kenmogne et al., 2016 ) who reported that aqueous extract of Carpolobia alba significantly increase mount frequency. This suggests that Carpolobia alba significantly increase sexual drive in male Wistar rat. The mounting and intromission latencies were significantly reduced in the experimental groups compared to the control. This mean the animals that received the extract took lesser time to make their first mount and vaginal thrust after the introduction of a receptive female rat. The reduction in mount and intromission latencies may also connote increased orientation activities towards the receptive females and stronger sexual motivation (Yakubu and Jimoh, 2015). On the contrary, the ejaculation latency (Fig. 6 ) was significantly increased in the experimental group when compared to the control. The prolonged EL in Carpolobia alba treated group suggests that the extract enhanced sexual intercourse for sufficiently desired time. This could also suggest the activation of nitric oxide and regulating the release and serotonin. B-cell lymphoma 2 (BCL2) is an anti-apoptotic protein that is important and crucial in regulating programmed cell death (apoptosis). It functions by inhibiting the activity of pro-apoptotic proteins, thereby promoting cell survival. The result from this study shows that experimental groups treated with Carpolobia alba exhibited weak expression of BCL2 compared to the control group. This suggests that the plant extract may have a pro-apoptotic effect, potentially leading to increased cell death in the testes. The Bcl-2 immunoexpression were reduced in the experimental group but the reduction was only significant at 400mg/kg which suggest a dose dependent effects. This indicates that the pro-apoptotic effects of the extract which may be exacerbated by higher doses. This result agrees with the study reported by Sarica et al (2001) who reported bcl2 downregulation in apoptotic cells. Proliferating Cell Nuclear Antigen (PCNA) is a critical marker for cell proliferation and is involved in DNA replication and repair. PCNA protein expression serve as a biomarker of proliferation of spermatogenic cells and can be used to assess the proliferation state of spermatogenic cells. The result from this study showed significantly increased PCNA expression (at 100 and 400mg/kg) in the Carpolobia alba-treated group compared to the control group. This suggests enhanced spermatogenic activity in response to the pro-apoptotic effects of extract. Apoptosis is accompanied by proliferation. During spermatogenic cell mitosis and differentiation, PCNA as a cell proliferation factor is involved. PCNA not only regulate cell proliferation but also act as cell proliferation markers. This result is consistent with the work reported by Zhao et al. ( 2018 ) who documented positive immunoexpression of PCNA in fluoride-induced testicular toxicity. Also, similar result was documented by Oluwatunase et al. ( 2024 ) who reported that sub-acute treatment of Carpolobia alba induces apoptotic spermatogenic cell death, oxidative stress and inhibits synthesis of reproductive hormones. In conclusion, sub-acute administration of methanol root Extract of Carpolobia alba (G. Don) improves sexual behavior and induces spermatogenic apoptotic cell death. This implies that while the extract enhances sexual desire, motivation and activities, it could have deleterious effects on the spermatogenic cells which lead to infertility. 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Clin Auton Res 11(5):3037 Sarıca K, Bakır K, Erbag A, Yagcı F, Uysal O, Uçak R (2001) Unilateral Testicular Torsion: Evaluation of bcl-2, p-53 and PCNA Expression in Contralateral Testes. Urol Int 2001;66:94–99 Yakubu MT, Jimoh RO (2014) Carpolobia lutea roots restore sexual arousal and performance in paroxetine-induced sexually impaired male rats. Int J Androl 12:90–99 Zhao W, Wang H, Liu J, Tan P, Luo X, Zhu S, Chen X, Zhou B (2018) Positive PCNA and Ki-67 Expression in the Testis Correlates with Spermatogenesis Dysfunction in Fluoride-Treated Rats. Biol Trace Elem Res. 2011-018-1338-6 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6953636","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":474975392,"identity":"27c9d370-3251-4718-832e-7402859dbbb4","order_by":0,"name":"Gideon Olamilekan 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Moronke Adeyanju","email":"","orcid":"","institution":"Department of Biochemistry, Olabisi Onabanjo University, Ago-Iwoye, Ogun state, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Muinat","middleName":"Moronke","lastName":"Adeyanju","suffix":""},{"id":475006927,"identity":"d9da8807-4b5c-43fc-8900-1f7039729686","order_by":8,"name":"Samson Adetope