Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats

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Black pepper (Piper nigrum L.) fruit extract has been reported to possess antidiabetic and reproductive benefits, yet its effect on DM-related ED remains underexplored. Objective To evaluate the effects of Piper nigrum fruit extract on erectile function, libido, sperm parameters, and testicular histology in alloxan-induced diabetic male rats. Methods Thirty male Sprague Dawley rats were divided into five groups: normal control, diabetic control, diabetic rats treated with black pepper extract (122.5 or 245 mg/kg BW), and diabetic rats treated with sildenafil citrate. Erectile function was assessed via penile reflexes, libido by mating behavior, sperm quality by concentration, motility, and morphology, and testicular histology by Leydig cell and spermatogonia counts. Data were analyzed using ANOVA with significance at p<0.05. Results Alloxan-induced diabetic rats showed significant impairment in erectile function, libido, sperm quality, and testicular histology (p<0.001 vs control). Black pepper extract at 122.5 mg/kg BW significantly improved total penile reflexes compared with diabetic controls (9.33±1.03 vs 6.00±1.26, p=0.02). Libido parameters including courtship latency (5.50±0.55 vs 21.00±9.47, p=0.013), mount latency (19.00±10.81 vs 37.17±6.31, p=0.009), and mount frequency (18.05±5.99 vs 7.17±1.83, p=0.002) were significantly improved. Sperm analysis revealed increases in sperm concentration (19.2±6.7 vs 12.6±1.3, though not significant, p=0.877), motility (31.8±23 vs 27±30, p=0.697), and normal morphology (40.9±7.8 vs 35±10.8, p=0.04). Testicular histology showed restoration of Leydig cell count (59.33±4.0 vs 30.50±3.86, p=0.035) and spermatogonia number (319.4±64.59 vs 491±37.0, p<0.001). The 245 mg/kg BW dose primarily improved sperm concentration (62.95±29.4 vs 12.6±1.3, p=0.01) and motility (36.6±23 vs 27±30, p=0.436). Sildenafil citrate significantly enhanced most parameters compared with diabetic controls (p<0.05). Conclusion Piper nigrum fruit extract ameliorates sexual dysfunction and reproductive impairment in alloxan-induced diabetic rats, particularly at 122.5 mg/kg BW, with significant improvements in erectile function, libido (p<0.05), sperm quality (p≤0.04), and testicular histology (p=0.035). These findings suggest its potential as a natural therapeutic agent for DM-related male reproductive dysfunction. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/15-38/v1", "name": "Black pepper (Piper nigrum L.) fruit extract ameliorates erectile..." } } ] } Home Browse Black pepper (Piper nigrum L.) fruit extract ameliorates erectile... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Hadibrata E, Sutyarso S, Busman H et al. Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.12688/f1000research.172637.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] Exsa Hadibrata https://orcid.org/0009-0003-0960-4521 1,2 , Sutyarso Sutyarso 3 , Hendri Busman 3 , [...] Syazili Mustofa https://orcid.org/0000-0002-7646-0869 4 , Wawan Abdullah Setiawan 3 , Ratna Dewi Puspita Sari 5 , Nuning Nurcahyani 3 Exsa Hadibrata https://orcid.org/0009-0003-0960-4521 1,2 , Sutyarso Sutyarso 3 , [...] Hendri Busman 3 , Syazili Mustofa https://orcid.org/0000-0002-7646-0869 4 , Wawan Abdullah Setiawan 3 , Ratna Dewi Puspita Sari 5 , Nuning Nurcahyani 3 PUBLISHED 09 Jan 2026 Author details Author details 1 Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Lampung, Indonesia 2 Department of Surgery, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia 3 Department of Biology, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Lampung, Indonesia 4 Department of Biochemistry and Biomolecular, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia 5 Department of Obstetric and Gynecology, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia Exsa Hadibrata Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Sutyarso Sutyarso Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Hendri Busman Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Syazili Mustofa Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization Wawan Abdullah Setiawan Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization Ratna Dewi Puspita Sari Roles: Formal Analysis, Methodology, Supervision, Validation, Visualization Nuning Nurcahyani Roles: Investigation, Methodology, Supervision, Validation, Visualization OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Diabetes mellitus (DM) is a chronic metabolic disorder frequently associated with male sexual and reproductive dysfunction, including erectile dysfunction (ED), reduced libido, and impaired spermatogenesis. Black pepper ( Piper nigrum L.) fruit extract has been reported to possess antidiabetic and reproductive benefits, yet its effect on DM-related ED remains underexplored. Objective To evaluate the effects of Piper nigrum fruit extract on erectile function, libido, sperm parameters, and testicular histology in alloxan-induced diabetic male rats. Methods Thirty male Sprague Dawley rats were divided into five groups: normal control, diabetic control, diabetic rats treated with black pepper extract (122.5 or 245 mg/kg BW), and diabetic rats treated with sildenafil citrate. Erectile function was assessed via penile reflexes, libido by mating behavior, sperm quality by concentration, motility, and morphology, and testicular histology by Leydig cell and spermatogonia counts. Data were analyzed using ANOVA with significance at p<0.05. Results Alloxan-induced diabetic rats showed significant impairment in erectile function, libido, sperm quality, and testicular histology (p<0.001 vs control). Black pepper extract at 122.5 mg/kg BW significantly improved total penile reflexes compared with diabetic controls (9.33±1.03 vs 6.00±1.26, p=0.02). Libido parameters including courtship latency (5.50±0.55 vs 21.00±9.47, p=0.013), mount latency (19.00±10.81 vs 37.17±6.31, p=0.009), and mount frequency (18.05±5.99 vs 7.17±1.83, p=0.002) were significantly improved. Sperm analysis revealed increases in sperm concentration (19.2±6.7 vs 12.6±1.3, though not significant, p=0.877), motility (31.8±23 vs 27±30, p=0.697), and normal morphology (40.9±7.8 vs 35±10.8, p=0.04). Testicular histology showed restoration of Leydig cell count (59.33±4.0 vs 30.50±3.86, p=0.035) and spermatogonia number (319.4±64.59 vs 491±37.0, p<0.001). The 245 mg/kg BW dose primarily improved sperm concentration (62.95±29.4 vs 12.6±1.3, p=0.01) and motility (36.6±23 vs 27±30, p=0.436). Sildenafil citrate significantly enhanced most parameters compared with diabetic controls (p<0.05). Conclusion Piper nigrum fruit extract ameliorates sexual dysfunction and reproductive impairment in alloxan-induced diabetic rats, particularly at 122.5 mg/kg BW, with significant improvements in erectile function, libido (p<0.05), sperm quality (p≤0.04), and testicular histology (p=0.035). These findings suggest its potential as a natural therapeutic agent for DM-related male reproductive dysfunction. READ ALL READ LESS Keywords Diabetes Mellitus, Hyperglycemia, Black Pepper, Piper Nigrum, Erectile Function, Libido Corresponding Author(s) Exsa Hadibrata ( [email protected] ) Close Corresponding author: Exsa Hadibrata Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2026 Hadibrata E et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Hadibrata E, Sutyarso S, Busman H et al. Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.12688/f1000research.172637.1 ) First published: 09 Jan 2026, 15 :38 ( https://doi.org/10.12688/f1000research.172637.1 ) Latest published: 29 Apr 2026, 15 :38 ( https://doi.org/10.12688/f1000research.172637.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Diabetes mellitus (DM) is one of the most common chronic diseases in the world characterized by carbohydrate metabolism disorders. 1 There were approximately 589 million people aged 20 to 79 years suffering from DM in 2025 that approximately 11.1% of the population as shown in Figure 1 . 1 , 2 Diabetes Mellitus (DM) can be caused by disorders in insulin production, cells resistance to insulin, or both. This chronic hyperglycemic condition can interact with other metabolic problems in people with DM causing organ damage and resulting in serious complications. 1 , 2 These complications include microvascular such as retinopathy, nephropathy, and neuropathy, as well as macrovascular causing coronary arteries, peripheral arteries, and cerebrovascular diseases. 3 Figure 1. The number of people diagnosed with DM as presented in millions. 1 Long-term complications of DM can cause serious health problems, one of which is sexual dysfunction in men and women. In men, sexual dysfunction due to DM is Erectile Dysfunction (ED) with a prevalence of 3.5 times higher than in men without DM. 4 Erectile Dysfunction (ED) in men with DM is also associated with decreased fertility in men due to hypothalamic-pituitary-gonadal axis dysfunction, spermatogenesis and maturation disorders. 5 Erectile Dysfunction (ED) and reproductive disorders in men are related to testosterone level. Many studies have shown that testosterone deficiency is common in men with DM, both Type 1 DM (T1DM) and Type 2 DM (T2DM). In people with T2DM, there is a decrease in free testosterone of up to 57%, while in people with T1DM, the decrease in free testosterone reaches 20.3%. Thus, it is not an exaggeration to say that total testosterone and free testosterone levels are risk factors for DM in men. 6 , 7 Apart from testosterone as a risk factor for DM, low testosterone levels themselves are known to cause decreased sexual function and fertility in men. The low of sexual function is characterized by loss of erection, decreased sexual desire, and decreased frequency of sexual intercourse. 8 Research on animal model has shown that testosterone is also a determining factor in male fertility because it affects spermatogenesis. The critical processes in spermatogenesis that are influenced by testosterone are maintaining the blood testes barrier, supporting the meiosis process, the adhesion of spermatids to Sertoli cells, and the release of sperms. 9 Currently, there are many types of drugs commonly used to treat DM, including metformin, sulfonylureas, meglitinides, thiazolidinediones, dpp-4 inhibitors. Unfortunately, all of these drugs have side effects. Metformin, for example, causes side effects such as nausea, vomiting, abdominal bloating, diarrhea, heartburn, headache, agitation, dizziness, tiredness, chills, abdominal cramps or pain, loss of appetite, asthenia, and myalgia. 10 Therefore, the search for DM drugs derived from plants continues to grow. One of the medicinal plants that has the potential to have anti-diabetic properties is black pepper ( Piper nigrum L. ). Tests on alloxan-induced diabetic rats showed that black pepper fruit extract was effective in lowering blood sugar levels and was also effective in lowering cholesterol levels. 11 , 12 Apart from having the potential as an anti-diabetic, black pepper fruit extract has also been proven to increase testosterone hormone levels, sexual function (libido), and spermatogenesis parameters in male rats. Mating tests on male rats given black pepper fruit extract showed a significant increase in libido, indicated by a shorter courtship latency. 13 The fertility parameters of male rats given black pepper fruit extract also increased, indicated by an increase in epididymal sperm concentration, spermatocyte counts, spermatid counts, and the weight of epididymis tubules. 14 Although black pepper has shown antidiabetic and reproductive benefits, its effect on DM-related ED is not well established. Especially, its influence on erectile function, libido parameters, spermatozoa parameters, and testicular histology parameters has not been evaluated. To our knowledge, this is the first to demonstrate the potential of black pepper ( Piper nigrum L. ) fruit extract to improve sexual function as depicted in libido parameters, spermatozoa parameters, and testicular histology parameters in alloxan-induced diabetic rats. Materials and methods Study design This study was an experimental, randomized controlled laboratory to assess the effects of black pepper ( Piper nigrum L. ) fruit extract on libido parameters, spermatozoa parameters, and testicular histology parameters in alloxan-induced diabetic rats. Rats were randomly sampled into control, diabetic, extract-treated, and positive control groups to allow direct comparison of treatment outcomes. Data obtained were collected and analyzed using the IBM SPSS Statistics version 31 software (New York, USA). Normality of the data was assessed with the Shapiro-Wilk test. Since the data was considered non-parametric, Kruskal-Wallis tests were used. Differences between groups were evaluated using One-Way Analysis of Variance (ANOVA), followed by a Least Significant Difference (LSD) post hoc test to identify specific intergroup differences. A p -value of <0.05 was considered statistically significant. Plant preparation Fresh black pepper fruit is collected from a farmer in Ngarip Village, Ulubelu District, Tanggamus Regency, Lampung Province. The fresh fruit is washed and rinsed until clean and then dried. After drying, the fruit is ground into powder using a blender. The extraction was done by soaking the black pepper powder in 95% ethanol at room temperature. The supernatant collected every 24 h for three days and filtered to remove unwanted components. The filtrate was concentrated using a rotary evaporator at a temperature of 40°C and a pressure of 60 mbar. The extract is stored in the refrigerator as stock until used. Animal preparation This study used 30 male Sprague Dawley rats as mentioned by Federer study before, Rattus norvegicus, aged 2.5-3 months weighing 100-150 grams obtained from the animal house at the IPB University, Bogor, Indonesia. All procedures were conducted in accordance and approved with the guidelines of the Health Research Ethics Commission of the Faculty of Medicine, Lampung University (No.3468/UN26.18/PP.05.02.00/2024). The rats were acclimated for a week at a temperature of 25°C with stable room humidity and lighting, and were given standard feed ad libitum. The studied rats were divided into five groups of 6 individuals each. Group 1 (I) was rats that were only given standard feed. Group 2 (II) was alloxan-induced hyperglycemic rats and given feed. Groups 3 (III) and group 4 (IV) were alloxan-induced hyperglycemic rats and given black pepper extract 122.5 and 245 mg/kg BW respectively for 8 days. Group 5 (V) is alloxan-induced hyperglycemic rats given Sildenafil therapy. Alloxan induction in studied rats was performed intraperitoneally using normal saline solvent at a dose of 150 mg/kg BW. To prevent alloxan-induced rats from suffering from severe hypoglycemia, the rats were given a 20% glucose solution (5-10 ml) orally after 6 hours. Furthermore, after being maintained for 24 hours, the rats were given further 5% glucose solution to prevent hypoglycemia. Rats are categorized to have DM if they experience glycosuria and hyperglycemia with blood glucose levels of 200 to 300 mg/dL. 15 Phosphodiesterase-5 inhibitor (PDE-5i) therapy was given one hour before observation of erectile function and libido assessment. The therapy was done using Sildenafil Citrate, a drug from the PDE-5i at a dose of 1 mg/kg BW as positive control for recovery of ED caused by DM. 16 Sildenafil treatment was done following Gurbuz et al. (2015) to decrease advanced glycation end products, Malondialdehyde (MDA) and Inducible Nitric Oxide Synthase (iNOS), to preserve Neuronal Nitric Oxide Synthase (nNOS) and Cyclic Guanosine Monophosphate (cGMP) contents in penile tissue. 17 Erectile function assessment Erectile function of the rats was assessed on the ninth day one hour before the mating test was performed. The animals were placed in supine position with partial restraint. The preputial sheath was pushed behind the gland penis and held in this manner for a period of 15 min to elicit penile reflex. Total Penile Reflexes (TPR) for each animal were calculated as the sum of Erection (E), Long Flips (LF) and Quick Flips (QF). Mathematically, it can be expressed using the following formula TPR = E+QF+LF. 18 , 19 Libido assessment To determine the libido level of the studied rats, a mating test was conducted on the ninth day. The mating test was conducted by placing male and female rats in a cage with a partition in the middle. The cage was placed in a room with low lighting. Observation and analysis of the mating behavior of rats were conducted through video recording. There were three mating behavior parameters assessed, namely courtship latency, mounting latency, and mounting frequency. Courtship latency is the time from the partition being opened until the male kisses the female’s genitals or other body parts. Mounting latency is the time from the partition being opened until the male mounts the female’s back. Mounting frequency is the number of times the male mounts the female’s back during the 30 minutes (1800 seconds) of the mating test. Sperm analysis and testis histology examination After the erectile function and mating behavior were assessed, sperm and histological analysis of the studied rats were performed. The sperm analysis and testis histology examination were performed in Anatomy Pathology Laboratorium of Faculty of Medicine, Lampung University. The rats were terminated by injection of ketamine at a dose of 100 mg/kg BW and cervical dislocation. A total of 30 right testes and cauda epididymis were removed using a dissecting kit. The cauda epididymis were pinched to squeeze the spermatozoa into the petri dish. Subsequently, a suspension of spermatozoa were formed after being mixed with NaCL 0.9%, as mentioned in previous study. 20 Spermatozoa were counted using a Neubauer’s hemocytometer under a light microscope (400x) and expressed as millions/ml. Sperm motility was evaluated by counting motile and immotile sperms. Sperm morphology was assessed from a smear of the epididymal filtrate prepared on a clean glass slide with 1% eosin staining. After the object dried, observation was done under Richter Optica HS-3B-3’s light microscope (China) at 400x magnification and abnormalities of either head or tail were noted. Spermatozoa parameters were only assessed in studied rats of group 1 to group 4. Testicular histology examination was performed using Haematoxylin & Eosin (HE) staining and spermatogonia were counted in 10 random seminiferous tubule cross-sections per testis, and Leydig cells were counted in 10 non-overlapping interstitial fields with thickness of 4-5 μm; counts were normalized to area using National Institute of Health ImageJ software version 1.8.0 (New York, USA) (calibrated to μm 2 ) at 400x magnification. 21 Results Erectile function Erectile function parameters of alloxan-induced rats treated with black pepper fruit extract are presented in Table 1 . Group I, consisting of normal rats with normal feeds, showed the highest average values for the quick flip and erection parameters. As the reference group in this study, Group I also had the highest average TPR values compared to the other groups. Among the alloxan-induced rats, Group III (treated with black pepper extract at a dose of 122.5 mg/kg BW) demonstrated average quick flip, long flip, and erection values that were comparable to those of Group I. The average TPR value in Group III was also similar to that of Group I. However, Group IV, which received a higher dose of black pepper extract (245 mg/kg BW), showed relatively lower average values for quick flip, long flip, and erection compared to Group III, with TPR values more comparable to those of Group II. As shown in Table 1 , Group III had relatively higher average TPR values compared to Group V. The treatment effect was observed only in the TPR parameter, whereas the differences in ER, QF, and LF parameters were not statistically significant. Diabetic rats given black pepper fruit extract at a dose of 122.5 mg/kg BW (Group III) showed a statistically significant differences in TPR values compared to alloxan-induced rats with normal feeds (Group II). Table 1. Erectile function parameters of studied rats. Group QF LF ER TPR TPR p -value # I (only fed) 3,67 ± 0,82 a 1.33 ± 0.52 a 4.67 ± 0.82 a 9.67 ± 1.03 a vs II = <0.001 ; vs III = 0.246; vs IV = 1.000; vs V = 0.135 II (alloxan) 1.83 ± 0.75 a 0.33 ± 0.52 a 3.83 ± 0.75 a 6.00 ± 1.26 a vs I = <0.001 ; vs III = 0.02 ; vs IV = < 0.001 ; vs V = 0.038 III (extract 122.5 mg/kg BW) 3.33 ± 1.03 a 1.67 ± 0.52 a 4.33 ± 0.52 a 9.33 ± 1.03 a vs I = 0.246; vs II = 0.02 ; vs IV = 0.747; vs V = 1.000 IV (extract 245 mg/kg BW) 2.83 ± 0.75 a 0.67 ± 0.82 a 3.33 ± 0.82 a 6.83 ± 0.98 a vs I = 1.000; vs II = <0.001 ; vs III = 0.747; vs V = 0.436 V (sildenafil citrate) 2.17 ± 0.75 a 1.33 ± 0.52 a 4.67 ± 0.52 a 8.17 ± 1.17 a vs I = 0.135; vs II = 0.038 ; vs III = 1.000; vs IV = 0.436 a = Presented in mean and Standard Deviation (SD), # = LSD Post Hoc test. Libido The effect of giving black pepper extract on the libido of rats using the parameters of courtship latency, mounting latency and mounting frequency can be seen in Table 2 . Based on the data in Table 2 , it can be seen that the administration of black pepper fruit extract significantly improved the decreased libido due to hyperglycemia caused by alloxan induction. Based on the values of the courtship latency, mounting latency and mounting frequency, it was revealed that the best dose of black pepper extract to restore the libido of hyperglycemic male rats was a dose of 122.5 mg/kg BW. The therapeutic effect with sildenafil citrate also significantly improved the libido of rats. Table 2. Libido parameters of Courtship Latency (CL), and Mount Latency (ML) of studied rats. Group CL CL p -value ^ ML ML p -value # I (only fed) 5.17 ± 0.41 a vs II = 0.003 ; vs III = 0.060; vs IV = 0.241; vs V = 0.005 8.50 ± 3.51 a vs II = <0.001 ; vs III = 0.033 ; vs IV = 0.006 ; vs V = <0.001 II (alloxan) 21.00 ± 9.47 a vs I = 0.003 ; vs III = 0.013 ; vs IV = 0.003 ; vs P2 = 0.003 37.17 ± 6.31 a vs I = <0.001 ; vs III = 0.009 ; vs IV = <0.001 ; vs V = 0.075 III (extract 122.5 mg/kg BW) 5.50 ± 0.55 a vs I = 0.06; vs II = 0.013 ; vs IV = 0.204; vs V = 1.000 19.00 ± 10.81 a vs I = 0.033 ; vs II = 0.009 ; vs IV = 0.464; vs V = 0.007 IV (extract 245 mg/kg BW) 7.00 ± 1.10 a vs I = 0.241; vs II = 0.003 ; vs III = 0.204; vs V = 0.019 27.67 ± 6.53 a vs I = 0.006 ; vs II = <0.001 ; vs III = 0.464; vs V = 0.026 V (sildenafil citrate) 9.00 ± 4.82 a vs I = 0.005 ; vs II = 0.003 ; vs III = 1.000; vs IV = 0.019 16.00 ± 8.27 a vs I = <0.001 ; vs II = 0.075; vs III = 0.007 ; vs IV = 0.026 a = Presented in mean and Standard