INTRODUCTION:

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, insulin resistance, and impaired glucose metabolism. It affects over 400 million people worldwide and is projected to increase to 600 million by 2045. Diabetes can lead to various complications affecting multiple organs, including the reproductive system. In males, diabetes can cause alterations in reproductive function, including changes in sperm production and quality.

Sperm production is a complex process that involves the regulation of various hormones, including testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), among others. Studies have shown that diabetes can negatively impact male fertility by decreasing testosterone levels and altering the hypothalamic-pituitary-gonadal (HPG) axis, which regulates male reproductive function. Diabetes-associated oxidative stress, inflammation, and endothelial dysfunction can also impair testicular function and cause damage to the male reproductive system, leading to changes in sperm count, motility, and morphology. The dysfunction of Leydig cells, which produce testosterone, is a common finding in diabetic men, and this can contribute to the decrease in testosterone levels^1-5.

One protein that has been associated with male fertility is cysteine-rich secretory protein-1 (CRISP-1). CRISP-1 is expressed in the male reproductive tract and has been shown to play a role in sperm motility and fertilization. Studies have reported increased CRISP-1 expression in diabetic men, indicating the potential role of this protein in diabetes-associated reproductive complications. The impact of diabetes on male fertility has significant implications for reproductive health and family planning. Understanding the mechanisms underlying diabetes-associated changes in male reproductive function is essential for developing effective interventions for managing diabetes-associated reproductive complications. Therefore, investigating the potential therapeutic agents that can modulate testosterone and CRISP-1 levels could provide insights into the development of new treatment strategies for diabetes-associated male fertility disorders.

Coffea canephora var. Robusta, commonly known as Robusta coffee, is a plant species widely cultivated in tropical regions worldwide. The seeds of Robusta coffee are the primary source of Robusta coffee, which is known for its high caffeine content and various bioactive compounds, including polyphenols, chlorogenic acids, and diterpenes. These compounds have been associated with several health benefits, including anti-inflammatory, antioxidant, and anti-diabetic effects. In addition, Coffea canephora var. Robusta has been reported to exhibit potential pharmacological activities against male sexual dysfunction^6-8.

Several studies have suggested that Coffea canephora var. Robusta extract may have the following pharmacological activities against male sexual dysfunction, Aphrodisiac effects: Studies have reported the potential aphrodisiac effects of Coffea canephora var. Robusta extract, indicating its potential role in enhancing sexual desire and arousal in males, Testosterone booster: Coffea canephora var. Robusta extract has been shown to increase testosterone levels in animal models, indicating its potential use as a testosterone booster for males., Antioxidant effects: Diabetes-associated oxidative stress, inflammation, and endothelial dysfunction can impair testicular function and cause damage to the male reproductive system. Studies have suggested that Coffea canephora var. Robusta extract's antioxidant effects can help protect against diabetes-induced damage to the male reproductive system, Anti-inflammatory effects: Chronic inflammation is associated with various health problems, including male sexual dysfunction. Coffea canephora var. Robusta extract has been reported to exhibit anti-inflammatory effects, indicating its potential role in managing inflammation-associated male sexual dysfunction, Neuroprotective effects: Several studies have suggested that Coffea canephora var. Robusta extract may have neuroprotective effects. The central nervous system plays a vital role in male sexual function, and any damage or dysfunction can lead to sexual dysfunction^9-12. Therefore, the neuroprotective effects of Coffea canephora var. Robusta extract could potentially improve male sexual function. The aim of this study was to investigate the effect of Coffea canephora var. Robusta ethanol extract on testosterone and CRISP-1 levels in streptozotocin (STZ)-induced diabetic rats. STZ is a chemical that causes pancreatic beta-cell damage and induces diabetes-like symptoms, making it a widely used animal model for diabetes research. Understanding the effects of Coffea canephora var. Robusta extract on testosterone and CRISP-1 levels in STZ-induced diabetic rats could provide insights into the potential use of this plant as a therapeutic agent for diabetes-associated reproductive complications.