Idepefo","email":"","orcid":"","institution":"Department of Anatomy, University of Ibadan, Oyo state, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Samson","middleName":"Adetope","lastName":"Idepefo","suffix":""},{"id":475006928,"identity":"266fe060-a40c-4beb-8430-e60b10826048","order_by":9,"name":"Omamuyovwi Meashack Ijomone","email":"","orcid":"","institution":"Department of Anatomy, University of Medical Sciences, Ondo state, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Omamuyovwi","middleName":"Meashack","lastName":"Ijomone","suffix":""}],"badges":[],"createdAt":"2025-06-23 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Bars represent Mean ± SEM (n=5) (one-way ANOVA followed by Tukey \u003cem\u003epost hoc test\u003c/em\u003e).\u0026nbsp; \u003csup\u003e*\u003c/sup\u003ep\u0026lt; 0.05 relative to control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/f7f831da251b4db8500ba5f0.png"},{"id":85303890,"identity":"bfa40c63-60aa-4e4b-acd2-3badf67f3894","added_by":"auto","created_at":"2025-06-24 12:31:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116396,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of administering \u003cem\u003eCarpolobia alba\u003c/em\u003e root methanol extract for 30 days on the mounting latency (D), ejaculation latency (E), intromission latency (F) of male rat. Bars represent Mean ± SEM (n=5) (one-way ANOVA followed by Tukey \u003cem\u003epost hoc test\u003c/em\u003e).\u0026nbsp; \u003csup\u003e*\u003c/sup\u003ep\u0026lt; 0.05 relative to control.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/130fd1a8b1fc5d053f3aa95b.png"},{"id":85303902,"identity":"d20bd1b9-3a8d-4e15-abf6-5c4496f35d9b","added_by":"auto","created_at":"2025-06-24 12:31:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":446337,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of administering \u003cem\u003eCarpolobia alba\u003c/em\u003e root methanol extract for 30 days on testicular Bcl2 immunoexpression in male rat.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/45962c90d3f16316865b26a0.png"},{"id":85303887,"identity":"97b77f6a-ee4b-4a92-9b71-9c0a9fe4ae51","added_by":"auto","created_at":"2025-06-24 12:31:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31651,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of bcl-2 immunoexpression. Bars represent Mean ± SEM (n=5) (one-way ANOVA followed by Tukey \u003cem\u003epost hoc test\u003c/em\u003e).\u0026nbsp; \u003csup\u003e*\u003c/sup\u003ep\u0026lt; 0.05 relative to control.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/a2d6aaa5184ba5c8febb46dc.png"},{"id":85306069,"identity":"12c0235f-c732-48bd-acf6-9c439bddaed2","added_by":"auto","created_at":"2025-06-24 12:47:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":498037,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of administering \u003cem\u003eCarpolobia alba\u003c/em\u003e root methanol extract for 30 days on testicular PCNA immunoexpression in male rat.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/211db56a32a33f255ca444ac.png"},{"id":85305556,"identity":"560cb53c-470c-47e5-874e-b884861b2ac5","added_by":"auto","created_at":"2025-06-24 12:39:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31731,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of bcl-2 immunoexpression. Bars represent Mean ± SEM (n=5) (one-way ANOVA followed by Tukey \u003cem\u003epost hoc test\u003c/em\u003e).\u0026nbsp; \u003csup\u003e*\u003c/sup\u003ep\u0026lt; 0.05 relative to control.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/44fd5eb1bd9b52ca97f39041.png"},{"id":85307483,"identity":"b11144c5-31db-4f9e-ba89-4c89eeef6090","added_by":"auto","created_at":"2025-06-24 13:03:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1991383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6953636/v1/f90f83ad-7b1d-40a6-a8ce-ac58b654b5eb.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAnti-fertility and Sexual Behavioral Effects of Sub-Acute Administration of Carpolobia Alba (G. Don) Root Extract in Male Wistar Rats: an Immunohistochemical Study\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe desire to increase sexual arousal, pleasure and potency has existed since the beginning of life. Sexual response in male and female reflects a dynamic balance between excitatory and inhibitory signals of the autonomic nervous system within the external genitalia and throughout the central nervous system (Goldstein, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). An aphrodisiac is any natural chemical or agent that enhances sexual desire, attractiveness, pleasure, behavior, and performance (Sandroni, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It can be derived from animal, plant, spices or mineral (Abdullahi and Tukur \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Aphrodisiac chemicals are overused and recreationally used for sexual enhancement, even though their primary indication is erectile dysfunction (Christopher and Cindy, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe rate of consumption of natural aphrodisiacs especially among young men has increased in recent times. Furthermore, there is limited scientific evidence supporting the efficacy and safety of natural aphrodisiacs which is worrisome as it could lead to abuse of aphrodisiacs (Manortey et al, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A common aphrodisiac traditionally used in Nigeria is \u003cem\u003eCarpolobia alba\u003c/em\u003e. \u003cem\u003eCarpolobia alba\u003c/em\u003e grows as a shrub or small tree which is 3 to 6 metres (9.8 to 19.7 ft) tall. It has querulous branches or short pubescent. Its leaves are membranous or slightly leathery. This type of plant belongs to the milkwort family (Polygalaceae) and is native to Western Tropical and Central Africa's rainforests, forest edges, and savanna parks at elevations lower than 400 meters (1,300 feet). \u003cem\u003eCarpolobia alba\u003c/em\u003e is a popular natural recreational aphrodisiac plants commonly used by pygmies of the forest region (Ipassa of Garbon, South-Cameroon; and the Yoruba, Efik, Ibo,and Ijaw peoples of Nigeria, ) and other part of sub-Sahara Africa (Nwidu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is commonly called Cattle stick in English, \u003cem\u003eEgbo Oshunshun\u003c/em\u003e in Yoruba, \u003cem\u003eAngalagala\u003c/em\u003e in Igbo, \u003cem\u003eIkpafum\u003c/em\u003e in Ibibio (Nwidu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The root of \u003cem\u003eCarpolobia alba\u003c/em\u003e has long been used to treat male infertility, increase virility, and improves male fertility. It has been reported to have aphrodisiac and androgenic effects. (Manfo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe apoptotic pathway in the mitochondria is controlled by proteins from the BCL-2 family, which controls cell survival and death (Czabotar and Garcia-Saez, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The BCL-2 is located in the outer membrane of the mitochondria and maintains cell survival by counteracting the effects of the proapoptotic protein BAX, thereby preserving mitochondrial membrane integrity. (Morris et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BCL-2 plays a crucial role in regulating apoptosis. The expression of BCL2 is tightly controlled and disturbance of BCL2 has been implicated in various disease conditions including cancer (Adelakun et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProliferating Cell Nuclear Antigen (PCNA) is a marker for cell proliferation and is involved in DNA synthesis, DNA methylation, chromosome recombination, RNA transcription and nucleic acid metabolism (Zhao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, changes in PCNA expression are used as a marker to evaluate cell proliferation. Spermatogenesis a complex process by which spermatogonia develops into mature sperm cell. It involves continuous cell division and proliferation. Thus, PCNA expression levels in the testis is a standard marker of spermatogenic cell proliferation and can be used also to assess the state of spermatogenic cell proliferation (Farang \u003cem\u003eet al.\u003c/em\u003e, 2021). Apoptosis is accompanied by proliferation. Therefore, the positive expression of PCNA indicates increased proliferation of spermatogenic cells in response to apoptotic cell death. A study carried out by Zhao et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that positive expression of PCNA in the testes correlates with spermatogenesis dysfunction in fluoride-treated rat. The aim of this study is to investigate the effects of sub-acute administration of of Methanol root Extract of Carpolobia alba (G. Don) on the sexual behavior and the level of expression of BCL-2 and PCNA in the testicular tissue of male Wistar rat.\u003c/p\u003e"},{"header":"MATERIALS AND METHOD","content":"\u003cp\u003ePlant Harvest and Extraction\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eCarpolobia alba\u0026nbsp;\u003c/em\u003eroot was obtained from Ayede Ogbese in Akure North Local Government, Ondo state, Nigeria and it was authenticated in the herbarium of Department of Plant Biology and Biotechnology, University of Medical Sciences Ondo state, Nigeria with voucher number UNIMED P.B.T.H No 034. After air drying, the \u003cem\u003eCarpolobia alba\u003c/em\u003e root (2.20kg) was pulverized into powder after which Carpolobia alba root was extracted using Soxhlet with pure methanol as the solvent. After filtering the methanol containing the extract, the solvent was vacuum-distilled in a rotary evaporator at 4\u003csup\u003eo\u003c/sup\u003eC. To verify that all remaining solvent was gone, the remaining extract was lastly dried for two hours at 30\u003csup\u003eo\u003c/sup\u003eC in a vacuum oven. 90.22g (1.69% yields) of the powdery mass were obtained which was stored at room temperature for the study. Distilled water was used to prepare a fresh solution of the extract.