Deviation (SD), ^ = Mann-Whitney test, # = LSD Post Hoc test. Courtship Latency (CL) and Mount Latency (ML) showed significant differences among groups as shown in Table 2 and Table 3 . The alloxan group (II) had the longest CL (21.00 ± 9.47, p = 0.003 vs group I) and ML (37.17 ± 6.31, p < 0.001 vs group I). In contrast, the extract 122.5 mg/kg BW group (III) demonstrated CL (5.50 ± 0.55) and ML (19.00 ± 10.81), both significantly different from group II (p = 0.013 and p = 0.009, respectively) and closer to group I. The extract 245 mg/kg BW group (IV) also showed shorter CL (7.00 ± 1.10) and ML (27.67 ± 6.53) compared with group II (p = 0.003 and p < 0.001, respectively). The sildenafil group (V) exhibited CL (9.00 ± 4.82) and ML (16.00 ± 8.27), with significant differences from group II (p = 0.003 and p = 0.075, respectively). Table 3. Mount Frequency (MF) of studied rats. Group MF MF p -value # I (only fed) 17.00 ± 3.74 a vs II = <0.001 ; vs III = 0.351; vs IV = 0.649; vs V = 0.157 II (alloxan) 7.17 ± 1.83 a vs I = <0.001 ; vs III = 0.002 ; vs IV = <0.001 ; vs V = 0.006 III (extract 122.5 mg/kg BW) 18.05 ± 5.99 a vs I = 0.351; vs II = 0.002 ; vs IV = 0.17; vs V = 0.615 IV (extract 245 mg/kg BW) 13.33 ± 3.45 a vs I = 0.649; vs II = <0.001 ; vs III = 0.17; vs IV = 0.066 V (sildenafil citrate) 14.83 ± 5.74 a vs I = 0.157; vs II = 0.006 ; vs III = 0.615; vs IV = 0.066 a = Presented in mean and Standard Deviation (SD), # = LSD Post Hoc test. Mount Frequency (MF) was lowest in group II (7.17 ± 1.83), significantly different from group I (17.00 ± 3.74, p < 0.001). Group III (18.05 ± 5.99) was not significantly different from group I (p = 0.351), while group IV (13.33 ± 3.45) and group V (14.83 ± 5.74) showed higher MF compared with group II (p < 0.001 and p = 0.006, respectively). Sperm analysis In sperm concentration (SC), the control group (I) recorded a mean of 75.81 ± 52.9, which was significantly higher than group II (12.6 ± 1.3; p = 0.002) and group III (19.2 ± 6.7; p = 0.004), but not different from group IV (62.95 ± 29.4; p = 0.483) as shown in Table 4 . Compared with group V (158.16 ± 29.8), group I was significantly lower (p < 0.001). Group II had significantly lower SC compared with group IV (p = 0.01) and group V (p < 0.001), but no significant difference from group III (p = 0.877). Group III showed significantly lower SC than group IV (p = 0.019) and group V (p < 0.001). Group IV exhibited significantly lower SC than group V (p < 0.001). Overall, group V demonstrated the highest SC among all groups with significant differences in all comparisons (p < 0.001). Table 4. Sperm analysis parameters of sperm concentration, and sperm progressive motility of studied rats. Group SC SC p -value # SPM SPM p -value # I (only fed) 75.81 ± 52.9 b vs II = 0.002 ; vs III = 0.004 *; vs IV = 0.483; vs V = <0.001 57 ± 33 a vs II = 0.024 ; vs III = 0.07; vs IV = 0.121; vs V = 0.556 II (alloxan) 12.6 ± 1.3 a vs I = 0.002 ; vs III = 0.877; vs IV= 0.01 ; vs V = <0.001 27 ± 30 a vs I = 0.024 ; vs III = 0.697; vs IV = 0.436; vs V = 0.006 III (extract 122.5 mg/kg BW) 19.2 ± 6.7 a vs I = 0.004 ; vs II = 0.877; vs IV = 0.019 ; vs V = <0.001 31.8 ± 23 a vs I = 0.07; vs II = 0.697; vs IV = 0.721; vs V = 0.021 IV (extract 245 mg/kg BW) 62.95 ± 29.4 a vs I = 0.483; vs II = 0.01 ; vs III = 0.019 ; vs V = < 0.001 36.6 ± 23 a vs I = 0.121; vs II = 0.436; vs III = 0.721; vs V = 0.037 V (sildenafil citrate) 158.16 ± 29.8 a vs I = <0.001 ; vs II = <0.001 ; vs III = <0.001 ; vs IV = <0.001 65 ± 35 a vs I = 0.556; vs II = 0.006 *; vs III = 0.021 ; vs IV = 0.037 a = Presented in mean and Standard Deviation (SD), # = LSD Post Hoc test. For sperm progressive motility (SPM) as shown in Table 4 , the control group (I) recorded 57 ± 33, which was significantly higher than group II (27 ± 30; p = 0.024), but not different from group III (31.8 ± 23; p = 0.07) or group IV (36.6 ± 23; p = 0.121). Compared with group V (65 ± 35), no significant difference was observed (p = 0.556). Group II showed significantly lower SPM than group V (p = 0.006) but not significantly different from group III (p = 0.697) or group IV (p = 0.436). Group III had significantly lower SPM compared with group V (p = 0.021) but did not differ significantly from group IV (p = 0.721). Group IV also showed significantly lower SPM than group V (p = 0.037). Thus, group V demonstrated the highest SPM, with significant superiority over groups II–IV. Regarding sperm normal morphology (SNM) as presented in Table 5 , the control group (I) recorded 75 ± 13.7, which was significantly higher than group II (35 ± 10.8; p < 0.001) and group III (40.9 ± 7.8; p < 0.001), but not different from group IV (62 ± 14.7; p = 0.119) or group V (82.9 ± 5.7; p = 0.273). Group II had significantly lower SNM than all other groups (p ≤ 0.04). Group III also showed significantly lower SNM compared with group IV (p = 0.017) and group V (p < 0.001). Group IV exhibited significantly lower SNM than group V (p = 0.011). Among all groups, group V demonstrated the highest SNM, with significant differences against groups II–IV. Table 5. Sperm analysis parameters of sperm normal morphology of studied rats. Group SNM SNM p -value # I (only fed) 75 ± 13.7 a vs II = <0.001 ; vs III = <0.001 ; vs IV = 0.119; vs V = 0.273 II (alloxan) 35 ± 10.8 a vs I = <0.001 ; vs III = 0.04 ; vs IV = <0.001 ; vs V = < 0.001 III (extract 122.5 mg/kg BW) 40.9 ± 7.8 a vs I = <0.001 ; vs II = 0.04 ; vs IV = 0.017 ; vs V = <0.001 IV (extract 245 mg/kg BW) 62 ± 14.7 a vs I = 0.119; vs II = <0.001 ; vs III = 0.017 ; vs V = 0.011 V (sildenafil citrate) 82.9 ± 5.7 a vs I = 0.273; vs II = <0.001 ; vs III = <0.001 ; vs IV = 0.011 a = Presented in mean and Standard Deviation (SD), # = LSD Post Hoc test. Testicular histology analysis Table 6 presents the examination results of the testicular histology of alloxan-induced diabetic rats treated with black pepper extract. The data in the table shows that black pepper extract at a dose of 122.5 mg/kg BW significantly restored the number of Leydig cells and spermatogonia count. As shown in Figure 2 , group III with black pepper extract of 122.5mg/kg BW showed a higher amounts of spermatogonia as compared with group II without black pepper extraction. Table 6. Testicular histology parameters. Group LC LC p -value SgC SgC p -value I (only fed) 60.83 ± 5.1 a vs II = <0.001 ; vs III = <0.001 ; vs IV = 0.581; vs V = 0.001 511 ± 73.4 a vs II = 0.593; vs III = <0.001 ; vs IV = 0.294; vs V = 0.013 II (alloxan) 30.50 ± 3.86 a vs I = <0.001 ; vs III = 0.035 ; vs IV = <0.001 ; vs V = 0.001 491 ± 37.0 a vs I = 0.593; vs III = <0.001 ; vs IV = 0.119; vs V = 0.042 III (extract 122.5 mg/kg BW) 59.33 ± 4.0 a vs I = <0.001 ; vs II = 0.035 ; vs IV = <0.001 ; vs V = <0.001 319.4 ± 64.59 a vs I = <0.001 ; vs II = <0.001 ; vs IV = <0.001 ; vs V = 0.014 IV (extract 245 mg/kg BW) 50.67 ± 2.73 a vs I = 0.581; vs II = <0.001 ; vs III = <0.001 ; vs IV = 0.003 640 ± 86.5 a vs I = 0.294; vs II = 0.119; vs III = <0.001 ; vs V = 0.001 V (sildenafil citrate) 32.60 ± 3.28 a vs I = 0.001 ; vs II = <0.001 ; vs III = <0.001 ; vs P1 = 0.003 414 ± 32.5 a vs I = 0.013 ; vs II = 0.042 ; vs III = 0.014 ; vs IV = 0.001 a = Presented in mean and Standard Deviation (SD), # = LSD Post Hoc test. Figure 2. Higher amounts of spermatogonia was observed in group III with black pepper extract 122.5 mg/kg BW (a) as compared in group II without black pepper extract (b). Discussion The present study demonstrated that administration of Piper nigrum fruit extract ameliorated reproductive parameters in alloxan-induced diabetic male rats, as evidenced by increased sperm concentration, motility, serum testosterone levels, and improved morphology of seminiferous tubules. These findings are consistent with previous reports showing that black pepper extract can enhance fertility potential and improve reproductive outcomes in diabetic or hyperglycemic animal models, most likely through attenuation of oxidative stress and restoration of metabolic status. 20 , 21 In Figure 3 , we presented the summary of our results based on erectile function, libido, sperm analysis, and testicular histology. Figure 3. Summary of results. The decline in erectile function in alloxan-induced diabetic rats may be related to insulin resistance. Insulin resistance is known to be the cause of erectile dysfunction and hypogonadism. 22 Persistent hyperglycemia triggers Endoplasmic Reticulum Stress (ERS) in Leydig cells of the testis, causing increased expression of ERS-related factors, resulting in Leydig cell apoptosis and disrupting testosterone production, resulting in decreased erectile function. 23 , 24 Insulin deficiency in diabetes mellitus also causes excessive secretion of arginase II in the corpus cavernosum so that Nitric Oxide (NO) cannot function properly, resulting in inadequate blood flow to the penis. This can trigger erectile dysfunction. 25 , 26 This study revealed that alloxan (Group II) induction significantly decreased libido parameters in male rats. This occurred, according to various reports, because hyperglycemic conditions caused severe gonadal dysfunction, reproductive dysfunction, and diminished body and reproductive and organ weight. 27 Male rats with alloxan-induced hypoglycemia treated with black pepper extract were found to have improved sexual function. This recovery is very likely due to the influence of piperine in black pepper fruit. Piperine is an active ingredient that is typically found in Piperaceae plants. Piperine extracted from the fruit plant of Piper retrofractum L. plant, for example, is known to have an aphrodisiac effect on the libido of male rats. 28 The restoration of sexual function by black pepper fruit extract is related to the effect of piperine which can inhibit the work of testosterone 5"-reductase so that testosterone levels remain high. In addition, black pepper fruit extract is also known to contain fatty acids, such as auric acid, myristic acid and palmitic acid which affect androgen secretion and metabolism. Black pepper fruit extract is also known to contain zinc which promotes increased sex hormones, including testosterone in serum. 29 Testosterone is a hormone that has been proven to increase sexual desire or libido in hypogonadal men (patients). 30 This study shows that alloxan induction in male rats (Group II) causes a significant decrease in spermatozoa concentration and Leydig cell count compared to normal rats (Group I). This may be related to endocrine imbalance and impaired glucose transport and metabolism in diabetic rats. Endocrine imbalance – dysfunction of HPG axis occurs in diabetic patients which can cause infertility. Hyperglycemia exposure causes decreased sensitivity of the pituitary to gonadotropin-releasing hormone (GnRH) stimulation, resulting in abnormal secretion of FSH and LH. FSH plays a role in stimulating testicular Sertoli cells to promote sperm maturation in the seminiferous tubules, LH plays a role in stimulating Leydig cells in increasing serum testosterone levels. This decreased sensitivity of pituitary to GnRH causes disorders in the male reproductive system. 30 This research showed that administration of black pepper fruit extract at a dose of 245 mg/kg to alloxan-induced diabetic rats significantly increased the number and motility of spermatozoa, but had no effect on the abnormal morphology of the spermatozoa. On the other hand, administration of black pepper extract at a concentration of 122.5 mg/kg significantly increased the number of Leydig cells and the number of spermatogonia. This result is not surprising because the study reported by Chinta et al. (2017) showed that administration of piperine to rats causes hormonal imbalance by altering the serum levels of follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, serum, and testicular testosterone. 31 Other research results showing piperine effects that are in line with the findings of this study were reported by Chen et al. (2018). Piperine increase serum testosterone, increased Leydig cell number, cell size, multiple steroidogenic pathway proteins, 3β-hydroxysteroid dehydrogenase 1, 17α-hydroxylase/20-lyase, and steroidogenic factor 1 expression levels. Piperine stimulates pubertal Leydig cell development by increasing the number of Leydig cell and promoting its maturation. However, because piperine reduce FSH, then it inhibits spermatogenesis in. 32 Other researcher (Ere et al., 2020) reported that administration of black pepper fruit extract to rabbits increased testicular morphometric parameters, but caused damage to the seminiferous tubules and decreased spermatogenesis. 33 Conclusion Administration of black pepper fruit extract to male alloxan-induced diabetic rats significantly increased erectile function, libido, spermatozoa count, and Leydig cell count and spermatogonia count. Therefore, it can be concluded that black pepper extract ( Piper nigrum L.) has potential as an ingredient to overcome sexual dysfunction and reproductive dysfunction in males. However, this topic still an early study, we still need bigger and randmoized sample, especially human subject. Data availability Underlying data Figshare: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats. Available at: https://doi.org/10.6084/m9.figshare.30456353 . 34 The project contains the following underlying data: • Erectile function parameters values on each group • Libido parameters values on each group • Sperm analysis parameters values on each group • Testicular histology parameters values on each group Extended data Figshare: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats. Available at: https://doi.org/10.6084/m9.figshare.30456353 . 34 The project contains the following underlying data: • qPCR eNOS values on the subjects • ARRIVE guidelines - Author checklist References 1. Inzucchi SE, Bergenstal RM, Buse JB, et al. : Management of Hyperglycemia in Type 2 Diabetes: A Patient-Centered Approach: Position Statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care. 2012; 35 (6): 1364–1379. PubMed Abstract | Publisher Full Text | Free Full Text 2. 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BMC Complement. Altern. Med. 2015; 15 (1). PubMed Abstract | Publisher Full Text | Free Full Text 20. Journal of Medula: The Effect Of Black Pepper (Piper nigrum L.) Extract on Male Reproductive Parameters in Alloxan-Induced Diabetic Rats. J. Dent. Med. 2025. 21. Kaleem M, et al. : Protective effects of Piper nigrum and Vinca rosea in alloxan induced diabetic rats. Indian J. Physiol. Pharmacol. 2005; 49 : 65–71. PubMed Abstract 22. He Z, Yin G, Li QQ, et al. : Diabetes Mellitus Causes Male Reproductive Dysfunction: A Review of the Evidence and Mechanisms. In Vivo. 2021; 35 : 2503–2511. PubMed Abstract | Publisher Full Text 23. Sheikh-Ali M, Sultan S, Alamir AR, et al. : Hyperglycemia-induced endoplasmic reticulum stress in endothelial cells. Nutrition. 2010; 26 : 1146–1150. PubMed Abstract | Publisher Full Text 24. Guo S, Zhao D, Zang Z, et al. : Effects of endoplasmic reticulum stress on erectile function in rats with cavernous nerve injury. Sex Med. 2023; 11 (11): qfad050. Publisher Full Text 25. Du Z, Xu S, Hu S, et al. : Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells. Cell Death Dis. 2018; 9 : 968. 26. Kashyap SR, Roman LJ, Lamont J, et al. : Insulin Resistance Is Associated with Impaired Nitric Oxide Synthase Activity in Skeletal Muscle of Type 2 Diabetic Subjects. J. Clin. Endocrinol. Metabol. 2005; 90 : 1100–1105. PubMed Abstract 27. Maresch CC, Stute DC, Alves MG, et al. : Diabetes-induced hyperglycemia impairs male reproductive function: a systematic review. Hum. Reprod. Update. 2018; 24 : 86–105. PubMed Abstract | Publisher Full Text 28. Rahmawati N, Bachri MS: The aphrodisiac effect and toxicity of combination Piper retrofractum L, Centella asiatica, and Curcuma domestica infusion. Health Science Indonesia. 2012; 3 : 19–22. 29. Obadia PM, Mulaji GK, Musambo TM, et al. : Natural aphrodisiacs consumption by male workers in the former Katanga province, DR Congo. medRxiv. 2024. preprint. Publisher Full Text 30. Rizk PJ, Kohn TP, Pastuszak AW, et al. : Testosterone therapy improves erectile function and libido in hypogonadal men. Curr. Opin. Urol. 2017; 27 : 511–515. PubMed Abstract | Publisher Full Text 31. Chinta G, Coumar MS, Periyasamy L: Reversible Testicular Toxicity of Piperine on Male Albino Rats. Pharmacogn. Mag. 2017; 13 (Suppl 3): S525–S532. PubMed Abstract | Publisher Full Text 32. Chen X, Ge F, Liu J, et al. : Diverged Effects of Piperine on Testicular Development: Stimulating Leydig Cell Development but Inhibiting Spermatogenesis in Rats. Front. Pharmacol. 2018; 9 (9): 244. Publisher Full Text 33. 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Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 09 Jan 2026 ADD YOUR COMMENT Comment Author details Author details 1 Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Lampung, Indonesia 2 Department of Surgery, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia 3 Department of Biology, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Lampung, Indonesia 4 Department of Biochemistry and Biomolecular, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia 5 Department of Obstetric and Gynecology, Universitas Lampung Fakultas Kedokteran, Bandar Lampung, Lampung, Indonesia Exsa Hadibrata Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Sutyarso Sutyarso Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Hendri Busman Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Syazili Mustofa Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization Wawan Abdullah Setiawan Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization Ratna Dewi Puspita Sari Roles: Formal Analysis, Methodology, Supervision, Validation, Visualization Nuning Nurcahyani Roles: Investigation, Methodology, Supervision, Validation, Visualization Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (2) version 2 Revised Published: 29 Apr 2026, 15:38 https://doi.org/10.12688/f1000research.172637.2 version 1 Published: 09 Jan 2026, 15:38 https://doi.org/10.12688/f1000research.172637.1 Copyright © 2026 Hadibrata E et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Hadibrata E, Sutyarso S, Busman H et al. Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.12688/f1000research.172637.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 09 Jan 2026 Views 0 Cite How to cite this report: Ajiboye OM. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458225 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458225 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 19 Mar 2026 Oluwapelumi Micheal Ajiboye , Babcock University, Ogun State, Nigeria Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.190377.r458225 Summary of the Article This study investigates the potential therapeutic effects of Piper nigrum (black pepper) fruit extract on erectile dysfunction (ED) and associated reproductive parameters in alloxan-induced diabetic male rats. The experimental design includes five groups: normal control, ... Continue reading READ ALL Summary of the Article This study investigates the potential therapeutic effects of Piper nigrum (black pepper) fruit extract on erectile dysfunction (ED) and associated reproductive parameters in alloxan-induced diabetic male rats. The experimental design includes five groups: normal control, diabetic control, extract-treated diabetic group, and a sildenafil-treated group as a positive control. Outcome measures include erectile function indices (mounting frequency, intromission frequency, total penile reflexes), libido parameters, sperm characteristics, and histological evaluation of testicular tissue. The authors report that the extract significantly improved certain erectile and libido parameters, particularly total penile reflexes, while effects on sperm indices were largely non-significant. Histological observations are interpreted as showing restoration of testicular architecture. While the topic is relevant and potentially publishable, the manuscript exhibits substantial methodological, statistical, interpretational, and editorial deficiencies. Significant revision is required before the work can meet publication standards. MAJOR COMMENTS 1. The manuscript states that data are non-parametric, yet both Kruskal–Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) 2. Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. 3. The term “gland penis” is incorrect. Should be "glans penis". 4. The term "male kisses" is non-scientific. Rephrase appropriately. 5. Include units for sperm count and fluid intake 6. Avoid overgeneralization. 7. There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. 8. "ddp-4" should be written as "DDP-4" 9. Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. 10. Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" 11. The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " 12. The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. 13. Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. 14. The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Phytomedicine; Functional Foods and Nutraceuticals; Endocrine Disorder (Diabetes mellitus and its complications); Neurodegeneration. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Ajiboye OM. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458225 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458225 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), ... Continue reading 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) Response: We sincerely thank the reviewer for identifying this inconsistency. We agree that the original statistical description contained a methodological contradiction. Although the manuscript incorrectly stated that one-way ANOVA with LSD post hoc testing was used, the analyses actually performed in this study were Kruskal-Wallis tests followed by Dunn’s multiple-comparison post hoc tests, consistent with the non-parametric nature of the data. This error has now been corrected in the revised manuscript, and the statistical methods have been rewritten to accurately reflect the analyses conducted. We have also carefully reviewed the Results section and tables to ensure consistency with the corrected statistical approach. 2. Comment: Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. Response: We sincerely thank the reviewer for this careful and important comment. We agree that it is not appropriate to interpret non-significant differences as definite improvements. In response, we have revised the Results and Discussion sections to ensure that only statistically significant findings are described as significant improvements, whereas outcomes with p > 0.05 are now reported more cautiously as numerical differences without statistical significance. For example, the statements regarding sperm parameters have been corrected accordingly. We have also rechecked the manuscript throughout to ensure consistency between the statistical results and their interpretation. 