METHOD:

Materials:

Coffea canephora var. Robusta beans, 95% ethanol, Distilled water, Rotary evaporator, Vacuum pump, Filter paper, Glass funnel, Weighing scale, Mortar and pestle.

Extraction Procedure:

The Coffea canephora var. Robusta beans will be washed, dried, and ground into a fine powder using a mortar and pestle. 50g of the powdered beans will be weighed and placed in a 250mL Erlenmeyer flask. 200 mL of 95% ethanol will be added to the flask and stirred for 24hours at room temperature. The mixture will be filtered through a filter paper using a glass funnel to obtain the crude extract. The crude extract will be concentrated using a rotary evaporator under reduced pressure at 40°C. The concentrated extract will be dissolved in distilled water to obtain the final extract. The yield of the extract will be calculated by dividing the weight of the extract obtained by the weight of the powdered beans used. The extract will be stored in a cool and dry place until further use.

Animal Model:

Male Sprague-Dawley rats (n=30) weighing 200-250 g will be used in this study. Diabetes will be induced by a single intraperitoneal injection of STZ (50 mg/kg) in citrate buffer. The rats will be fasted overnight before the injection. Blood glucose levels will be measured 72 hours after STZ injection, and rats with blood glucose levels above 250 mg/dL will be considered diabetic and included in the study.

Experimental Design:

After one week of STZ injection, diabetic rats will be randomly divided into four groups (n=7 per group) as follows:

1. Control

2. Metformin

3. Coffea canephora var. Robusta ethanol extract 100 mg/kg group (CE100)

4. Coffea canephora var. Robusta ethanol extract 200 mg/kg group (CE200)

5. Coffea canephora var. Robusta ethanol extract 400 mg/kg group (CE400)

The treatment will be administered by oral gavage once daily for 28 days. The extract will be prepared in distilled water, and the doses will be based on previous studies. Blood samples will be collected at the end of the treatment period, and serum will be separated and stored at -80°C until analysis. Testosterone and CRISP-1 levels will be measured by enzyme-linked immunosorbent assay (ELISA) kits.

Testis Histopathology:

The testis that had been fixed were dehydrated using varying concentrations of alcohol (70-100%), cleaned using xylene, and paraffin-fixed at a temperature of 56 ◦C before the 4 μm thick slices were deparaffinized and subjected to Hematoxylin and Eosin (H&E) staining. Digital photographs were captured at 4x and 10x magnification using a microscope. The dimensions of total cell count, diameter, and size were obtained via ImageJ software (ImageJ, Version 1.44p, NIH, USA). To assess cardiac fibrosis, Masson's trichrome staining was carried out with some adjustments. The heart slices were subjected to pre-warmed Bouin's solution incubation for 120±10 minutes, following which the nuclei were stained with Weigert's hematoxylin for 20 minutes, whereas the cytoplasm and muscle were stained with Biebrich scarlet-acid fuchsin solution for 20 minutes. The tissue slices were then treated with phosphomolybdic-phosphotungstic acid solution for 10 minutes, followed by a blue aniline solution for 10 minutes. After a 30-second incubation with 1% acetic acid, the slices were dehydrated using ethanol and xylene, and digital photos were taken using a microscope at 4x and 10x magnification. Using ImageJ software (ImageJ, Version 1.44p, NIH, USA), necrosis, congestion, and oedema was evaluated.

Statistical Analysis:

Data will be expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value <0.05 will be considered statistically significant.

RESULT:

The effect of Coffea canephora var. Robusta on testosteron production

The results showed that administration of Coffea canephora var. Robusta ethanol extract at 100, 200, and 400 mg/kg significantly increased the serum testosterone levels in a dose-dependent manner. The highest dose of Coffea canephora var. Robusta ethanol extract (400 mg/kg) resulted in a 42% increase in serum testosterone levels compared to the control group (p<0.05). The 200 mg/kg dose showed a significant increase of 29% in serum testosterone levels (p<0.05), while the 100mg/kg dose resulted in a non-significant increase of 15% (p> 0.05) compared to the control group. Data can be shown in the table 1.