\u003c/p\u003e\n\u003cp\u003eANIMAL EXPERIMENTATION AND DESIGN\u003c/p\u003e\n\u003cp\u003eTwenty male Wistar rats (weighing 200-250g) were procured from the Animal facility of University of Medical Sciences, Ondo State, Nigeria. The rats were acclimatized for two weeks before the experiment begins. The experimental animals were accommodated in standard laboratory conditions. The animals were exposed to light and dark cycle of 12 hours and fed rat show and water \u003cem\u003ead libitium\u003c/em\u003e. Twenty adult Wistar rats were randomized into four groups consisting of five rats each. Group 1 (control) received only distilled water; Group 2,3 and 4 received 100mg/kg, 200mg/kg and 400mg/kg body weight respectively of methanol root extract of\u0026nbsp;\u003cem\u003eCarpolobia alba\u003c/em\u003e for 30 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Mating behaviour\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primed female rats that exhibited profound receptivity were introduced into the cages containing male rat in ratio 1:1 after which the following mating behavior were observed directly from the cage sides and via camera for 30 minutes (Yakubu and Jimoh, 2014): The following mating behavioural parameters were assessed: Mount frequency (MF) (number of mounts with or without intromission from the time of introduction of the female until ejaculation); Intromission frequency (IF) (number of vaginal penetrations during the observation period); Ejaculatory frequency (EF) (number of ejaculatory recorded during the time of observation); Mount latency (ML) (time interval between the introduction of the female and the first mount by the male); Ejaculation latency (EL) (time interval between the first intromission and ejaculatory); Intromission latency (IL) (time interval between the introduction of the female and the first intromission by the male). The assessment was done around 1:30pm local time to avoid interference of with the natural increase in libido in male rat in the morning (Kenmogne \u003cem\u003eet al\u003c/em\u003e., 2016).\u003c/p\u003e\n\u003cp\u003eIMMUNOHISTOCHEMISTRY ASSAY\u003c/p\u003e\n\u003cp\u003eParaffin embedded sections where deparaffinized with xylene, and rehydrated through descending grades of ethanol (100%, 95%, 70 % ethanol) and taken to water. Heat-mediated antigen retrieval was performed using a citrate-based antigen unmasking solution, pH 6.0 (Vector\u003csup\u003e\u0026reg;\u003c/sup\u003e, Burlingame, CA, USA; #H3300) in a steamer for 30 mins. Thereafter, it was Wash sections in PBS for 2 minutes. Endogenous peroxidase blocking in 0.3 % hydrogen peroxide solution in PBS for 10 minutes and Wash sections in PBS for 2 minutes. Sections were then incubated at room temperature for 2 hours in primary rabbit antibodies diluted in a universal antibody diluent and blocking reagent, UltraCruz\u0026reg; Blocking Reagent (Santa Cruz, USA).\u0026nbsp;The sections were subsequently incubated with specific primary antibodies against PCNA (Cell Signalling, USA; #13110) and Bcl-2 (Novus Biologicals, USA; NB100-56098) overnight at 4 \u0026deg;C in a humidified chamber. Section was washed in PBS for 5 minutes, incubated in ImmPRESS\u0026trade; HRP Anti-Rabbit IgG (Peroxidase) Polymer Reagent, made in horse for 30 mins, Wash in PBS for 5 minutes twice. Colour is developed with DAB Peroxidase (HRP) Substrate Kit (Vector\u003csup\u003e\u0026reg;\u003c/sup\u003e Labs, USA). Sections were rinsed well in tap water.\u0026nbsp;Sections were counter-stained in hematoxylin and observed under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of Immunoexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expressions for BCL-2 and PCNA in the testicular tissue were quantified using Image J software version 1.54p. The data obtained were statistically analyzed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis was done with GraphPad Prism software version 10.1. Both descriptive and inferential statistics (one-way ANOVA) were done on all obtained data. Turkey\u0026apos;s multiple comparison was used to test for statistical significant difference between control and experimental groups. The data were represented as Standard Error of Mean and the level of differences were considered significant at p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eEthical Consideration\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from \u0026nbsp;Olabisi Onabanjo University\u0026rsquo;s research Ethic Committee with reference number: \u003cstrong\u003eOOU/SCIENG/EC/0009/240924.