3. Comment: The term “gland penis” is incorrect. Should be "glans penis". Response: We sincerely thank the reviewer for this valuable comment. We agree that the correct anatomical term is “glans penis” rather than “gland penis.” The manuscript has been revised accordingly in the Materials and Methods section to ensure anatomical accuracy. 4. Comment: The term "male kisses" is non-scientific. Rephrase appropriately. Response: We sincerely thank the reviewer for this valuable comment. We agree that the expression “male kisses” is not appropriate for scientific reporting of animal sexual behavior. In the revised manuscript, we have replaced this phrase with more formal and objective terminology to describe the observed behavior in the Libido Assessment subsection. We changed the sentence into “Courtship latency was defined as the time from opening of the partition until the male initiated investigatory or courtship behavior toward the female, including anogenital sniffing or contact with the female’s body.” 5. Comment: Include units for sperm count and fluid intake. Response: We sincerely thank the reviewer for this valuable observation. We agree that the omission of units for sperm count and fluid intake reduced the clarity of data presentation. In response, we have revised the manuscript into units of millions/mL to include the appropriate units for these variables throughout the text and tables. 6. Comment: Avoid overgeneralization. Response : We sincerely thank the reviewer for this valuable observation. We agree that the original manuscript contained several statements that were overly broad in relation to the data presented. In response, we have revised the manuscript throughout to ensure that the conclusions are stated more cautiously and are fully supported by the results. Specifically, we avoided definitive claims, reduced overstatement of non-significant findings, and clarified that the observed effects should be interpreted as dose-dependent, endpoint-specific, and preliminary findings in a short-term alloxan-induced diabetic rat model. 7. Comment: There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. Response : We sincerely thank the reviewer for this valuable comment. We agree that the earlier wording may have oversimplified the published evidence. In the revised manuscript, we clarified that the available literature suggests complex and context-dependent effects of piperine, rather than uniformly beneficial or uniformly harmful reproductive effects. We now explicitly state that piperine may enhance Leydig-cell function and steroidogenic activity while, under different experimental conditions, adversely affecting spermatogenesis. We therefore revised the Discussion to reflect that these findings represent different reproductive endpoints and may vary according to dose, exposure duration, and study conditions. 8. Comment : "ddp-4" should be written as "DDP-4" Response: We sincerely thank the reviewer for this careful observation. We agree that the abbreviation should be written consistently in uppercase. In the revised manuscript, “dpp-4” has been corrected to “DPP-4” throughout the text. 9. Comment : Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. Response: We sincerely thank the reviewer for this important comment. In response, we further revised the Discussion to more explicitly establish the relevance of the oxidative stress pathway and nitric oxide signaling to the findings of the present study. Specifically, we clarified that hyperglycemia-induced oxidative stress can reduce nitric oxide bioavailability and impair eNOS-mediated endothelial signaling in the corpus cavernosum, which contributes to diabetic erectile dysfunction. We also strengthened the discussion linking the directional increase in eNOS mRNA expression after black pepper treatment with the possibility of partial restoration of endothelial NO signaling. In addition, we added relevant recent literature to support the mechanistic relationship among oxidative stress, endothelial dysfunction, and impaired erectile function in diabetes. 10. Comment: Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" Response: We sincerely thank the reviewer for this valuable observation. We agree that the previous Conclusion was somewhat overstated. In the revised manuscript, we have rewritten the Conclusion to refer specifically to the measured outcomes, including total penile reflexes, libido-related parameters, intratesticular testosterone, sperm concentration, sperm morphology, Leydig cell count, and eNOS expression, rather than making broad claims about diabetic male reproductive dysfunction overall. We have also revised the language to ensure that the findings are interpreted as dose-dependent, endpoint-specific, and preliminary. However, we don’t used the term “randomized” in our manuscript anymore. 11. Comment: The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " Response: We sincerely thank the reviewer for this valuable comment. We agree that the original phrasing implied a stronger causal interpretation than was warranted. In the revised Discussion, we have replaced “known to be the cause of” with newer sentences in our revised manuscript. 12. Comment: The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. Response: We sincerely thank the reviewer for this valuable observation. We agree that the original Discussion contained mechanistic statements that could be interpreted as stronger than warranted by the available data. In the revised manuscript, we have clarified that intratesticular testosterone was directly measured and may therefore be discussed as an observed endpoint, whereas oxidative stress-related mechanisms were not directly evaluated, as no assays such as SOD, catalase, or MDA were performed. We therefore revised the Discussion to remove over-speculative language, qualify mechanistic interpretations more carefully, and restrict causal inferences to the measured outcomes. Where oxidative stress or antioxidant effects are mentioned, these are now presented explicitly as literature-supported hypotheses rather than direct findings of the present study. We have also included this into our limitation. 13. Comment: Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. Response: We sincerely thank the reviewer for this valuable comment. We acknowledge that some outcomes, particularly sperm count, showed relatively large standard deviations. We carefully re-examined the raw data and confirmed that these values were accurate and not due to transcription error. We elected to retain the original values to preserve transparency and to avoid underreporting biological variability. We agree that the relatively small group size may have contributed to the wide dispersion observed. Accordingly, we used non-parametric statistical analysis (Kruskal-Wallis followed by Dunn’s post hoc test) and revised the text to ensure that the findings are interpreted cautiously. However, we also include this topic into our limitation to keep our study transparent. 14. Comment: The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this important comment. We have carefully re-examined the entire reference list and corrected the identified errors, including the duplicated title in Reference 3, the incomplete author and journal information in Reference 20, and inconsistencies in journal-name formatting. The references have now been standardized throughout the manuscript to ensure consistency and accuracy. Furthermore, we revised the Discussion extensively to strengthen the scientific presentation of the work and to ensure that the findings are interpreted in light of relevant and current literature. 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) Response: We sincerely thank the reviewer for identifying this inconsistency. We agree that the original statistical description contained a methodological contradiction. Although the manuscript incorrectly stated that one-way ANOVA with LSD post hoc testing was used, the analyses actually performed in this study were Kruskal-Wallis tests followed by Dunn’s multiple-comparison post hoc tests, consistent with the non-parametric nature of the data. This error has now been corrected in the revised manuscript, and the statistical methods have been rewritten to accurately reflect the analyses conducted. We have also carefully reviewed the Results section and tables to ensure consistency with the corrected statistical approach. 2. Comment: Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. Response: We sincerely thank the reviewer for this careful and important comment. We agree that it is not appropriate to interpret non-significant differences as definite improvements. In response, we have revised the Results and Discussion sections to ensure that only statistically significant findings are described as significant improvements, whereas outcomes with p > 0.05 are now reported more cautiously as numerical differences without statistical significance. For example, the statements regarding sperm parameters have been corrected accordingly. We have also rechecked the manuscript throughout to ensure consistency between the statistical results and their interpretation. 3. Comment: The term “gland penis” is incorrect. Should be "glans penis". Response: We sincerely thank the reviewer for this valuable comment. We agree that the correct anatomical term is “glans penis” rather than “gland penis.” The manuscript has been revised accordingly in the Materials and Methods section to ensure anatomical accuracy. 4. Comment: The term "male kisses" is non-scientific. Rephrase appropriately. Response: We sincerely thank the reviewer for this valuable comment. We agree that the expression “male kisses” is not appropriate for scientific reporting of animal sexual behavior. In the revised manuscript, we have replaced this phrase with more formal and objective terminology to describe the observed behavior in the Libido Assessment subsection. We changed the sentence into “Courtship latency was defined as the time from opening of the partition until the male initiated investigatory or courtship behavior toward the female, including anogenital sniffing or contact with the female’s body.” 5. Comment: Include units for sperm count and fluid intake. Response: We sincerely thank the reviewer for this valuable observation. We agree that the omission of units for sperm count and fluid intake reduced the clarity of data presentation. In response, we have revised the manuscript into units of millions/mL to include the appropriate units for these variables throughout the text and tables. 6. Comment: Avoid overgeneralization. Response : We sincerely thank the reviewer for this valuable observation. We agree that the original manuscript contained several statements that were overly broad in relation to the data presented. In response, we have revised the manuscript throughout to ensure that the conclusions are stated more cautiously and are fully supported by the results. Specifically, we avoided definitive claims, reduced overstatement of non-significant findings, and clarified that the observed effects should be interpreted as dose-dependent, endpoint-specific, and preliminary findings in a short-term alloxan-induced diabetic rat model. 7. Comment: There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. Response : We sincerely thank the reviewer for this valuable comment. We agree that the earlier wording may have oversimplified the published evidence. In the revised manuscript, we clarified that the available literature suggests complex and context-dependent effects of piperine, rather than uniformly beneficial or uniformly harmful reproductive effects. We now explicitly state that piperine may enhance Leydig-cell function and steroidogenic activity while, under different experimental conditions, adversely affecting spermatogenesis. We therefore revised the Discussion to reflect that these findings represent different reproductive endpoints and may vary according to dose, exposure duration, and study conditions. 8. Comment : "ddp-4" should be written as "DDP-4" Response: We sincerely thank the reviewer for this careful observation. We agree that the abbreviation should be written consistently in uppercase. In the revised manuscript, “dpp-4” has been corrected to “DPP-4” throughout the text. 9. Comment : Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. Response: We sincerely thank the reviewer for this important comment. In response, we further revised the Discussion to more explicitly establish the relevance of the oxidative stress pathway and nitric oxide signaling to the findings of the present study. Specifically, we clarified that hyperglycemia-induced oxidative stress can reduce nitric oxide bioavailability and impair eNOS-mediated endothelial signaling in the corpus cavernosum, which contributes to diabetic erectile dysfunction. We also strengthened the discussion linking the directional increase in eNOS mRNA expression after black pepper treatment with the possibility of partial restoration of endothelial NO signaling. In addition, we added relevant recent literature to support the mechanistic relationship among oxidative stress, endothelial dysfunction, and impaired erectile function in diabetes. 10. Comment: Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" Response: We sincerely thank the reviewer for this valuable observation. We agree that the previous Conclusion was somewhat overstated. In the revised manuscript, we have rewritten the Conclusion to refer specifically to the measured outcomes, including total penile reflexes, libido-related parameters, intratesticular testosterone, sperm concentration, sperm morphology, Leydig cell count, and eNOS expression, rather than making broad claims about diabetic male reproductive dysfunction overall. We have also revised the language to ensure that the findings are interpreted as dose-dependent, endpoint-specific, and preliminary. However, we don’t used the term “randomized” in our manuscript anymore. 11. Comment: The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " Response: We sincerely thank the reviewer for this valuable comment. We agree that the original phrasing implied a stronger causal interpretation than was warranted. In the revised Discussion, we have replaced “known to be the cause of” with newer sentences in our revised manuscript. 12. Comment: The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. Response: We sincerely thank the reviewer for this valuable observation. We agree that the original Discussion contained mechanistic statements that could be interpreted as stronger than warranted by the available data. In the revised manuscript, we have clarified that intratesticular testosterone was directly measured and may therefore be discussed as an observed endpoint, whereas oxidative stress-related mechanisms were not directly evaluated, as no assays such as SOD, catalase, or MDA were performed. We therefore revised the Discussion to remove over-speculative language, qualify mechanistic interpretations more carefully, and restrict causal inferences to the measured outcomes. Where oxidative stress or antioxidant effects are mentioned, these are now presented explicitly as literature-supported hypotheses rather than direct findings of the present study. We have also included this into our limitation. 13. Comment: Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. Response: We sincerely thank the reviewer for this valuable comment. We acknowledge that some outcomes, particularly sperm count, showed relatively large standard deviations. We carefully re-examined the raw data and confirmed that these values were accurate and not due to transcription error. We elected to retain the original values to preserve transparency and to avoid underreporting biological variability. We agree that the relatively small group size may have contributed to the wide dispersion observed. Accordingly, we used non-parametric statistical analysis (Kruskal-Wallis followed by Dunn’s post hoc test) and revised the text to ensure that the findings are interpreted cautiously. However, we also include this topic into our limitation to keep our study transparent. 14. Comment: The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this important comment. We have carefully re-examined the entire reference list and corrected the identified errors, including the duplicated title in Reference 3, the incomplete author and journal information in Reference 20, and inconsistencies in journal-name formatting. The references have now been standardized throughout the manuscript to ensure consistency and accuracy. Furthermore, we revised the Discussion extensively to strengthen the scientific presentation of the work and to ensure that the findings are interpreted in light of relevant and current literature. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), ... Continue reading 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) Response: We sincerely thank the reviewer for identifying this inconsistency. We agree that the original statistical description contained a methodological contradiction. Although the manuscript incorrectly stated that one-way ANOVA with LSD post hoc testing was used, the analyses actually performed in this study were Kruskal-Wallis tests followed by Dunn’s multiple-comparison post hoc tests, consistent with the non-parametric nature of the data. This error has now been corrected in the revised manuscript, and the statistical methods have been rewritten to accurately reflect the analyses conducted. We have also carefully reviewed the Results section and tables to ensure consistency with the corrected statistical approach. 2. Comment: Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. Response: We sincerely thank the reviewer for this careful and important comment. We agree that it is not appropriate to interpret non-significant differences as definite improvements. In response, we have revised the Results and Discussion sections to ensure that only statistically significant findings are described as significant improvements, whereas outcomes with p > 0.05 are now reported more cautiously as numerical differences without statistical significance. For example, the statements regarding sperm parameters have been corrected accordingly. We have also rechecked the manuscript throughout to ensure consistency between the statistical results and their interpretation. 3. Comment: The term “gland penis” is incorrect. Should be "glans penis". Response: We sincerely thank the reviewer for this valuable comment. We agree that the correct anatomical term is “glans penis” rather than “gland penis.” The manuscript has been revised accordingly in the Materials and Methods section to ensure anatomical accuracy. 4. Comment: The term "male kisses" is non-scientific. Rephrase appropriately. Response: We sincerely thank the reviewer for this valuable comment. We agree that the expression “male kisses” is not appropriate for scientific reporting of animal sexual behavior. In the revised manuscript, we have replaced this phrase with more formal and objective terminology to describe the observed behavior in the Libido Assessment subsection. We changed the sentence into “Courtship latency was defined as the time from opening of the partition until the male initiated investigatory or courtship behavior toward the female, including anogenital sniffing or contact with the female’s body.” 5. Comment: Include units for sperm count and fluid intake. Response: We sincerely thank the reviewer for this valuable observation. We agree that the omission of units for sperm count and fluid intake reduced the clarity of data presentation. In response, we have revised the manuscript into units of millions/mL to include the appropriate units for these variables throughout the text and tables. 6. Comment: Avoid overgeneralization. Response : We sincerely thank the reviewer for this valuable observation. We agree that the original manuscript contained several statements that were overly broad in relation to the data presented. In response, we have revised the manuscript throughout to ensure that the conclusions are stated more cautiously and are fully supported by the results. Specifically, we avoided definitive claims, reduced overstatement of non-significant findings, and clarified that the observed effects should be interpreted as dose-dependent, endpoint-specific, and preliminary findings in a short-term alloxan-induced diabetic rat model. 7. Comment: There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. Response : We sincerely thank the reviewer for this valuable comment. We agree that the earlier wording may have oversimplified the published evidence. In the revised manuscript, we clarified that the available literature suggests complex and context-dependent effects of piperine, rather than uniformly beneficial or uniformly harmful reproductive effects. We now explicitly state that piperine may enhance Leydig-cell function and steroidogenic activity while, under different experimental conditions, adversely affecting spermatogenesis. We therefore revised the Discussion to reflect that these findings represent different reproductive endpoints and may vary according to dose, exposure duration, and study conditions. 8. Comment : "ddp-4" should be written as "DDP-4" Response: We sincerely thank the reviewer for this careful observation. We agree that the abbreviation should be written consistently in uppercase. In the revised manuscript, “dpp-4” has been corrected to “DPP-4” throughout the text. 9. Comment : Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. Response: We sincerely thank the reviewer for this important comment. In response, we further revised the Discussion to more explicitly establish the relevance of the oxidative stress pathway and nitric oxide signaling to the findings of the present study. Specifically, we clarified that hyperglycemia-induced oxidative stress can reduce nitric oxide bioavailability and impair eNOS-mediated endothelial signaling in the corpus cavernosum, which contributes to diabetic erectile dysfunction. We also strengthened the discussion linking the directional increase in eNOS mRNA expression after black pepper treatment with the possibility of partial restoration of endothelial NO signaling. In addition, we added relevant recent literature to support the mechanistic relationship among oxidative stress, endothelial dysfunction, and impaired erectile function in diabetes. 