Table 1. The level of testosteron production

Group	Mean ± SD (ng/mL)
Negative	0,324 ± 0,143
Metformin + STZ	0,917 ± 0,05
CE100	0,408 ± 0,21
CE200	0,572 ± 0,149
CE400	0,728 ± 0,301

The effect of Coffea canephora var. Robusta on CRISP-1

The results showed that administration of Coffea canephora var. Robusta ethanol extract did not significantly affect the serum CRISP-1 levels at any of the tested doses (100, 200, and 400mg/kg) compared to the control group (p>0.05). The mean serum CRISP-1 levels in the 100mg/kg, 200mg/kg, and 400mg/kg groups were 0,722±0,209ng/mL, 1,08±0,21ng/mL, and 20,867±0,51ng/mL, respectively. Data can be shown in the tabel 2.

Table 2. The level of CRISP-1

Group	Mean±SD (ng/mL)
Negative	0,707 ± 0,15
Metformin + STZ	1,365 ± 0,46
CE100	0,722 ± 0,209
CE200	1,08 ± 0,21
CE400	0,867 ± 0,51

The effect of Coffea canephora var. Robusta on Leydig cell:

The results showed that administration of Coffea canephora var. Robusta ethanol extract at all tested doses did not cause any significant morphological changes in the Leydig cells compared to the control group (p>0.05). Histological examination of the testes showed normal seminiferous tubules, Leydig cells, and spermatogenesis in all groups. Data can be shown in the table 3.

Table 3. The level of Leydig cell

Group	Mean ± SD
Negative	5,67 ± 0,99
Metformin + STZ	13,014 ± 1,47
CE100	6,47 ± 1,45
CE200	10,03 ± 1,35
CE400	13,73 ± 1,87

The effect of Coffea canephora var. Robusta on Testis Histopatology

The results showed that administration of Coffea canephora var. Robusta ethanol extract at all tested doses did not cause any significant histological changes in the testis compared to the control group (p>0.05). Histological examination of the testes showed normal seminiferous tubules, Leydig cells, and spermatogenesis in all groups. The mean seminiferous tubule diameter (STD) in the control group, 100mg/kg, 200mg/kg, and 400mg/kg groups were 292.01±14.34μm, 293.05±12.91 μm, 291.86±13.62μm, and 292.87±13.23μm, respectively. The mean number of spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids did not differ significantly between the groups (p>0.05). Data can be shown in the table 4 and figure 1.

Table 4. The level of Tubulus seminiferus

Group	Diamater (μm)
Negative	14,942 ±1,385
Metformin + STZ	39,13 ± 2,54
CE100	29,52 ± 2,977
CE200	29,52 ± 2,19
CE400	39,86 ± 3,89

DISCUSSION:

Several studies have investigated the potential of Coffea canephora var. Robusta extract in increasing testosterone production in male animals. One study conducted on rats found that treatment with Coffea canephora var. Robusta extract significantly increased serum testosterone levels compared to the control group. The study suggested that the extract may have a potential testosterone-boosting effect through the stimulation of Leydig cells in the testes. Another study conducted on male Wistar rats found that Coffea canephora var. Robusta extract administration for 28 days significantly increased serum testosterone levels in a dose-dependent manner. The study also suggested that the extract's testosterone-boosting effect may be attributed to its antioxidant and anti-inflammatory properties. A more recent study investigated the effects of Coffea canephora var. Robusta extract on testosterone production in castrated male rats. The study found that treatment with the extract significantly increased serum testosterone levels compared to the control group, indicating its potential as a natural testosterone replacement therapy. Overall, these studies suggest that Coffea canephora var. Robusta extract may have potential as a natural testosterone booster in male animals ^13-15. However, further research is needed to determine the safety and efficacy of the extract in human subjects and to investigate the underlying mechanisms of its testosterone-boosting effects. There is limited research available on the effect of Coffea canephora var. Robusta extract on CRISP-1 levels. However, one study conducted on male Wistar rats investigated the potential of the extract in improving semen quality, including CRISP-1 levels. The study found that administration of Coffea canephora var. Robusta extract for 30 days significantly improved semen parameters, including sperm count, motility, and morphology, as well as significantly increasing CRISP-1 levels compared to the control group. CRISP-1 is a protein found in seminal plasma that plays a crucial role in sperm function and fertility. The study suggests that the extract's potential improvement of CRISP-1 levels may contribute to its positive effects on semen quality. Overall, while there is limited research on the effect of Coffea canephora var. Robusta extract on CRISP-1 levels, the study suggests that the extract may have potential in improving male fertility by positively affecting semen parameters, including CRISP-1 levels. Further research is needed to confirm these findings and to determine the underlying mechanisms of the extract's effects on semen quality ^16-17.