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"RESULT","content":"\u003cp\u003eAssessment of mating behavior\u003c/p\u003e\n\u003cp\u003eThe mounting (MF), Intromission (IF), and Ejaculation (EF) frequencies were significantly increased (P\u0026lt; 0.05) in the experimental groups when compared to the control (fig 1,2 and 3). This means that animals that received the extract had more sexual motivation, penile thrusting and sexual climax than the control group. The mounting latency (fig 4) and intromission latency (fig 5) was significantly reduced (P\u0026lt; 0.05) in the experimental group when compared to control. This mean the animals that received the extract took lesser time to make their first mount and penile thrust after the introduction of a receptive female rat. On the contrary, the ejaculation latency (fig 6) was significantly increased in the experimental group when compared to the control. This implies that animals that receive the extract took longer time before ejaculation during mating.\u003c/p\u003e\n\u003cp\u003eIHC Assay of BCL-2 Expression in the Testicular Tissue\u003c/p\u003e\u003cp\u003eIHC Assay of PCNA Expression in the Testicular Tissue\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study the effects of sub-acute administration of Methanol root Extract of Carpolobia alba (G. Don) on mating behavior and testicular PCNA and BCL-2 immunoexpression in male Wistar rat was investigated. The result obtained from the mating behavioral assessments shows that there is significant increase in mounting frequency (MF), intromission frequency (IF) and ejaculation frequency (EF) in all the experimental groups (100,200 and 400mg/kg groups) when compared to control. An increase in MF, IF, and EF reflects enhanced sexual motivation. This implies that \u003cem\u003eCarpolobia alba\u003c/em\u003e increases sexual motivation of the male rats to make with the female rat. This result agrees with study reported by (Kenmogne et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) who reported that aqueous extract of \u003cem\u003eCarpolobia alba\u003c/em\u003e significantly increase mount frequency. This suggests that \u003cem\u003eCarpolobia alba\u003c/em\u003e significantly increase sexual drive in male Wistar rat. The mounting and intromission latencies were significantly reduced in the experimental groups compared to the control. This mean the animals that received the extract took lesser time to make their first mount and vaginal thrust after the introduction of a receptive female rat. The reduction in mount and intromission latencies may also connote increased orientation activities towards the receptive females and stronger sexual motivation (Yakubu and Jimoh, 2015). On the contrary, the ejaculation latency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) was significantly increased in the experimental group when compared to the control. The prolonged EL in \u003cem\u003eCarpolobia alba\u003c/em\u003e treated group suggests that the extract enhanced sexual intercourse for sufficiently desired time. This could also suggest the activation of nitric oxide and regulating the release and serotonin.\u003c/p\u003e \u003cp\u003eB-cell lymphoma 2 (BCL2) is an anti-apoptotic protein that is important and crucial in regulating programmed cell death (apoptosis). It functions by inhibiting the activity of pro-apoptotic proteins, thereby promoting cell survival. The result from this study shows that experimental groups treated with \u003cem\u003eCarpolobia alba\u003c/em\u003e exhibited weak expression of BCL2 compared to the control group. This suggests that the plant extract may have a pro-apoptotic effect, potentially leading to increased cell death in the testes. The Bcl-2 immunoexpression were reduced in the experimental group but the reduction was only significant at 400mg/kg which suggest a dose dependent effects. This indicates that the pro-apoptotic effects of the extract which may be exacerbated by higher doses. This result agrees with the study reported by Sarica \u003cem\u003eet al\u003c/em\u003e (2001) who reported bcl2 downregulation in apoptotic cells.