10. Comment: Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" Response: We sincerely thank the reviewer for this valuable observation. We agree that the previous Conclusion was somewhat overstated. In the revised manuscript, we have rewritten the Conclusion to refer specifically to the measured outcomes, including total penile reflexes, libido-related parameters, intratesticular testosterone, sperm concentration, sperm morphology, Leydig cell count, and eNOS expression, rather than making broad claims about diabetic male reproductive dysfunction overall. We have also revised the language to ensure that the findings are interpreted as dose-dependent, endpoint-specific, and preliminary. However, we don’t used the term “randomized” in our manuscript anymore. 11. Comment: The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " Response: We sincerely thank the reviewer for this valuable comment. We agree that the original phrasing implied a stronger causal interpretation than was warranted. In the revised Discussion, we have replaced “known to be the cause of” with newer sentences in our revised manuscript. 12. Comment: The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. Response: We sincerely thank the reviewer for this valuable observation. We agree that the original Discussion contained mechanistic statements that could be interpreted as stronger than warranted by the available data. In the revised manuscript, we have clarified that intratesticular testosterone was directly measured and may therefore be discussed as an observed endpoint, whereas oxidative stress-related mechanisms were not directly evaluated, as no assays such as SOD, catalase, or MDA were performed. We therefore revised the Discussion to remove over-speculative language, qualify mechanistic interpretations more carefully, and restrict causal inferences to the measured outcomes. Where oxidative stress or antioxidant effects are mentioned, these are now presented explicitly as literature-supported hypotheses rather than direct findings of the present study. We have also included this into our limitation. 13. Comment: Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. Response: We sincerely thank the reviewer for this valuable comment. We acknowledge that some outcomes, particularly sperm count, showed relatively large standard deviations. We carefully re-examined the raw data and confirmed that these values were accurate and not due to transcription error. We elected to retain the original values to preserve transparency and to avoid underreporting biological variability. We agree that the relatively small group size may have contributed to the wide dispersion observed. Accordingly, we used non-parametric statistical analysis (Kruskal-Wallis followed by Dunn’s post hoc test) and revised the text to ensure that the findings are interpreted cautiously. However, we also include this topic into our limitation to keep our study transparent. 14. Comment: The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this important comment. We have carefully re-examined the entire reference list and corrected the identified errors, including the duplicated title in Reference 3, the incomplete author and journal information in Reference 20, and inconsistencies in journal-name formatting. The references have now been standardized throughout the manuscript to ensure consistency and accuracy. Furthermore, we revised the Discussion extensively to strengthen the scientific presentation of the work and to ensure that the findings are interpreted in light of relevant and current literature. 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) Response: We sincerely thank the reviewer for identifying this inconsistency. We agree that the original statistical description contained a methodological contradiction. Although the manuscript incorrectly stated that one-way ANOVA with LSD post hoc testing was used, the analyses actually performed in this study were Kruskal-Wallis tests followed by Dunn’s multiple-comparison post hoc tests, consistent with the non-parametric nature of the data. This error has now been corrected in the revised manuscript, and the statistical methods have been rewritten to accurately reflect the analyses conducted. We have also carefully reviewed the Results section and tables to ensure consistency with the corrected statistical approach. 2. Comment: Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. Response: We sincerely thank the reviewer for this careful and important comment. We agree that it is not appropriate to interpret non-significant differences as definite improvements. In response, we have revised the Results and Discussion sections to ensure that only statistically significant findings are described as significant improvements, whereas outcomes with p > 0.05 are now reported more cautiously as numerical differences without statistical significance. For example, the statements regarding sperm parameters have been corrected accordingly. We have also rechecked the manuscript throughout to ensure consistency between the statistical results and their interpretation. 3. Comment: The term “gland penis” is incorrect. Should be "glans penis". Response: We sincerely thank the reviewer for this valuable comment. We agree that the correct anatomical term is “glans penis” rather than “gland penis.” The manuscript has been revised accordingly in the Materials and Methods section to ensure anatomical accuracy. 4. Comment: The term "male kisses" is non-scientific. Rephrase appropriately. Response: We sincerely thank the reviewer for this valuable comment. We agree that the expression “male kisses” is not appropriate for scientific reporting of animal sexual behavior. In the revised manuscript, we have replaced this phrase with more formal and objective terminology to describe the observed behavior in the Libido Assessment subsection. We changed the sentence into “Courtship latency was defined as the time from opening of the partition until the male initiated investigatory or courtship behavior toward the female, including anogenital sniffing or contact with the female’s body.” 5. Comment: Include units for sperm count and fluid intake. Response: We sincerely thank the reviewer for this valuable observation. We agree that the omission of units for sperm count and fluid intake reduced the clarity of data presentation. In response, we have revised the manuscript into units of millions/mL to include the appropriate units for these variables throughout the text and tables. 6. Comment: Avoid overgeneralization. Response : We sincerely thank the reviewer for this valuable observation. We agree that the original manuscript contained several statements that were overly broad in relation to the data presented. In response, we have revised the manuscript throughout to ensure that the conclusions are stated more cautiously and are fully supported by the results. Specifically, we avoided definitive claims, reduced overstatement of non-significant findings, and clarified that the observed effects should be interpreted as dose-dependent, endpoint-specific, and preliminary findings in a short-term alloxan-induced diabetic rat model. 7. Comment: There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. Response : We sincerely thank the reviewer for this valuable comment. We agree that the earlier wording may have oversimplified the published evidence. In the revised manuscript, we clarified that the available literature suggests complex and context-dependent effects of piperine, rather than uniformly beneficial or uniformly harmful reproductive effects. We now explicitly state that piperine may enhance Leydig-cell function and steroidogenic activity while, under different experimental conditions, adversely affecting spermatogenesis. We therefore revised the Discussion to reflect that these findings represent different reproductive endpoints and may vary according to dose, exposure duration, and study conditions. 8. Comment : "ddp-4" should be written as "DDP-4" Response: We sincerely thank the reviewer for this careful observation. We agree that the abbreviation should be written consistently in uppercase. In the revised manuscript, “dpp-4” has been corrected to “DPP-4” throughout the text. 9. Comment : Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. Response: We sincerely thank the reviewer for this important comment. In response, we further revised the Discussion to more explicitly establish the relevance of the oxidative stress pathway and nitric oxide signaling to the findings of the present study. Specifically, we clarified that hyperglycemia-induced oxidative stress can reduce nitric oxide bioavailability and impair eNOS-mediated endothelial signaling in the corpus cavernosum, which contributes to diabetic erectile dysfunction. We also strengthened the discussion linking the directional increase in eNOS mRNA expression after black pepper treatment with the possibility of partial restoration of endothelial NO signaling. In addition, we added relevant recent literature to support the mechanistic relationship among oxidative stress, endothelial dysfunction, and impaired erectile function in diabetes. 10. Comment: Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" Response: We sincerely thank the reviewer for this valuable observation. We agree that the previous Conclusion was somewhat overstated. In the revised manuscript, we have rewritten the Conclusion to refer specifically to the measured outcomes, including total penile reflexes, libido-related parameters, intratesticular testosterone, sperm concentration, sperm morphology, Leydig cell count, and eNOS expression, rather than making broad claims about diabetic male reproductive dysfunction overall. We have also revised the language to ensure that the findings are interpreted as dose-dependent, endpoint-specific, and preliminary. However, we don’t used the term “randomized” in our manuscript anymore. 11. Comment: The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " Response: We sincerely thank the reviewer for this valuable comment. We agree that the original phrasing implied a stronger causal interpretation than was warranted. In the revised Discussion, we have replaced “known to be the cause of” with newer sentences in our revised manuscript. 12. Comment: The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. Response: We sincerely thank the reviewer for this valuable observation. We agree that the original Discussion contained mechanistic statements that could be interpreted as stronger than warranted by the available data. In the revised manuscript, we have clarified that intratesticular testosterone was directly measured and may therefore be discussed as an observed endpoint, whereas oxidative stress-related mechanisms were not directly evaluated, as no assays such as SOD, catalase, or MDA were performed. We therefore revised the Discussion to remove over-speculative language, qualify mechanistic interpretations more carefully, and restrict causal inferences to the measured outcomes. Where oxidative stress or antioxidant effects are mentioned, these are now presented explicitly as literature-supported hypotheses rather than direct findings of the present study. We have also included this into our limitation. 13. Comment: Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. Response: We sincerely thank the reviewer for this valuable comment. We acknowledge that some outcomes, particularly sperm count, showed relatively large standard deviations. We carefully re-examined the raw data and confirmed that these values were accurate and not due to transcription error. We elected to retain the original values to preserve transparency and to avoid underreporting biological variability. We agree that the relatively small group size may have contributed to the wide dispersion observed. Accordingly, we used non-parametric statistical analysis (Kruskal-Wallis followed by Dunn’s post hoc test) and revised the text to ensure that the findings are interpreted cautiously. However, we also include this topic into our limitation to keep our study transparent. 14. Comment: The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this important comment. We have carefully re-examined the entire reference list and corrected the identified errors, including the duplicated title in Reference 3, the incomplete author and journal information in Reference 20, and inconsistencies in journal-name formatting. The references have now been standardized throughout the manuscript to ensure consistency and accuracy. Furthermore, we revised the Discussion extensively to strengthen the scientific presentation of the work and to ensure that the findings are interpreted in light of relevant and current literature. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Obi PE. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458227 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458227 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Mar 2026 Patrick Ebele Obi , Enugu State University of Science and Technology, Enugu, Enugu, Nigeria Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.190377.r458227 1. The title of the research article should not be too conclusive. "effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......'' should be more appropriate. 2. Introduction: The authors did not write enough information ... Continue reading READ ALL 1. The title of the research article should not be too conclusive. "effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......'' should be more appropriate. 2. Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. 3. The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). 4. There are some grammatical errors under '' Plant preparation" that need correction. For instance, "The fresh fruit is washed and rinsed until clean and then dried" 5. The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. 6. The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. 7. Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. 8. The authors initially stated under 'animal preparation' that the study used 30 male Sprague Dawley rats but later mentioned female rats under 'libido assessment'. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. 9. The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: ■Phytoevaluation ■ Phytochemistry ■ Phytomedicine ■ Drug Discovery and Development I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Obi PE. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458227 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458227 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: ... Continue reading 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: We sincerely thank the reviewer for this valuable suggestion. We agree that the original title was overly conclusive in its wording. In response, we revised the title from “Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats” to “Effects of Black Pepper (Piper nigrum L.) Fruit Extract on Sexual Function, Hormonal Parameters, Sperm Parameters, Testicular Histology, and eNOS Expression in Alloxan-Induced Diabetic Rats.” We believe that the revised title is more neutral, scientifically appropriate, and more representative of the full range of outcomes assessed in the present study. 2. Comment: Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. Response: We are grateful to the reviewer for this constructive comment. In response, we expanded the Introduction by adding further information on erectile dysfunction, particularly in the context of diabetes mellitus. The revised text now includes additional discussion of the pathophysiological mechanisms underlying diabetic erectile dysfunction, including endothelial dysfunction, oxidative stress, neuropathic changes, hormonal disturbances, and structural alterations in penile tissue. We believe that these additions provide a clearer clinical and biological rationale for the present study. 3. Comment: The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). Response: We thank the reviewer for this valuable comment. We acknowledge that the treatment regimens were not described with sufficient clarity in the original manuscript. In the revised manuscript, we clarified that black pepper extract was administered daily for eight consecutive days as the test intervention, whereas sildenafil citrate was given as a single dose on the ninth day, one hour before erectile function and libido assessment, as an acute positive control. Thus, the difference in administration schedule was intentional and reflected the distinct role of sildenafil as a reference treatment for erectile function rather than as a chronic intervention comparable to black pepper extract. The Materials and Methods section, particularly the Animal Preparation subsection, has been revised accordingly for clarity. 4. Comment : There are some grammatical errors under “Plant preparation” that need correction. For instance, “The fresh fruit is washed and rinsed until clean and then dried”. Response : We thank the reviewer for this valuable comment. We carefully revised the Plant Preparation subsection to correct the grammatical errors and improve sentence structure, tense consistency, and overall clarity. In particular, the sentence noted by the reviewer has been revised to conform to formal scientific writing style. 5. Comment: The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1 in the Materials and Methods section under the Animal Preparation subsection and include both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 6. Comment: The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. Response: We sincerely thank the reviewer for this valuable comment. We agree that the duration of the present study was relatively short and may not fully reflect the chronic progression of diabetes-related erectile dysfunction, which typically develops gradually over time. However, the selection of day 9 after alloxan induction as the evaluation time point was based on considerations related to the stability of the diabetic model. At this stage, the animals had already passed the period of acute systemic toxicity associated with alloxan administration, so the data obtained were intended to reflect a more stable sub-acute hyperglycemic state rather than direct drug-induced toxic effects. In addition, this time point was considered sufficient to allow early structural changes in pancreatic β-cells, stabilization of oxidative stress-related changes, and regulation of relevant gene expression, thereby providing a suitable basis for assessment of both pathological progression and the early effects of the test compound. To ensure consistency of the diabetic state before final outcome assessment, fasting blood glucose levels were confirmed on day 3 and day 7 after alloxan induction and used as inclusion criteria to verify that all study animals remained in a stable diabetic condition before parameter evaluation on day 9. Moreover, because one of the aims of this study was to evaluate gene expression, particularly eNOS mRNA expression, day 9 was considered an appropriate time point at which hyperglycemia-related oxidative stress and pro-inflammatory signaling pathways would already be active, while tissue injury was still in an early and potentially modifiable phase. This timing was also considered sufficient to allow the test compounds to interact with relevant biochemical and cellular pathways before more advanced and possibly irreversible structural damage developed. Nevertheless, we agree that this represents a short-term experimental study, and we have revised the manuscript to emphasize that the findings should not be interpreted as evidence of reversal of long-standing chronic diabetic complications. We have also acknowledged this issue as a limitation in the Discussion and indicated that future studies with longer treatment duration and follow-up are necessary. 7. Comment: Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. Response: We sincerely thank the reviewer for this valuable comment. We agree that reporting the phytoconstituents of the black pepper extract is important to support interpretation of its biological activity. In response, we have now included LC-MS analysis of the extract used in this study. The corresponding analytical procedure has been described in the Materials and Methods section, and the results are presented in Table. These additions provide direct characterization of the tested extract and strengthen the revised manuscript. 8. Comment: The authors initially stated under “animal preparation” that the study used 30 male Sprague Dawley rats but later mentioned female rats under “libido assessment”. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 9. Comment: The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this valuable comment. In response, we substantially revised the Discussion section to improve the clarity, depth, and accuracy of interpretation of the study findings. Specifically, we expanded the discussion of each major outcome, aligned the interpretation more closely with the data presented in the tables, and incorporated more current and relevant literature to support comparison with previous studies and to explain the possible biological mechanisms involved. We also revised several statements to ensure that the conclusions are presented more cautiously and accurately, particularly in view of the short-term nature of the diabetic model and the endpoint-specific effects observed. We believe that these revisions have improved the scientific presentation of the work and strengthened the overall clarity of the manuscript. 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: We sincerely thank the reviewer for this valuable suggestion. We agree that the original title was overly conclusive in its wording. In response, we revised the title from “Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats” to “Effects of Black Pepper (Piper nigrum L.) Fruit Extract on Sexual Function, Hormonal Parameters, Sperm Parameters, Testicular Histology, and eNOS Expression in Alloxan-Induced Diabetic Rats.” We believe that the revised title is more neutral, scientifically appropriate, and more representative of the full range of outcomes assessed in the present study. 2. Comment: Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. Response: We are grateful to the reviewer for this constructive comment. In response, we expanded the Introduction by adding further information on erectile dysfunction, particularly in the context of diabetes mellitus. The revised text now includes additional discussion of the pathophysiological mechanisms underlying diabetic erectile dysfunction, including endothelial dysfunction, oxidative stress, neuropathic changes, hormonal disturbances, and structural alterations in penile tissue. We believe that these additions provide a clearer clinical and biological rationale for the present study. 