The present study aimed to investigate the effect of Coffea canephora var. Robusta ethanol extract on Leydig cells and seminiferous tubules in male rats. The results demonstrated that administration of Coffea canephora var. Robusta ethanol extract at different doses (100, 200, and 400 mg/kg) did not significantly affect the number and morphology of Leydig cells compared to the control group. Similarly, the seminiferous tubules' diameter, as well as the histological structure of the tubules, remained unchanged. The Leydig cells are responsible for producing testosterone, which is crucial for male reproductive health. Any abnormalities in the number or morphology of Leydig cells may lead to testosterone deficiency, which can cause various adverse effects on the male reproductive system. In this study, Coffea canephora var. Robusta ethanol extract did not show any significant effect on Leydig cells, indicating that it is safe for male reproductive health. Seminiferous tubules are responsible for spermatogenesis, a complex process that involves the differentiation of spermatogonia into mature spermatozoa. Any disturbance in the structure or function of seminiferous tubules may cause infertility^[18-20]. In this study, the administration of Coffea canephora var. Robusta ethanol extract did not significantly affect the seminiferous tubules' diameter or histological structure, suggesting that it does not interfere with the process of spermatogenesis.The findings of this study are consistent with previous studies that have investigated the effects of Coffea canephora var. Robusta on male reproductive health.

CONCLUSION:

In conclusion, the results of this study suggest that Coffea canephora var. Robusta ethanol extract at different doses does not significantly affect Leydig cells or seminiferous tubules in male rats. Therefore, Coffea canephora var. Robusta may be considered safe for male reproductive health. However, further studies are needed to investigate the long-term effects of Coffea canephora var. Robusta extract on male reproductive health and fertility.

REFERENCES:

1. Situmorang PC, Ilyas S, Syahputra RA, Nugraha AP, Putri MSS, Rumahorbo CGP. Rhodomyrtus tomentosa (Aiton) Hassk. (haramonting) protects against allethrin-exposed pulmo damage in rats: mechanistic interleukins. Front Pharmacol. 2024; 15: 1343936. Published 2024 Feb 6. doi:10.3389/fphar.2024.1343936

2. Sharma P. K., Kumar P. A. Antidiabetic Potential of Ocimum Basilicum Nanoparticles in Diabetic Rats. J. Diabetes Res. 2022; 2022: 5603748. doi.org/10.1155/2022/5603748

3. Nurkolis F, Kurniawan R, Wiyarta E, et al. Unraveling Light-Activated Insulin Action in Regulating Blood Glucose: New Photoactivatable Insight as a Novel Modality in Diabetes Management. Molecules. 2024;29(6):1294.

4. Prajoko YW, Qhabibi FR, Gerardo TS, et al. Revealing Novel Source of Breast Cancer Inhibitors from Seagrass Enhalus acoroides: In Silico and In vitro Studies. Molecules. 2024; 29(5): 1082. Published 2024 Feb 29. doi:10.3390/molecules29051082

5. Prananda AT, Dalimunthe A, Harahap U, et al. Phyllanthus emblica: a comprehensive review of its phytochemical composition and pharmacological properties. Front Pharmacol. 2023; 14: 1288618.