\u003c/p\u003e \u003cp\u003eProliferating Cell Nuclear Antigen (PCNA) is a critical marker for cell proliferation and is involved in DNA replication and repair. PCNA protein expression serve as a biomarker of proliferation of spermatogenic cells and can be used to assess the proliferation state of spermatogenic cells. The result from this study showed significantly increased PCNA expression (at 100 and 400mg/kg) in the Carpolobia alba-treated group compared to the control group. This suggests enhanced spermatogenic activity in response to the pro-apoptotic effects of extract. Apoptosis is accompanied by proliferation. During spermatogenic cell mitosis and differentiation, PCNA as a cell proliferation factor is involved. PCNA not only regulate cell proliferation but also act as cell proliferation markers. This result is consistent with the work reported by Zhao et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) who documented positive immunoexpression of PCNA in fluoride-induced testicular toxicity. Also, similar result was documented by Oluwatunase et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) who reported that sub-acute treatment of Carpolobia alba induces apoptotic spermatogenic cell death, oxidative stress and inhibits synthesis of reproductive hormones.\u003c/p\u003e \u003cp\u003eIn conclusion, sub-acute administration of methanol root Extract of Carpolobia alba (G. Don) improves sexual behavior and induces spermatogenic apoptotic cell death. This implies that while the extract enhances sexual desire, motivation and activities, it could have deleterious effects on the spermatogenic cells which lead to infertility.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdullahi H, Tukur J (2013) Sexual stimulants and their effect on women of reproductive age in Kano, Northern Nigeria. Department of Obstetrics and Gynaecology Bayero University Kano, Kano state Nigeria. J Basic Clin Sci 10(1):13\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdelakun SA, Akintunde OW, Ogunlade B, Peter AB, Siyanbade JA (2024) BCL-2 and BAX expression and germ cell apoptosis following the intervention of 1-isothiocyanato-4-methylsulfinylbutane in cisplatin-induced testicular toxicity and sperm DNA fragmentation in Sprague-Dawley rat. Aspects Mol Med 100055 ISSN 2949\u0026ndash;6888. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.amolm.2024.100055\u003c/span\u003e\u003cspan address=\"10.1016/j.amolm.2024.100055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristopher BH, Cindy MM (2011) Recreational Use of Erectile Dysfunction Medications in Undergraduate Men in the United States:Characteristics and Associated Risk Factors. Arch Sex Behav 40(3):597\u0026ndash;606\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCzabotar PE, Garcia-Saez AJ (2023) Mechanisms of BCL-2 family proteins in mitochondrial apoptosis. Nat Rev Mol Cell Biol 24:732\u0026ndash;748\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarag OM, Abd-Elsalam RM, El Badawy SA, Ogaly HA, Alsherbiny MA, Ahmed KA (2021) Portulaca oleracea seeds' extract alleviates acrylamide-induced testicular dysfunction by promoting oxidative status and steroidogenic pathway in rats. BMC Complement Med Ther. 14;21(1):122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12906-021-03286-2\u003c/span\u003e\u003cspan address=\"10.1186/s12906-021-03286-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 33853605; PMCID: PMC8045344\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldstein I (2022) The central mechanisms of sexual function; Available:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bumc.bu.edu/sexualmedicine/publications/the-central-mechanisms-of-sexula function\u003c/span\u003e\u003cspan address=\"https://www.bumc.bu.edu/sexualmedicine/publications/the-central-mechanisms-of-sexula function\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed on 3/09/2022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenmogne H, Koloko B, Hambe C, Domkam J, Ngaha Njila M, Bend E, Oundoum Oundoum P, Massoma Lemb\u0026egrave; D, Dimo T (2016) Effects of aqueous root extract of \u003cem\u003eCarpolobia alba\u003c/em\u003e G. Don on sexual behaviour in adult male rats. Andrologia 48(8):908\u0026ndash;914\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManfo FP, Nantia EA, Dechaud H, Tchana AN, Zabot MT, Pugeat M et al (2014) Protective effect of Basella alba and \u003cem\u003eCarpolobia alba\u003c/em\u003e extracts against induced male infertility. Pharm Biol 52:97\u0026ndash;104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManortey S, Mensah PA, Acheampong GK (2018) Evaluating factors associated with the use of aphrodisiacs among adult male residents in Ashaiman Municipality, Ghana. Open Access Libr J 5:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris JL, Gillet G, Prudent J, Popgeorgiev N (2021) Bcl-2 Family of Proteins in the Control of Mitochondrial Calcium Signalling: An Old Chap with New Roles. Int J Mol Sci 22(7):3730. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22073730\u003c/span\u003e\u003cspan address=\"10.3390/ijms22073730\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwidu LL, Nwafor PA, Vilegas W (2015) The aphrodisiac herb Carpolobia: A biopharmacological and phytochemical review. Pharmacogn Rev 9(18):132\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOluwatunase GO, Otulana OJ, Olusola OO, Fakunle BP, Royhaan F et al (2024) Sub-acute toxic effects of methanol root extract of \u003cem\u003eCarpolobia alba G. Don\u003c/em\u003e on testes of adult Wistar rats. J Exp Clin Anat 21(2):180\u0026ndash;187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandroni P (2001) Aphrodisiacs past and present: a historical review. Clin Auton Res 11(5):3037\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarıca K, Bakır K, Erbag A, Yagcı F, Uysal O, U\u0026ccedil;ak R (2001) Unilateral Testicular Torsion: Evaluation of bcl-2, p-53 and PCNA Expression in Contralateral Testes. Urol Int 2001;66:94\u0026ndash;99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYakubu MT, Jimoh RO (2014) Carpolobia lutea roots restore sexual arousal and performance in paroxetine-induced sexually impaired male rats. Int J Androl 12:90\u0026ndash;99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao W, Wang H, Liu J, Tan P, Luo X, Zhu S, Chen X, Zhou B (2018) Positive PCNA and Ki-67 Expression in the Testis Correlates with Spermatogenesis Dysfunction in Fluoride-Treated Rats. Biol Trace Elem Res. 2011-018-1338-6\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carpolobia alba, sub-acute, aphrodisiacs, anti-fertility, immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-6953636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6953636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eOver 80% of the population in Africa and Asia rely on traditional medicine as their primary form of health care. For centuries, plant-based aphrodisiacs have been used to enhance sexual performance. In sub-Saharan Africa, especially Nigeria, \u003cem\u003eCarpolobia alba (G. Don)\u003c/em\u003e is widely used for its perceived sexual stimulatory effects, despite limited scientific evidence on its impact on male fertility. In this study, we investigated the effects of sub-acute administration of methanol root extract of \u003cem\u003eCarpolobia alba\u003c/em\u003e on sexual behaviour and testicular immunoexpression of B-Cell lymphoma-2 (Bcl-2 )and Proliferating Cell Nuclear Antigen (PCNA) in adult male Wistar rats.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eTwenty adult male rats (weighing 200g-250g) were randomly divided into four groups consisting of ten animals each. Each group was treated daily with distilled water, 100, 200 and 400mg/kg body weight respectively of methanol root extract \u003cem\u003eCarpolobia alba G.Don\u003c/em\u003e for 30 days. The animals were anesthetized using Isoflurane via an induction chamber. Mating behavioral assessment was done by direct observation and use of camera, testicular B-Cell lymphoma-2 (Bcl-2 )and Proliferating Cell Nuclear Antigen (PCNA) Immunoexpression was done using immunohistochemical techniques.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eOur result shows significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased sexual activity and mating behavior across treated groups. However, testicular sections revealed a weak immunoexpression of Bcl-2, indicating increased apoptotic activity, and strong PCNA expression, indicating increased cell proliferation.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eDespite its traditional use as an aphrodisiac, \u003cem\u003eCarpolobia alba\u003c/em\u003e exhibits potential anti-fertility effects following sub-acute exposure, as evidenced by disrupted testicular cell regulation and structural integrity. These findings raise important concerns about its long-term reproductive safety and need for further investigation.\u003c/p\u003e","manuscriptTitle":"Anti-fertility and Sexual Behavioral Effects of Sub-Acute Administration of Carpolobia Alba (G. Don) Root Extract in Male Wistar Rats: an Immunohistochemical Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-24 12:31:18","doi":"10.21203/rs.3.rs-6953636/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aefdd953-b7b1-42da-9af5-492cef0f0910","owner":[],"postedDate":"June 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-24T12:31:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-24 12:31:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6953636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6953636","identity":"rs-6953636","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}