3. Comment: The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). Response: We thank the reviewer for this valuable comment. We acknowledge that the treatment regimens were not described with sufficient clarity in the original manuscript. In the revised manuscript, we clarified that black pepper extract was administered daily for eight consecutive days as the test intervention, whereas sildenafil citrate was given as a single dose on the ninth day, one hour before erectile function and libido assessment, as an acute positive control. Thus, the difference in administration schedule was intentional and reflected the distinct role of sildenafil as a reference treatment for erectile function rather than as a chronic intervention comparable to black pepper extract. The Materials and Methods section, particularly the Animal Preparation subsection, has been revised accordingly for clarity. 4. Comment : There are some grammatical errors under “Plant preparation” that need correction. For instance, “The fresh fruit is washed and rinsed until clean and then dried”. Response : We thank the reviewer for this valuable comment. We carefully revised the Plant Preparation subsection to correct the grammatical errors and improve sentence structure, tense consistency, and overall clarity. In particular, the sentence noted by the reviewer has been revised to conform to formal scientific writing style. 5. Comment: The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1 in the Materials and Methods section under the Animal Preparation subsection and include both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 6. Comment: The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. Response: We sincerely thank the reviewer for this valuable comment. We agree that the duration of the present study was relatively short and may not fully reflect the chronic progression of diabetes-related erectile dysfunction, which typically develops gradually over time. However, the selection of day 9 after alloxan induction as the evaluation time point was based on considerations related to the stability of the diabetic model. At this stage, the animals had already passed the period of acute systemic toxicity associated with alloxan administration, so the data obtained were intended to reflect a more stable sub-acute hyperglycemic state rather than direct drug-induced toxic effects. In addition, this time point was considered sufficient to allow early structural changes in pancreatic β-cells, stabilization of oxidative stress-related changes, and regulation of relevant gene expression, thereby providing a suitable basis for assessment of both pathological progression and the early effects of the test compound. To ensure consistency of the diabetic state before final outcome assessment, fasting blood glucose levels were confirmed on day 3 and day 7 after alloxan induction and used as inclusion criteria to verify that all study animals remained in a stable diabetic condition before parameter evaluation on day 9. Moreover, because one of the aims of this study was to evaluate gene expression, particularly eNOS mRNA expression, day 9 was considered an appropriate time point at which hyperglycemia-related oxidative stress and pro-inflammatory signaling pathways would already be active, while tissue injury was still in an early and potentially modifiable phase. This timing was also considered sufficient to allow the test compounds to interact with relevant biochemical and cellular pathways before more advanced and possibly irreversible structural damage developed. Nevertheless, we agree that this represents a short-term experimental study, and we have revised the manuscript to emphasize that the findings should not be interpreted as evidence of reversal of long-standing chronic diabetic complications. We have also acknowledged this issue as a limitation in the Discussion and indicated that future studies with longer treatment duration and follow-up are necessary. 7. Comment: Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. Response: We sincerely thank the reviewer for this valuable comment. We agree that reporting the phytoconstituents of the black pepper extract is important to support interpretation of its biological activity. In response, we have now included LC-MS analysis of the extract used in this study. The corresponding analytical procedure has been described in the Materials and Methods section, and the results are presented in Table. These additions provide direct characterization of the tested extract and strengthen the revised manuscript. 8. Comment: The authors initially stated under “animal preparation” that the study used 30 male Sprague Dawley rats but later mentioned female rats under “libido assessment”. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 9. Comment: The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this valuable comment. In response, we substantially revised the Discussion section to improve the clarity, depth, and accuracy of interpretation of the study findings. Specifically, we expanded the discussion of each major outcome, aligned the interpretation more closely with the data presented in the tables, and incorporated more current and relevant literature to support comparison with previous studies and to explain the possible biological mechanisms involved. We also revised several statements to ensure that the conclusions are presented more cautiously and accurately, particularly in view of the short-term nature of the diabetic model and the endpoint-specific effects observed. We believe that these revisions have improved the scientific presentation of the work and strengthened the overall clarity of the manuscript. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: ... Continue reading 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: We sincerely thank the reviewer for this valuable suggestion. We agree that the original title was overly conclusive in its wording. In response, we revised the title from “Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats” to “Effects of Black Pepper (Piper nigrum L.) Fruit Extract on Sexual Function, Hormonal Parameters, Sperm Parameters, Testicular Histology, and eNOS Expression in Alloxan-Induced Diabetic Rats.” We believe that the revised title is more neutral, scientifically appropriate, and more representative of the full range of outcomes assessed in the present study. 2. Comment: Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. Response: We are grateful to the reviewer for this constructive comment. In response, we expanded the Introduction by adding further information on erectile dysfunction, particularly in the context of diabetes mellitus. The revised text now includes additional discussion of the pathophysiological mechanisms underlying diabetic erectile dysfunction, including endothelial dysfunction, oxidative stress, neuropathic changes, hormonal disturbances, and structural alterations in penile tissue. We believe that these additions provide a clearer clinical and biological rationale for the present study. 3. Comment: The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). Response: We thank the reviewer for this valuable comment. We acknowledge that the treatment regimens were not described with sufficient clarity in the original manuscript. In the revised manuscript, we clarified that black pepper extract was administered daily for eight consecutive days as the test intervention, whereas sildenafil citrate was given as a single dose on the ninth day, one hour before erectile function and libido assessment, as an acute positive control. Thus, the difference in administration schedule was intentional and reflected the distinct role of sildenafil as a reference treatment for erectile function rather than as a chronic intervention comparable to black pepper extract. The Materials and Methods section, particularly the Animal Preparation subsection, has been revised accordingly for clarity. 4. Comment : There are some grammatical errors under “Plant preparation” that need correction. For instance, “The fresh fruit is washed and rinsed until clean and then dried”. Response : We thank the reviewer for this valuable comment. We carefully revised the Plant Preparation subsection to correct the grammatical errors and improve sentence structure, tense consistency, and overall clarity. In particular, the sentence noted by the reviewer has been revised to conform to formal scientific writing style. 5. Comment: The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1 in the Materials and Methods section under the Animal Preparation subsection and include both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 6. Comment: The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. Response: We sincerely thank the reviewer for this valuable comment. We agree that the duration of the present study was relatively short and may not fully reflect the chronic progression of diabetes-related erectile dysfunction, which typically develops gradually over time. However, the selection of day 9 after alloxan induction as the evaluation time point was based on considerations related to the stability of the diabetic model. At this stage, the animals had already passed the period of acute systemic toxicity associated with alloxan administration, so the data obtained were intended to reflect a more stable sub-acute hyperglycemic state rather than direct drug-induced toxic effects. In addition, this time point was considered sufficient to allow early structural changes in pancreatic β-cells, stabilization of oxidative stress-related changes, and regulation of relevant gene expression, thereby providing a suitable basis for assessment of both pathological progression and the early effects of the test compound. To ensure consistency of the diabetic state before final outcome assessment, fasting blood glucose levels were confirmed on day 3 and day 7 after alloxan induction and used as inclusion criteria to verify that all study animals remained in a stable diabetic condition before parameter evaluation on day 9. Moreover, because one of the aims of this study was to evaluate gene expression, particularly eNOS mRNA expression, day 9 was considered an appropriate time point at which hyperglycemia-related oxidative stress and pro-inflammatory signaling pathways would already be active, while tissue injury was still in an early and potentially modifiable phase. This timing was also considered sufficient to allow the test compounds to interact with relevant biochemical and cellular pathways before more advanced and possibly irreversible structural damage developed. Nevertheless, we agree that this represents a short-term experimental study, and we have revised the manuscript to emphasize that the findings should not be interpreted as evidence of reversal of long-standing chronic diabetic complications. We have also acknowledged this issue as a limitation in the Discussion and indicated that future studies with longer treatment duration and follow-up are necessary. 7. Comment: Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. Response: We sincerely thank the reviewer for this valuable comment. We agree that reporting the phytoconstituents of the black pepper extract is important to support interpretation of its biological activity. In response, we have now included LC-MS analysis of the extract used in this study. The corresponding analytical procedure has been described in the Materials and Methods section, and the results are presented in Table. These additions provide direct characterization of the tested extract and strengthen the revised manuscript. 8. Comment: The authors initially stated under “animal preparation” that the study used 30 male Sprague Dawley rats but later mentioned female rats under “libido assessment”. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 9. Comment: The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this valuable comment. In response, we substantially revised the Discussion section to improve the clarity, depth, and accuracy of interpretation of the study findings. Specifically, we expanded the discussion of each major outcome, aligned the interpretation more closely with the data presented in the tables, and incorporated more current and relevant literature to support comparison with previous studies and to explain the possible biological mechanisms involved. We also revised several statements to ensure that the conclusions are presented more cautiously and accurately, particularly in view of the short-term nature of the diabetic model and the endpoint-specific effects observed. We believe that these revisions have improved the scientific presentation of the work and strengthened the overall clarity of the manuscript. 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: We sincerely thank the reviewer for this valuable suggestion. We agree that the original title was overly conclusive in its wording. In response, we revised the title from “Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats” to “Effects of Black Pepper (Piper nigrum L.) Fruit Extract on Sexual Function, Hormonal Parameters, Sperm Parameters, Testicular Histology, and eNOS Expression in Alloxan-Induced Diabetic Rats.” We believe that the revised title is more neutral, scientifically appropriate, and more representative of the full range of outcomes assessed in the present study. 2. Comment: Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. Response: We are grateful to the reviewer for this constructive comment. In response, we expanded the Introduction by adding further information on erectile dysfunction, particularly in the context of diabetes mellitus. The revised text now includes additional discussion of the pathophysiological mechanisms underlying diabetic erectile dysfunction, including endothelial dysfunction, oxidative stress, neuropathic changes, hormonal disturbances, and structural alterations in penile tissue. We believe that these additions provide a clearer clinical and biological rationale for the present study. 3. Comment: The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). Response: We thank the reviewer for this valuable comment. We acknowledge that the treatment regimens were not described with sufficient clarity in the original manuscript. In the revised manuscript, we clarified that black pepper extract was administered daily for eight consecutive days as the test intervention, whereas sildenafil citrate was given as a single dose on the ninth day, one hour before erectile function and libido assessment, as an acute positive control. Thus, the difference in administration schedule was intentional and reflected the distinct role of sildenafil as a reference treatment for erectile function rather than as a chronic intervention comparable to black pepper extract. The Materials and Methods section, particularly the Animal Preparation subsection, has been revised accordingly for clarity. 4. Comment : There are some grammatical errors under “Plant preparation” that need correction. For instance, “The fresh fruit is washed and rinsed until clean and then dried”. Response : We thank the reviewer for this valuable comment. We carefully revised the Plant Preparation subsection to correct the grammatical errors and improve sentence structure, tense consistency, and overall clarity. In particular, the sentence noted by the reviewer has been revised to conform to formal scientific writing style. 5. Comment: The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1 in the Materials and Methods section under the Animal Preparation subsection and include both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 6. Comment: The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. Response: We sincerely thank the reviewer for this valuable comment. We agree that the duration of the present study was relatively short and may not fully reflect the chronic progression of diabetes-related erectile dysfunction, which typically develops gradually over time. However, the selection of day 9 after alloxan induction as the evaluation time point was based on considerations related to the stability of the diabetic model. At this stage, the animals had already passed the period of acute systemic toxicity associated with alloxan administration, so the data obtained were intended to reflect a more stable sub-acute hyperglycemic state rather than direct drug-induced toxic effects. In addition, this time point was considered sufficient to allow early structural changes in pancreatic β-cells, stabilization of oxidative stress-related changes, and regulation of relevant gene expression, thereby providing a suitable basis for assessment of both pathological progression and the early effects of the test compound. To ensure consistency of the diabetic state before final outcome assessment, fasting blood glucose levels were confirmed on day 3 and day 7 after alloxan induction and used as inclusion criteria to verify that all study animals remained in a stable diabetic condition before parameter evaluation on day 9. Moreover, because one of the aims of this study was to evaluate gene expression, particularly eNOS mRNA expression, day 9 was considered an appropriate time point at which hyperglycemia-related oxidative stress and pro-inflammatory signaling pathways would already be active, while tissue injury was still in an early and potentially modifiable phase. This timing was also considered sufficient to allow the test compounds to interact with relevant biochemical and cellular pathways before more advanced and possibly irreversible structural damage developed. Nevertheless, we agree that this represents a short-term experimental study, and we have revised the manuscript to emphasize that the findings should not be interpreted as evidence of reversal of long-standing chronic diabetic complications. We have also acknowledged this issue as a limitation in the Discussion and indicated that future studies with longer treatment duration and follow-up are necessary. 7. Comment: Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. Response: We sincerely thank the reviewer for this valuable comment. We agree that reporting the phytoconstituents of the black pepper extract is important to support interpretation of its biological activity. In response, we have now included LC-MS analysis of the extract used in this study. The corresponding analytical procedure has been described in the Materials and Methods section, and the results are presented in Table. These additions provide direct characterization of the tested extract and strengthen the revised manuscript. 8. Comment: The authors initially stated under “animal preparation” that the study used 30 male Sprague Dawley rats but later mentioned female rats under “libido assessment”. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 9. Comment: The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this valuable comment. In response, we substantially revised the Discussion section to improve the clarity, depth, and accuracy of interpretation of the study findings. Specifically, we expanded the discussion of each major outcome, aligned the interpretation more closely with the data presented in the tables, and incorporated more current and relevant literature to support comparison with previous studies and to explain the possible biological mechanisms involved. We also revised several statements to ensure that the conclusions are presented more cautiously and accurately, particularly in view of the short-term nature of the diabetic model and the endpoint-specific effects observed. We believe that these revisions have improved the scientific presentation of the work and strengthened the overall clarity of the manuscript. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Nwagwe OR. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r455996 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-455996 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 12 Mar 2026 Onyinyechi Ruth Nwagwe , Federal University, Oye-Ekiti,, Oye-Ekiti,, Nigeria Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.190377.r455996 The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So ... Continue reading READ ALL The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. I didn't see the anaesthesia used for the rats after the treatment . at the Methods area, Kindly recast the first sentence. Specify the kind of rats u used whether male/female. Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Which type of DMwas identified in the rats 24 hours after the alloxan induction? 8days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Why was there no graph of the rats blood glucose levels? For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Again , why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Also, why did you not carry out the bioactive composition of the plant? Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Nwagwe OR. Reviewer Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r455996 ) The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-455996 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer ... Continue reading 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer for this valuable suggestion. In response, we revised the Introduction to incorporate the most recent epidemiological data on diabetes mellitus from the International Diabetes Federation (IDF) Diabetes Atlas, 11th edition, published in 2025. We clarified that the latest global estimate available in this report refers to 2024, as no separate worldwide estimate specifically for 2025 is currently provided. In addition, we included the projected peak global burden of diabetes reported in the same source, which estimates that the number of adults living with diabetes will increase to 853 million by 2050. 2. Comment: There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. Response: We are grateful to the reviewer for this important and constructive comment. In response, we substantially revised the Introduction by adding a dedicated paragraph to strengthen the scientific rationale for the use of black pepper in the present animal model. Specifically, we incorporated additional in vitro and in vivo evidence regarding piperine, the principal bioactive alkaloid of Piper nigrum, to support the biological plausibility of black pepper extract as a candidate intervention. We also clarified in the revised text that these preclinical studies were included to provide mechanistic and experimental justification for the intervention, rather than to imply definitive therapeutic superiority. 