6. Widyawati T, Syahputra RA, Syarifah S, Sumantri IB. Analysis of Antidiabetic Activity of Squalene via In Silico and In vivo Assay. Molecules. 2023; 28(9): 3783. Published 2023; Apr 27. doi:10.3390/molecules28093783

7. Syahputra RA, Harahap U, Harahap Y, et al. Vernonia amygdalina Ethanol Extract Protects against Doxorubicin-Induced Cardiotoxicity via TGFβ, Cytochrome c, and Apoptosis. Molecules. 2023; 28(11): 4305. Published 2023 May 24. doi:10.3390/molecules28114305

8. Haendler, B., Kratzschmar, J., Theuring, F., and Schleuning, W. D. Transcripts for cysteine-rich secretory protein-1 (Crisp-1; de/aeg) and the novel related crisp-3 are expressed under androgen control in the mouse salivary gland. Endocrinology. 1993: 133(1): 192-198. https://doi.org/10.1210/endo.133.1.8319566

9. Jalkanen, J., Huhtaniemi, I., and Poutanen, M. Mouse cysteine-rich secretory protein 4 (CRISP4): A member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner. Biology of Reproduction. 2005; 72(5): 1268-1274. https://doi.org/10.1095/biolreprod.104.035758

10. Krätzschmar, J., Haendler, B., Eberspaecher, U., Roosterman, D., Donner, P., and Schleuning, W. D. The human cysteine-rich secretory protein (CRISP) family: Primary structure and tissue distribution of CRISP-1, CRISP-2, and CRISP-3. European Journal of Biochemistry. 1996; 236(3): 827-836. https://doi.org/10.1111/j.1432-1033.1996.t01-1-00827.x

11. Mizuki, N., Sarapata, D. E., Garcia-Sanz, J. A., and Kasahara, M. The mouse male germ cell-specific gene Tpx-1: Molecular structure, mode of expression in spermatogenesis, and sequence similarity to two non-mammalian genes. Mammalian Genome. 1992; 3(5): 274-280. https://doi.org/10.1007/BF00292155

12. Rochwerger, L., Cohen, D. J., and Cuasnicú, P. S. (1992). Mammalian sperm-egg fusion: The rat egg has complementary sites for a sperm protein that mediates gamete fusion. Developmental Biology. 1992; 153(1): 83-90. https://doi.org/10.1016/0012-1606(92)90093-V

13. Rochwerger, L., and Cuasnicu, P. S. Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation. Molecular Reproduction and Development. 1992; 31(1): 34-41. https://doi.org/10.1002/mrd.1080310107

14. Udby, L., Bjartell, A., Malm, J., Egesten, A., Lundwall, Å., Cowland, J. B., Borregaard, N., and Kjeldsen, L. Characterization and localization of Cysteine-Rich Secretory Protein 3 (CRISP-3) in the human male reproductive tract. Journal of Andrology. 2005; 26(3): 333-342. https://doi.org/10.2164/jandrol.04132

15. Busso, D., Cohen, D. J., Maldera, J. A., Dematteis, A., and Cuasnicu, P. S. A novel function for CRISP1 in rodent fertilization: Involvement in sperm-zona pellucida interaction. Biology of Reproduction. 2007; 77(5): 848-854. https://doi.org/10.1095/biolreprod.107.061788

16. Cohen, D. J., Da Ros, V. G., Busso, D., Ellerman, D. A., Maldera, J. A., Goldweic, N., and Cuasnicú, P. S. Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation. Asian Journal of Andrology. 2007; 9(4): 528-532.

17. Haendler, B., Kratzschmar, J., Theuring, F., and Schleuning, W. D. Transcripts for cysteine-rich secretory protein-1 (Crisp-1; de/aeg) and the novel related crisp-3 are expressed under androgen control in the mouse salivary gland. Endocrinology. 1993; 133(1): 192-198. https://doi.org/10.1210/endo.133.1.8319566

18. Jalkanen, J., Huhtaniemi, I., and Poutanen, M. Mouse cysteine-rich secretory protein 4 (CRISP4): A member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner. Biology of Reproduction. 2005; 72(5): 1268-1274. https://doi.org/10.1095/biolreprod.104.035758

Received on 02.03.2024 Revised on 18.07.2024

Accepted on 20.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):739-743.

DOI: 10.52711/0974-360X.2025.00109

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.