3. Comment: I didn't see the anaesthesia used for the rats after the treatment. at the Methods area, Kindly recast the first sentence. Response: We thank the reviewer for this valuable comment. We apologize that the procedure was not clearly described in the original manuscript. In the revised Methods section, we clarified the terminal procedure used after completion of the treatment period and all functional assessments. Specifically, the text has been revised to clearly state that the animals were euthanized in accordance with the approved institutional ethics protocol. 4. Comment: Specify the kind of rats u used whether male/female. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused some confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 5. Comment : Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Response: We thank the reviewer for this valuable comment. We agree that inclusion of the full identifier name, herbarium deposition details, and voucher specimen number would improve the botanical documentation of the study. However, these records were not available for the present work. We therefore clarified the available plant source information in the revised manuscript and acknowledged the absence of formal voucher documentation as a limitation of the study. 6. Comment: Which type of DM was identified in the rats 24 hours after the alloxan induction? 8 days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Response: We sincerely thank the reviewer for this important and thoughtful comment. We clarified in the revised manuscript that the alloxan-induced diabetic rat model used in this study represents an insulin-deficient, type 1-like diabetes model, because alloxan selectively damages pancreatic β-cells and is widely used to induce experimental insulin-deficient diabetes in rodents. We also agree that an 8-day treatment period is relatively short and may not fully reproduce the chronic pathophysiology of long-standing diabetes-related sexual dysfunction. Therefore, we revised the manuscript to emphasize that the present work should be interpreted as a short-term experimental study designed to evaluate early changes in hyperglycemia and sexual/reproductive parameters after alloxan induction, rather than as evidence of complete reversal of established chronic diabetic complications. In addition, we added a more cautious statement in the Discussion noting that, although early improvements in blood glucose and functional parameters may be observed in experimental animal studies, these findings should be interpreted carefully and require confirmation in studies with longer treatment duration and follow-up. 7. Comment: Why was there no graph of the rats blood glucose levels? Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1, which includes both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 8. Comment: For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. Response: We sincerely thank the reviewer for this valuable comment. We agree that intromission number and intromission latency are relevant parameters in sexual behavior studies. In the present study, however, the behavioral assessment was prospectively focused on courtship latency, mount latency, and mount frequency as the predefined libido-related outcomes, and intromission-related parameters were therefore not systematically collected for quantitative analysis. We acknowledge this as a limitation and have clarified this point in the revised manuscript. In addition, the behavioral observations were performed using CCTV-based recording, and in response to the reviewer’s suggestion, we have provided representative recording examples and images in our Figshare (https://doi.org/10.6084/m9.figshare.30456353) as supporting documentation. 9. Comment: The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Response: We sincerely thank the reviewer for this valuable comment. The histological evaluation in the present study was intentionally limited to the testis, because the primary objective of the study was to assess the effects of black pepper extract on male sexual and reproductive function rather than on general diabetic organ injury. Since sperm parameters, testosterone levels, and sexual behavior were central outcomes of this study, testicular histology was considered the most relevant tissue-level endpoint. Nevertheless, we agree that examination of other organs commonly affected by diabetes, including the pancreas, kidney, liver, and penile tissue, would have strengthened the study by providing additional evidence on the systemic effects of the extract. We have therefore acknowledged this as a limitation in the revised manuscript. 10. Comment: Again, why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Response: We sincerely thank the reviewer for this valuable comment. We agree that inclusion of additional sexual hormones and biochemical parameters would have strengthened the study. In the present work, testosterone was chosen as the principal hormonal marker because it is closely related to libido, erectile function, spermatogenesis, and Leydig cell activity, which were central outcomes of this study. We acknowledge that additional hormones, such as LH and FSH, as well as biochemical markers related to erectile function and diabetes, including PDE5 activity and other relevant enzymatic assays, were not evaluated. This limitation has now been stated in the revised manuscript, and we indicated that future studies should investigate these parameters to provide a more comprehensive mechanistic understanding. 11. Comment: Also, why did you not carry out the bioactive composition of the plant? Response: We sincerely thank the reviewer for this valuable comment. We agree that analysis of the bioactive composition of the black pepper extract is important for strengthening the mechanistic interpretation of the study. In response, we have revised the manuscript to include LC-MS characterization of the extract used in this study. The LC-MS procedure is now described in the Materials and Methods section with results presented in table. These additions improve the phytochemical documentation of the tested extract and provide direct characterization of the plant material used in the experiment. 12. Comment: Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Response: We sincerely thank the reviewer for this valuable comment. In response, we carefully re-examined all tables in the manuscript and corrected the identified errors. In particular, the value reported for Sperm Morphology in Group 1 has been revised to include the appropriate decimal point. We also performed an additional review of all tabulated results to minimize formatting and transcription errors in the revised version. 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer for this valuable suggestion. In response, we revised the Introduction to incorporate the most recent epidemiological data on diabetes mellitus from the International Diabetes Federation (IDF) Diabetes Atlas, 11th edition, published in 2025. We clarified that the latest global estimate available in this report refers to 2024, as no separate worldwide estimate specifically for 2025 is currently provided. In addition, we included the projected peak global burden of diabetes reported in the same source, which estimates that the number of adults living with diabetes will increase to 853 million by 2050. 2. Comment: There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. Response: We are grateful to the reviewer for this important and constructive comment. In response, we substantially revised the Introduction by adding a dedicated paragraph to strengthen the scientific rationale for the use of black pepper in the present animal model. Specifically, we incorporated additional in vitro and in vivo evidence regarding piperine, the principal bioactive alkaloid of Piper nigrum, to support the biological plausibility of black pepper extract as a candidate intervention. We also clarified in the revised text that these preclinical studies were included to provide mechanistic and experimental justification for the intervention, rather than to imply definitive therapeutic superiority. 3. Comment: I didn't see the anaesthesia used for the rats after the treatment. at the Methods area, Kindly recast the first sentence. Response: We thank the reviewer for this valuable comment. We apologize that the procedure was not clearly described in the original manuscript. In the revised Methods section, we clarified the terminal procedure used after completion of the treatment period and all functional assessments. Specifically, the text has been revised to clearly state that the animals were euthanized in accordance with the approved institutional ethics protocol. 4. Comment: Specify the kind of rats u used whether male/female. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused some confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 5. Comment : Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Response: We thank the reviewer for this valuable comment. We agree that inclusion of the full identifier name, herbarium deposition details, and voucher specimen number would improve the botanical documentation of the study. However, these records were not available for the present work. We therefore clarified the available plant source information in the revised manuscript and acknowledged the absence of formal voucher documentation as a limitation of the study. 6. Comment: Which type of DM was identified in the rats 24 hours after the alloxan induction? 8 days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Response: We sincerely thank the reviewer for this important and thoughtful comment. We clarified in the revised manuscript that the alloxan-induced diabetic rat model used in this study represents an insulin-deficient, type 1-like diabetes model, because alloxan selectively damages pancreatic β-cells and is widely used to induce experimental insulin-deficient diabetes in rodents. We also agree that an 8-day treatment period is relatively short and may not fully reproduce the chronic pathophysiology of long-standing diabetes-related sexual dysfunction. Therefore, we revised the manuscript to emphasize that the present work should be interpreted as a short-term experimental study designed to evaluate early changes in hyperglycemia and sexual/reproductive parameters after alloxan induction, rather than as evidence of complete reversal of established chronic diabetic complications. In addition, we added a more cautious statement in the Discussion noting that, although early improvements in blood glucose and functional parameters may be observed in experimental animal studies, these findings should be interpreted carefully and require confirmation in studies with longer treatment duration and follow-up. 7. Comment: Why was there no graph of the rats blood glucose levels? Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1, which includes both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 8. Comment: For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. Response: We sincerely thank the reviewer for this valuable comment. We agree that intromission number and intromission latency are relevant parameters in sexual behavior studies. In the present study, however, the behavioral assessment was prospectively focused on courtship latency, mount latency, and mount frequency as the predefined libido-related outcomes, and intromission-related parameters were therefore not systematically collected for quantitative analysis. We acknowledge this as a limitation and have clarified this point in the revised manuscript. In addition, the behavioral observations were performed using CCTV-based recording, and in response to the reviewer’s suggestion, we have provided representative recording examples and images in our Figshare (https://doi.org/10.6084/m9.figshare.30456353) as supporting documentation. 9. Comment: The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Response: We sincerely thank the reviewer for this valuable comment. The histological evaluation in the present study was intentionally limited to the testis, because the primary objective of the study was to assess the effects of black pepper extract on male sexual and reproductive function rather than on general diabetic organ injury. Since sperm parameters, testosterone levels, and sexual behavior were central outcomes of this study, testicular histology was considered the most relevant tissue-level endpoint. Nevertheless, we agree that examination of other organs commonly affected by diabetes, including the pancreas, kidney, liver, and penile tissue, would have strengthened the study by providing additional evidence on the systemic effects of the extract. We have therefore acknowledged this as a limitation in the revised manuscript. 10. Comment: Again, why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Response: We sincerely thank the reviewer for this valuable comment. We agree that inclusion of additional sexual hormones and biochemical parameters would have strengthened the study. In the present work, testosterone was chosen as the principal hormonal marker because it is closely related to libido, erectile function, spermatogenesis, and Leydig cell activity, which were central outcomes of this study. We acknowledge that additional hormones, such as LH and FSH, as well as biochemical markers related to erectile function and diabetes, including PDE5 activity and other relevant enzymatic assays, were not evaluated. This limitation has now been stated in the revised manuscript, and we indicated that future studies should investigate these parameters to provide a more comprehensive mechanistic understanding. 11. Comment: Also, why did you not carry out the bioactive composition of the plant? Response: We sincerely thank the reviewer for this valuable comment. We agree that analysis of the bioactive composition of the black pepper extract is important for strengthening the mechanistic interpretation of the study. In response, we have revised the manuscript to include LC-MS characterization of the extract used in this study. The LC-MS procedure is now described in the Materials and Methods section with results presented in table. These additions improve the phytochemical documentation of the tested extract and provide direct characterization of the plant material used in the experiment. 12. Comment: Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Response: We sincerely thank the reviewer for this valuable comment. In response, we carefully re-examined all tables in the manuscript and corrected the identified errors. In particular, the value reported for Sperm Morphology in Group 1 has been revised to include the appropriate decimal point. We also performed an additional review of all tabulated results to minimize formatting and transcription errors in the revised version. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 29 Apr 2026 Exsa Hardibrata , Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 29 Apr 2026 Author Response 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer ... Continue reading 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer for this valuable suggestion. In response, we revised the Introduction to incorporate the most recent epidemiological data on diabetes mellitus from the International Diabetes Federation (IDF) Diabetes Atlas, 11th edition, published in 2025. We clarified that the latest global estimate available in this report refers to 2024, as no separate worldwide estimate specifically for 2025 is currently provided. In addition, we included the projected peak global burden of diabetes reported in the same source, which estimates that the number of adults living with diabetes will increase to 853 million by 2050. 2. Comment: There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. Response: We are grateful to the reviewer for this important and constructive comment. In response, we substantially revised the Introduction by adding a dedicated paragraph to strengthen the scientific rationale for the use of black pepper in the present animal model. Specifically, we incorporated additional in vitro and in vivo evidence regarding piperine, the principal bioactive alkaloid of Piper nigrum, to support the biological plausibility of black pepper extract as a candidate intervention. We also clarified in the revised text that these preclinical studies were included to provide mechanistic and experimental justification for the intervention, rather than to imply definitive therapeutic superiority. 3. Comment: I didn't see the anaesthesia used for the rats after the treatment. at the Methods area, Kindly recast the first sentence. Response: We thank the reviewer for this valuable comment. We apologize that the procedure was not clearly described in the original manuscript. In the revised Methods section, we clarified the terminal procedure used after completion of the treatment period and all functional assessments. Specifically, the text has been revised to clearly state that the animals were euthanized in accordance with the approved institutional ethics protocol. 4. Comment: Specify the kind of rats u used whether male/female. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused some confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 5. Comment : Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Response: We thank the reviewer for this valuable comment. We agree that inclusion of the full identifier name, herbarium deposition details, and voucher specimen number would improve the botanical documentation of the study. However, these records were not available for the present work. We therefore clarified the available plant source information in the revised manuscript and acknowledged the absence of formal voucher documentation as a limitation of the study. 6. Comment: Which type of DM was identified in the rats 24 hours after the alloxan induction? 8 days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Response: We sincerely thank the reviewer for this important and thoughtful comment. We clarified in the revised manuscript that the alloxan-induced diabetic rat model used in this study represents an insulin-deficient, type 1-like diabetes model, because alloxan selectively damages pancreatic β-cells and is widely used to induce experimental insulin-deficient diabetes in rodents. We also agree that an 8-day treatment period is relatively short and may not fully reproduce the chronic pathophysiology of long-standing diabetes-related sexual dysfunction. Therefore, we revised the manuscript to emphasize that the present work should be interpreted as a short-term experimental study designed to evaluate early changes in hyperglycemia and sexual/reproductive parameters after alloxan induction, rather than as evidence of complete reversal of established chronic diabetic complications. In addition, we added a more cautious statement in the Discussion noting that, although early improvements in blood glucose and functional parameters may be observed in experimental animal studies, these findings should be interpreted carefully and require confirmation in studies with longer treatment duration and follow-up. 7. Comment: Why was there no graph of the rats blood glucose levels? Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1, which includes both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 8. Comment: For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. Response: We sincerely thank the reviewer for this valuable comment. We agree that intromission number and intromission latency are relevant parameters in sexual behavior studies. In the present study, however, the behavioral assessment was prospectively focused on courtship latency, mount latency, and mount frequency as the predefined libido-related outcomes, and intromission-related parameters were therefore not systematically collected for quantitative analysis. We acknowledge this as a limitation and have clarified this point in the revised manuscript. In addition, the behavioral observations were performed using CCTV-based recording, and in response to the reviewer’s suggestion, we have provided representative recording examples and images in our Figshare (https://doi.org/10.6084/m9.figshare.30456353) as supporting documentation. 9. Comment: The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Response: We sincerely thank the reviewer for this valuable comment. The histological evaluation in the present study was intentionally limited to the testis, because the primary objective of the study was to assess the effects of black pepper extract on male sexual and reproductive function rather than on general diabetic organ injury. Since sperm parameters, testosterone levels, and sexual behavior were central outcomes of this study, testicular histology was considered the most relevant tissue-level endpoint. Nevertheless, we agree that examination of other organs commonly affected by diabetes, including the pancreas, kidney, liver, and penile tissue, would have strengthened the study by providing additional evidence on the systemic effects of the extract. We have therefore acknowledged this as a limitation in the revised manuscript. 10. Comment: Again, why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Response: We sincerely thank the reviewer for this valuable comment. We agree that inclusion of additional sexual hormones and biochemical parameters would have strengthened the study. In the present work, testosterone was chosen as the principal hormonal marker because it is closely related to libido, erectile function, spermatogenesis, and Leydig cell activity, which were central outcomes of this study. We acknowledge that additional hormones, such as LH and FSH, as well as biochemical markers related to erectile function and diabetes, including PDE5 activity and other relevant enzymatic assays, were not evaluated. This limitation has now been stated in the revised manuscript, and we indicated that future studies should investigate these parameters to provide a more comprehensive mechanistic understanding. 11. Comment: Also, why did you not carry out the bioactive composition of the plant? Response: We sincerely thank the reviewer for this valuable comment. We agree that analysis of the bioactive composition of the black pepper extract is important for strengthening the mechanistic interpretation of the study. In response, we have revised the manuscript to include LC-MS characterization of the extract used in this study. The LC-MS procedure is now described in the Materials and Methods section with results presented in table. These additions improve the phytochemical documentation of the tested extract and provide direct characterization of the plant material used in the experiment. 12. Comment: Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Response: We sincerely thank the reviewer for this valuable comment. In response, we carefully re-examined all tables in the manuscript and corrected the identified errors. In particular, the value reported for Sperm Morphology in Group 1 has been revised to include the appropriate decimal point. We also performed an additional review of all tabulated results to minimize formatting and transcription errors in the revised version. 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer for this valuable suggestion. In response, we revised the Introduction to incorporate the most recent epidemiological data on diabetes mellitus from the International Diabetes Federation (IDF) Diabetes Atlas, 11th edition, published in 2025. We clarified that the latest global estimate available in this report refers to 2024, as no separate worldwide estimate specifically for 2025 is currently provided. In addition, we included the projected peak global burden of diabetes reported in the same source, which estimates that the number of adults living with diabetes will increase to 853 million by 2050. 2. Comment: There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. Response: We are grateful to the reviewer for this important and constructive comment. In response, we substantially revised the Introduction by adding a dedicated paragraph to strengthen the scientific rationale for the use of black pepper in the present animal model. Specifically, we incorporated additional in vitro and in vivo evidence regarding piperine, the principal bioactive alkaloid of Piper nigrum, to support the biological plausibility of black pepper extract as a candidate intervention. We also clarified in the revised text that these preclinical studies were included to provide mechanistic and experimental justification for the intervention, rather than to imply definitive therapeutic superiority. 3. Comment: I didn't see the anaesthesia used for the rats after the treatment. at the Methods area, Kindly recast the first sentence. Response: We thank the reviewer for this valuable comment. We apologize that the procedure was not clearly described in the original manuscript. In the revised Methods section, we clarified the terminal procedure used after completion of the treatment period and all functional assessments. Specifically, the text has been revised to clearly state that the animals were euthanized in accordance with the approved institutional ethics protocol. 4. Comment: Specify the kind of rats u used whether male/female. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused some confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 5. Comment : Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Response: We thank the reviewer for this valuable comment. We agree that inclusion of the full identifier name, herbarium deposition details, and voucher specimen number would improve the botanical documentation of the study. However, these records were not available for the present work. We therefore clarified the available plant source information in the revised manuscript and acknowledged the absence of formal voucher documentation as a limitation of the study. 6. Comment: Which type of DM was identified in the rats 24 hours after the alloxan induction? 8 days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Response: We sincerely thank the reviewer for this important and thoughtful comment. We clarified in the revised manuscript that the alloxan-induced diabetic rat model used in this study represents an insulin-deficient, type 1-like diabetes model, because alloxan selectively damages pancreatic β-cells and is widely used to induce experimental insulin-deficient diabetes in rodents. We also agree that an 8-day treatment period is relatively short and may not fully reproduce the chronic pathophysiology of long-standing diabetes-related sexual dysfunction. Therefore, we revised the manuscript to emphasize that the present work should be interpreted as a short-term experimental study designed to evaluate early changes in hyperglycemia and sexual/reproductive parameters after alloxan induction, rather than as evidence of complete reversal of established chronic diabetic complications. In addition, we added a more cautious statement in the Discussion noting that, although early improvements in blood glucose and functional parameters may be observed in experimental animal studies, these findings should be interpreted carefully and require confirmation in studies with longer treatment duration and follow-up. 7. Comment: Why was there no graph of the rats blood glucose levels? Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1, which includes both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 8. Comment: For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. Response: We sincerely thank the reviewer for this valuable comment. We agree that intromission number and intromission latency are relevant parameters in sexual behavior studies. In the present study, however, the behavioral assessment was prospectively focused on courtship latency, mount latency, and mount frequency as the predefined libido-related outcomes, and intromission-related parameters were therefore not systematically collected for quantitative analysis. We acknowledge this as a limitation and have clarified this point in the revised manuscript. In addition, the behavioral observations were performed using CCTV-based recording, and in response to the reviewer’s suggestion, we have provided representative recording examples and images in our Figshare (https://doi.org/10.6084/m9.figshare.30456353) as supporting documentation. 9. Comment: The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Response: We sincerely thank the reviewer for this valuable comment. The histological evaluation in the present study was intentionally limited to the testis, because the primary objective of the study was to assess the effects of black pepper extract on male sexual and reproductive function rather than on general diabetic organ injury. Since sperm parameters, testosterone levels, and sexual behavior were central outcomes of this study, testicular histology was considered the most relevant tissue-level endpoint. Nevertheless, we agree that examination of other organs commonly affected by diabetes, including the pancreas, kidney, liver, and penile tissue, would have strengthened the study by providing additional evidence on the systemic effects of the extract. We have therefore acknowledged this as a limitation in the revised manuscript. 10. Comment: Again, why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Response: We sincerely thank the reviewer for this valuable comment. We agree that inclusion of additional sexual hormones and biochemical parameters would have strengthened the study. In the present work, testosterone was chosen as the principal hormonal marker because it is closely related to libido, erectile function, spermatogenesis, and Leydig cell activity, which were central outcomes of this study. We acknowledge that additional hormones, such as LH and FSH, as well as biochemical markers related to erectile function and diabetes, including PDE5 activity and other relevant enzymatic assays, were not evaluated. This limitation has now been stated in the revised manuscript, and we indicated that future studies should investigate these parameters to provide a more comprehensive mechanistic understanding. 11. Comment: Also, why did you not carry out the bioactive composition of the plant? Response: We sincerely thank the reviewer for this valuable comment. We agree that analysis of the bioactive composition of the black pepper extract is important for strengthening the mechanistic interpretation of the study. In response, we have revised the manuscript to include LC-MS characterization of the extract used in this study. The LC-MS procedure is now described in the Materials and Methods section with results presented in table. These additions improve the phytochemical documentation of the tested extract and provide direct characterization of the plant material used in the experiment. 12. Comment: Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Response: We sincerely thank the reviewer for this valuable comment. In response, we carefully re-examined all tables in the manuscript and corrected the identified errors. In particular, the value reported for Sperm Morphology in Group 1 has been revised to include the appropriate decimal point. We also performed an additional review of all tabulated results to minimize formatting and transcription errors in the revised version. Competing Interests: The authors declare that they have no conflict of interest. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 09 Jan 2026 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 29 Apr 26 read read Version 1 09 Jan 26 read read read Onyinyechi Ruth Nwagwe , Federal University, Oye-Ekiti,, Oye-Ekiti,, Nigeria Patrick Ebele Obi , Enugu State University of Science and Technology, Enugu, Nigeria Oluwapelumi Micheal Ajiboye , Babcock University, Ogun State, Nigeria Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Nwagwe O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 18 May 2026 | for Version 2 Onyinyechi Ruth Nwagwe , Federal University, Oye-Ekiti,, Oye-Ekiti,, Nigeria 0 Views copyright © 2026 Nwagwe O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have gone through the revised version, the authors really adhered to the suggestions given to them, so in essence, the work is making a lot of sense now. Again, the authors should check their grammatical expressions of each sentence they make. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Nwagwe OR. Peer Review Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.198879.r480266) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/15-38/v2#referee-response-480266 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Obi P. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 May 2026 | for Version 2 Patrick Ebele Obi , Enugu State University of Science and Technology, Enugu, Enugu, Nigeria 0 Views copyright © 2026 Obi P. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 1. Fasting blood glucose levels of 500mg/dl and above are unrealistic. 2. Grammatical error: ''Fresh black pepper fruit is collected from a farmer...'' should be corrected to '' Fresh black pepper fruits were collected from a farmer....'' Competing Interests No competing interests were disclosed. Reviewer Expertise ■Phytoevaluation ■ Phytochemistry ■ Phytomedicine ■ Drug Discovery and Development I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Obi PE. Peer Review Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.198879.r480267) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/15-38/v2#referee-response-480267 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Ajiboye O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 19 Mar 2026 | for Version 1 Oluwapelumi Micheal Ajiboye , Babcock University, Ogun State, Nigeria 0 Views copyright © 2026 Ajiboye O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Summary of the Article This study investigates the potential therapeutic effects of Piper nigrum (black pepper) fruit extract on erectile dysfunction (ED) and associated reproductive parameters in alloxan-induced diabetic male rats. The experimental design includes five groups: normal control, diabetic control, extract-treated diabetic group, and a sildenafil-treated group as a positive control. Outcome measures include erectile function indices (mounting frequency, intromission frequency, total penile reflexes), libido parameters, sperm characteristics, and histological evaluation of testicular tissue. The authors report that the extract significantly improved certain erectile and libido parameters, particularly total penile reflexes, while effects on sperm indices were largely non-significant. Histological observations are interpreted as showing restoration of testicular architecture. While the topic is relevant and potentially publishable, the manuscript exhibits substantial methodological, statistical, interpretational, and editorial deficiencies. Significant revision is required before the work can meet publication standards. MAJOR COMMENTS 1. The manuscript states that data are non-parametric, yet both Kruskal–Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) 2. Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. 3. The term “gland penis” is incorrect. Should be "glans penis". 4. The term "male kisses" is non-scientific. Rephrase appropriately. 5. Include units for sperm count and fluid intake 6. Avoid overgeneralization. 7. There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. 8. "ddp-4" should be written as "DDP-4" 9. Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. 10. Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" 11. The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " 12. The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. 13. Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. 14. The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Phytomedicine; Functional Foods and Nutraceuticals; Endocrine Disorder (Diabetes mellitus and its complications); Neurodegeneration. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 29 Apr 2026 Exsa Hardibrata, Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 1. Comment: The manuscript states that data are non-parametric, yet both Kruskal-Wallis test and ANOVA are reported. This represents a fundamental methodological contradiction. (ANOVA + Tukey post hoc (if parametric), OR Kruskal–Wallis + Dunn’s test (if non-parametric)) Response: We sincerely thank the reviewer for identifying this inconsistency. We agree that the original statistical description contained a methodological contradiction. Although the manuscript incorrectly stated that one-way ANOVA with LSD post hoc testing was used, the analyses actually performed in this study were Kruskal-Wallis tests followed by Dunn’s multiple-comparison post hoc tests, consistent with the non-parametric nature of the data. This error has now been corrected in the revised manuscript, and the statistical methods have been rewritten to accurately reflect the analyses conducted. We have also carefully reviewed the Results section and tables to ensure consistency with the corrected statistical approach. 2. Comment: Several results are described as “increased” or “improved” despite p > 0.05. E.g. sperm parameters (p = 0.877) are interpreted as improvements. Response: We sincerely thank the reviewer for this careful and important comment. We agree that it is not appropriate to interpret non-significant differences as definite improvements. In response, we have revised the Results and Discussion sections to ensure that only statistically significant findings are described as significant improvements, whereas outcomes with p > 0.05 are now reported more cautiously as numerical differences without statistical significance. For example, the statements regarding sperm parameters have been corrected accordingly. We have also rechecked the manuscript throughout to ensure consistency between the statistical results and their interpretation. 3. Comment: The term “gland penis” is incorrect. Should be "glans penis". Response: We sincerely thank the reviewer for this valuable comment. We agree that the correct anatomical term is “glans penis” rather than “gland penis.” The manuscript has been revised accordingly in the Materials and Methods section to ensure anatomical accuracy. 4. Comment: The term "male kisses" is non-scientific. Rephrase appropriately. Response: We sincerely thank the reviewer for this valuable comment. We agree that the expression “male kisses” is not appropriate for scientific reporting of animal sexual behavior. In the revised manuscript, we have replaced this phrase with more formal and objective terminology to describe the observed behavior in the Libido Assessment subsection. We changed the sentence into “Courtship latency was defined as the time from opening of the partition until the male initiated investigatory or courtship behavior toward the female, including anogenital sniffing or contact with the female’s body.” 5. Comment: Include units for sperm count and fluid intake. Response: We sincerely thank the reviewer for this valuable observation. We agree that the omission of units for sperm count and fluid intake reduced the clarity of data presentation. In response, we have revised the manuscript into units of millions/mL to include the appropriate units for these variables throughout the text and tables. 6. Comment: Avoid overgeneralization. Response : We sincerely thank the reviewer for this valuable observation. We agree that the original manuscript contained several statements that were overly broad in relation to the data presented. In response, we have revised the manuscript throughout to ensure that the conclusions are stated more cautiously and are fully supported by the results. Specifically, we avoided definitive claims, reduced overstatement of non-significant findings, and clarified that the observed effects should be interpreted as dose-dependent, endpoint-specific, and preliminary findings in a short-term alloxan-induced diabetic rat model. 7. Comment: There is contradictory literature use, i.e. "piperine increases testosterone", and "piperine inhibits spermatogenesis". These two statements contradict each other. Response : We sincerely thank the reviewer for this valuable comment. We agree that the earlier wording may have oversimplified the published evidence. In the revised manuscript, we clarified that the available literature suggests complex and context-dependent effects of piperine, rather than uniformly beneficial or uniformly harmful reproductive effects. We now explicitly state that piperine may enhance Leydig-cell function and steroidogenic activity while, under different experimental conditions, adversely affecting spermatogenesis. We therefore revised the Discussion to reflect that these findings represent different reproductive endpoints and may vary according to dose, exposure duration, and study conditions. 8. Comment : "ddp-4" should be written as "DDP-4" Response: We sincerely thank the reviewer for this careful observation. We agree that the abbreviation should be written consistently in uppercase. In the revised manuscript, “dpp-4” has been corrected to “DPP-4” throughout the text. 9. Comment : Establish the relevance of the oxidative stress pathway and nitric oxide signalling in your discussion. Response: We sincerely thank the reviewer for this important comment. In response, we further revised the Discussion to more explicitly establish the relevance of the oxidative stress pathway and nitric oxide signaling to the findings of the present study. Specifically, we clarified that hyperglycemia-induced oxidative stress can reduce nitric oxide bioavailability and impair eNOS-mediated endothelial signaling in the corpus cavernosum, which contributes to diabetic erectile dysfunction. We also strengthened the discussion linking the directional increase in eNOS mRNA expression after black pepper treatment with the possibility of partial restoration of endothelial NO signaling. In addition, we added relevant recent literature to support the mechanistic relationship among oxidative stress, endothelial dysfunction, and impaired erectile function in diabetes. 10. Comment: Conclusion is overstated. Conclude using the specific parameters evaluated. Correct typo such as "randmoized" to "randomized" Response: We sincerely thank the reviewer for this valuable observation. We agree that the previous Conclusion was somewhat overstated. In the revised manuscript, we have rewritten the Conclusion to refer specifically to the measured outcomes, including total penile reflexes, libido-related parameters, intratesticular testosterone, sperm concentration, sperm morphology, Leydig cell count, and eNOS expression, rather than making broad claims about diabetic male reproductive dysfunction overall. We have also revised the language to ensure that the findings are interpreted as dose-dependent, endpoint-specific, and preliminary. However, we don’t used the term “randomized” in our manuscript anymore. 11. Comment: The phrase "known to be the cause of....." used in the second paragraph of the discussion section should be rephrased as "....is associated with..... " Response: We sincerely thank the reviewer for this valuable comment. We agree that the original phrasing implied a stronger causal interpretation than was warranted. In the revised Discussion, we have replaced “known to be the cause of” with newer sentences in our revised manuscript. 12. Comment: The discussion attributes effects to increased testosterone and antioxidant activity. However, no biochemical assays (e.g., testosterone, SOD, catalase, MDA) were performed. Remove or clearly qualify speculative statements and limit discussion to observed data unless supported by references. Response: We sincerely thank the reviewer for this valuable observation. We agree that the original Discussion contained mechanistic statements that could be interpreted as stronger than warranted by the available data. In the revised manuscript, we have clarified that intratesticular testosterone was directly measured and may therefore be discussed as an observed endpoint, whereas oxidative stress-related mechanisms were not directly evaluated, as no assays such as SOD, catalase, or MDA were performed. We therefore revised the Discussion to remove over-speculative language, qualify mechanistic interpretations more carefully, and restrict causal inferences to the measured outcomes. Where oxidative stress or antioxidant effects are mentioned, these are now presented explicitly as literature-supported hypotheses rather than direct findings of the present study. We have also included this into our limitation. 13. Comment: Extremely large standard deviations (e.g., sperm count: ±52.9), which may indicate poor experimental control or insufficient sample size. Response: We sincerely thank the reviewer for this valuable comment. We acknowledge that some outcomes, particularly sperm count, showed relatively large standard deviations. We carefully re-examined the raw data and confirmed that these values were accurate and not due to transcription error. We elected to retain the original values to preserve transparency and to avoid underreporting biological variability. We agree that the relatively small group size may have contributed to the wide dispersion observed. Accordingly, we used non-parametric statistical analysis (Kruskal-Wallis followed by Dunn’s post hoc test) and revised the text to ensure that the findings are interpreted cautiously. However, we also include this topic into our limitation to keep our study transparent. 14. Comment: The title was duplicated in reference 3. The author's name(s) are not included in reference 20, and two journal names were mentioned within the same reference. Some references have the journal names abbreviated, while some do not (be consistent). Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this important comment. We have carefully re-examined the entire reference list and corrected the identified errors, including the duplicated title in Reference 3, the incomplete author and journal information in Reference 20, and inconsistencies in journal-name formatting. The references have now been standardized throughout the manuscript to ensure consistency and accuracy. Furthermore, we revised the Discussion extensively to strengthen the scientific presentation of the work and to ensure that the findings are interpreted in light of relevant and current literature. View more View less Competing Interests The authors declare that they have no conflict of interest. reply Respond Report a concern Ajiboye OM. Peer Review Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458225) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458225 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Obi P. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Mar 2026 | for Version 1 Patrick Ebele Obi , Enugu State University of Science and Technology, Enugu, Enugu, Nigeria 0 Views copyright © 2026 Obi P. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 1. The title of the research article should not be too conclusive. "effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......'' should be more appropriate. 2. Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. 3. The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). 4. There are some grammatical errors under '' Plant preparation" that need correction. For instance, "The fresh fruit is washed and rinsed until clean and then dried" 5. The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. 6. The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. 7. Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. 8. The authors initially stated under 'animal preparation' that the study used 30 male Sprague Dawley rats but later mentioned female rats under 'libido assessment'. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. 9. The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise ■Phytoevaluation ■ Phytochemistry ■ Phytomedicine ■ Drug Discovery and Development I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 29 Apr 2026 Exsa Hardibrata, Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 1. Comment: The title of the research article should not be too conclusive. “effects of black pepper fruit extract on erectile dysfunction in alloxan-induced .......” should be more appropriate. Response: We sincerely thank the reviewer for this valuable suggestion. We agree that the original title was overly conclusive in its wording. In response, we revised the title from “Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats” to “Effects of Black Pepper (Piper nigrum L.) Fruit Extract on Sexual Function, Hormonal Parameters, Sperm Parameters, Testicular Histology, and eNOS Expression in Alloxan-Induced Diabetic Rats.” We believe that the revised title is more neutral, scientifically appropriate, and more representative of the full range of outcomes assessed in the present study. 2. Comment: Introduction: The authors did not write enough information about erectile dysfunction under the introduction. A little more should be written. Response: We are grateful to the reviewer for this constructive comment. In response, we expanded the Introduction by adding further information on erectile dysfunction, particularly in the context of diabetes mellitus. The revised text now includes additional discussion of the pathophysiological mechanisms underlying diabetic erectile dysfunction, including endothelial dysfunction, oxidative stress, neuropathic changes, hormonal disturbances, and structural alterations in penile tissue. We believe that these additions provide a clearer clinical and biological rationale for the present study. 3. Comment: The preparation of experimental animal across groups is not consistent or not clearly stated. From the manuscript, black pepper extract was administered daily for eight days while the Sildenafil Citrate was administered only on the ninth day (ie one hour before observation of erectile function and libido assessment). Response: We thank the reviewer for this valuable comment. We acknowledge that the treatment regimens were not described with sufficient clarity in the original manuscript. In the revised manuscript, we clarified that black pepper extract was administered daily for eight consecutive days as the test intervention, whereas sildenafil citrate was given as a single dose on the ninth day, one hour before erectile function and libido assessment, as an acute positive control. Thus, the difference in administration schedule was intentional and reflected the distinct role of sildenafil as a reference treatment for erectile function rather than as a chronic intervention comparable to black pepper extract. The Materials and Methods section, particularly the Animal Preparation subsection, has been revised accordingly for clarity. 4. Comment : There are some grammatical errors under “Plant preparation” that need correction. For instance, “The fresh fruit is washed and rinsed until clean and then dried”. Response : We thank the reviewer for this valuable comment. We carefully revised the Plant Preparation subsection to correct the grammatical errors and improve sentence structure, tense consistency, and overall clarity. In particular, the sentence noted by the reviewer has been revised to conform to formal scientific writing style. 5. Comment: The authors should include the fasting blood sugar levels of the animals before and after inducing diabetes. Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1 in the Materials and Methods section under the Animal Preparation subsection and include both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 6. Comment: The duration of the research (9 days) is too short giving that diabetes complications such as erectile dysfunction are not instantaneous but gradual. Response: We sincerely thank the reviewer for this valuable comment. We agree that the duration of the present study was relatively short and may not fully reflect the chronic progression of diabetes-related erectile dysfunction, which typically develops gradually over time. However, the selection of day 9 after alloxan induction as the evaluation time point was based on considerations related to the stability of the diabetic model. At this stage, the animals had already passed the period of acute systemic toxicity associated with alloxan administration, so the data obtained were intended to reflect a more stable sub-acute hyperglycemic state rather than direct drug-induced toxic effects. In addition, this time point was considered sufficient to allow early structural changes in pancreatic β-cells, stabilization of oxidative stress-related changes, and regulation of relevant gene expression, thereby providing a suitable basis for assessment of both pathological progression and the early effects of the test compound. To ensure consistency of the diabetic state before final outcome assessment, fasting blood glucose levels were confirmed on day 3 and day 7 after alloxan induction and used as inclusion criteria to verify that all study animals remained in a stable diabetic condition before parameter evaluation on day 9. Moreover, because one of the aims of this study was to evaluate gene expression, particularly eNOS mRNA expression, day 9 was considered an appropriate time point at which hyperglycemia-related oxidative stress and pro-inflammatory signaling pathways would already be active, while tissue injury was still in an early and potentially modifiable phase. This timing was also considered sufficient to allow the test compounds to interact with relevant biochemical and cellular pathways before more advanced and possibly irreversible structural damage developed. Nevertheless, we agree that this represents a short-term experimental study, and we have revised the manuscript to emphasize that the findings should not be interpreted as evidence of reversal of long-standing chronic diabetic complications. We have also acknowledged this issue as a limitation in the Discussion and indicated that future studies with longer treatment duration and follow-up are necessary. 7. Comment: Report on phytoconstituents of the black pepper extract is completely missing. Knowledge of the phytoconstituents will be needed. Response: We sincerely thank the reviewer for this valuable comment. We agree that reporting the phytoconstituents of the black pepper extract is important to support interpretation of its biological activity. In response, we have now included LC-MS analysis of the extract used in this study. The corresponding analytical procedure has been described in the Materials and Methods section, and the results are presented in Table. These additions provide direct characterization of the tested extract and strengthen the revised manuscript. 8. Comment: The authors initially stated under “animal preparation” that the study used 30 male Sprague Dawley rats but later mentioned female rats under “libido assessment”. I understand that erectile dysfunction is male related complication but all animal used for this study should be clearly listed. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 9. Comment: The research results were poorly discussed. Is the work clearly and accurately presented and does it cite the current literature? Response: We sincerely thank the reviewer for this valuable comment. In response, we substantially revised the Discussion section to improve the clarity, depth, and accuracy of interpretation of the study findings. Specifically, we expanded the discussion of each major outcome, aligned the interpretation more closely with the data presented in the tables, and incorporated more current and relevant literature to support comparison with previous studies and to explain the possible biological mechanisms involved. We also revised several statements to ensure that the conclusions are presented more cautiously and accurately, particularly in view of the short-term nature of the diabetic model and the endpoint-specific effects observed. We believe that these revisions have improved the scientific presentation of the work and strengthened the overall clarity of the manuscript. View more View less Competing Interests The authors declare that they have no conflict of interest. reply Respond Report a concern Obi PE. Peer Review Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r458227) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-458227 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Nwagwe O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Mar 2026 | for Version 1 Onyinyechi Ruth Nwagwe , Federal University, Oye-Ekiti,, Oye-Ekiti,, Nigeria 0 Views copyright © 2026 Nwagwe O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. I didn't see the anaesthesia used for the rats after the treatment . at the Methods area, Kindly recast the first sentence. Specify the kind of rats u used whether male/female. Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Which type of DMwas identified in the rats 24 hours after the alloxan induction? 8days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Why was there no graph of the rats blood glucose levels? For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Again , why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Also, why did you not carry out the bioactive composition of the plant? Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 29 Apr 2026 Exsa Hardibrata, Doctoral Program, Universitas Lampung Fakultas Matematika dan Ilmu Pengetahuan Alam, Bandar Lampung, Indonesia 1. Comment: The Fig 1 did not capture the year 2025 and the peak number of people having Diabetes, so it must be stated. Response: We sincerely thank the reviewer for this valuable suggestion. In response, we revised the Introduction to incorporate the most recent epidemiological data on diabetes mellitus from the International Diabetes Federation (IDF) Diabetes Atlas, 11th edition, published in 2025. We clarified that the latest global estimate available in this report refers to 2024, as no separate worldwide estimate specifically for 2025 is currently provided. In addition, we included the projected peak global burden of diabetes reported in the same source, which estimates that the number of adults living with diabetes will increase to 853 million by 2050. 2. Comment: There was no significant evidence presented in the authors background section to justify pepperine in an animal model.So more referenced evidence to explain why the treatment is better. And it should come from invitro/invivo works. Response: We are grateful to the reviewer for this important and constructive comment. In response, we substantially revised the Introduction by adding a dedicated paragraph to strengthen the scientific rationale for the use of black pepper in the present animal model. Specifically, we incorporated additional in vitro and in vivo evidence regarding piperine, the principal bioactive alkaloid of Piper nigrum, to support the biological plausibility of black pepper extract as a candidate intervention. We also clarified in the revised text that these preclinical studies were included to provide mechanistic and experimental justification for the intervention, rather than to imply definitive therapeutic superiority. 3. Comment: I didn't see the anaesthesia used for the rats after the treatment. at the Methods area, Kindly recast the first sentence. Response: We thank the reviewer for this valuable comment. We apologize that the procedure was not clearly described in the original manuscript. In the revised Methods section, we clarified the terminal procedure used after completion of the treatment period and all functional assessments. Specifically, the text has been revised to clearly state that the animals were euthanized in accordance with the approved institutional ethics protocol. 4. Comment: Specify the kind of rats u used whether male/female. Response: We thank the reviewer for this comment. In the original Methods section, under the Animal Preparation subsection, we stated that the experimental animals were 30 male Sprague Dawley rats. However, we recognize that the later description of the mating test, which involved female rats as stimulus animals, may have caused some confusion. Therefore, we revised the Methods section to clarify that all experimental and treated animals were male rats, whereas female rats were used only as stimulus animals during the libido assessment. 5. Comment : Provide the full name of the individual who undertook identification of the plant material. Was a Voucher specimen deposited in a publicly available Hebarium?A deposition number should be included and full name of the hebarium. Response: We thank the reviewer for this valuable comment. We agree that inclusion of the full identifier name, herbarium deposition details, and voucher specimen number would improve the botanical documentation of the study. However, these records were not available for the present work. We therefore clarified the available plant source information in the revised manuscript and acknowledged the absence of formal voucher documentation as a limitation of the study. 6. Comment: Which type of DM was identified in the rats 24 hours after the alloxan induction? 8 days treatment could it lower blood glucose and improve sexual function? Could it be too good to believe? Response: We sincerely thank the reviewer for this important and thoughtful comment. We clarified in the revised manuscript that the alloxan-induced diabetic rat model used in this study represents an insulin-deficient, type 1-like diabetes model, because alloxan selectively damages pancreatic β-cells and is widely used to induce experimental insulin-deficient diabetes in rodents. We also agree that an 8-day treatment period is relatively short and may not fully reproduce the chronic pathophysiology of long-standing diabetes-related sexual dysfunction. Therefore, we revised the manuscript to emphasize that the present work should be interpreted as a short-term experimental study designed to evaluate early changes in hyperglycemia and sexual/reproductive parameters after alloxan induction, rather than as evidence of complete reversal of established chronic diabetic complications. In addition, we added a more cautious statement in the Discussion noting that, although early improvements in blood glucose and functional parameters may be observed in experimental animal studies, these findings should be interpreted carefully and require confirmation in studies with longer treatment duration and follow-up. 7. Comment: Why was there no graph of the rats blood glucose levels? Response: We sincerely thank the reviewer for this helpful suggestion. In response, we added the fasting blood glucose levels of the animals before and after alloxan induction to the revised manuscript. These data are presented as Table 1, which includes both individual values and mean ± SD for each group. We believe that this tabular presentation provides a clear and detailed description of the glycemic changes and confirms successful induction of hyperglycemia in the experimental animals. 8. Comment: For the sexual behavioral study, Why didn’t you check for the intromission number and Latency. Please let the video recording be shown. Response: We sincerely thank the reviewer for this valuable comment. We agree that intromission number and intromission latency are relevant parameters in sexual behavior studies. In the present study, however, the behavioral assessment was prospectively focused on courtship latency, mount latency, and mount frequency as the predefined libido-related outcomes, and intromission-related parameters were therefore not systematically collected for quantitative analysis. We acknowledge this as a limitation and have clarified this point in the revised manuscript. In addition, the behavioral observations were performed using CCTV-based recording, and in response to the reviewer’s suggestion, we have provided representative recording examples and images in our Figshare (https://doi.org/10.6084/m9.figshare.30456353) as supporting documentation. 9. Comment: The Histological aspect, Why did you work only on the Testis? What of other organs wouldn’t you have checked them since your work is on Diabetes to know the effect of Pepperine on them? Response: We sincerely thank the reviewer for this valuable comment. The histological evaluation in the present study was intentionally limited to the testis, because the primary objective of the study was to assess the effects of black pepper extract on male sexual and reproductive function rather than on general diabetic organ injury. Since sperm parameters, testosterone levels, and sexual behavior were central outcomes of this study, testicular histology was considered the most relevant tissue-level endpoint. Nevertheless, we agree that examination of other organs commonly affected by diabetes, including the pancreas, kidney, liver, and penile tissue, would have strengthened the study by providing additional evidence on the systemic effects of the extract. We have therefore acknowledged this as a limitation in the revised manuscript. 10. Comment: Again, why was other sexual hormones measured apart from Testosterone. Some Biochemical parameters was not assessed like PDE5 activity, SOME ENZYMATIC ASSAYS ON Diabetes. Response: We sincerely thank the reviewer for this valuable comment. We agree that inclusion of additional sexual hormones and biochemical parameters would have strengthened the study. In the present work, testosterone was chosen as the principal hormonal marker because it is closely related to libido, erectile function, spermatogenesis, and Leydig cell activity, which were central outcomes of this study. We acknowledge that additional hormones, such as LH and FSH, as well as biochemical markers related to erectile function and diabetes, including PDE5 activity and other relevant enzymatic assays, were not evaluated. This limitation has now been stated in the revised manuscript, and we indicated that future studies should investigate these parameters to provide a more comprehensive mechanistic understanding. 11. Comment: Also, why did you not carry out the bioactive composition of the plant? Response: We sincerely thank the reviewer for this valuable comment. We agree that analysis of the bioactive composition of the black pepper extract is important for strengthening the mechanistic interpretation of the study. In response, we have revised the manuscript to include LC-MS characterization of the extract used in this study. The LC-MS procedure is now described in the Materials and Methods section with results presented in table. These additions improve the phytochemical documentation of the tested extract and provide direct characterization of the plant material used in the experiment. 12. Comment: Check your tables well to avoid mistakes. Example, Sperm Morphology, Group 1: 75 should have a decimal point. Response: We sincerely thank the reviewer for this valuable comment. In response, we carefully re-examined all tables in the manuscript and corrected the identified errors. In particular, the value reported for Sperm Morphology in Group 1 has been revised to include the appropriate decimal point. We also performed an additional review of all tabulated results to minimize formatting and transcription errors in the revised version. View more View less Competing Interests The authors declare that they have no conflict of interest. reply Respond Report a concern Nwagwe OR. Peer Review Report For: Black pepper (Piper nigrum L.) fruit extract ameliorates erectile dysfunction in alloxan-induced diabetic rats [version 1; peer review: 2 approved with reservations, 1 not approved] . F1000Research 2026, 15 :38 ( https://doi.org/10.5256/f1000research.190377.r455996) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/15-38/v1#referee-response-455996 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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