Research Article

The Effect of Telang Flower (Clitoria ternatea L.) Extract on Thickness of Seminiferous Tubule Epithelium and Sertoli Cells in Male Rats (Rattus norvegicus) Exposed to Cigarette Smoke

Putra Aliffiansyah Farhanudin1, Widjiati Widjiati2*, Wiwiek Tyasningsih3, Suherni Susilowati4, Nove Hidajati5, Sri Pantja Madyawati4, Iwan Sahrial Hamid5, Viski Fitri Hendrawan4

1Master’s Degree of Biology Reproduction Student Program, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia; 2Anatomy Division, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia; 3Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia; 4Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia; 5Department of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia.

Received | September 24, 2025; Accepted | January 12, 2026; Published | February 11, 2026

*Correspondence | Widjiati, Anatomy Division, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia; Email: [email protected]

Citation | Farhanudin PA, Widjiati W, Tyasningsih W, Susilowati S, Hidajati N, Madyawati SP, Hamid IS, Hendrawan VF (2026). The effect of telang flower (Clitoria ternatea L.) extract on thickness of seminiferous tubule epithelium and sertoli cells in male rats (Rattus norvegicus) exposed to cigarette smoke. J. Anim. Health Prod. 14(1): 372-378.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.1.372.378

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Currently in Indonesia there are many active smokers from various groups ranging from adults to teenagers. Cigarettes can have a negative impact on individual and community health (Batubara et al., 2013). The number of deaths in the world population caused by smoking is 30% or the equivalent of 17.3 million people, while in Indonesia alone 225,700 people died due to cigarettes, cigarette smoke or other related tobacco products (World Health Organization, 2022). Exposure to cigarette smoke causes damage to organs in the body caused by free radicals. Very high amounts of free radicals in cigarette smoke will cause damage such as edema and infiltrated inflammatory cells resulting from the effects of cigarette smoke (Herdiani and Budi, 2018).

Exposure to cigarette smoke can disrupt the normal development of spermatozoa, cause damage to the DNA of testicular cells, and cause problems with the reproductive organs (Liu et al., 2022). The impact of cigarette smoke can cause damage to the seminiferous tubules and Sertoli cells, as well as reducing the number of spermatogenic cells which can result in infertility in men. Damage is characterized by a decrease in the number of Sertoli cells and a decrease in the diameter of the seminiferous tubules after exposure to cigarette smoke (Angelia et al., 2022).

The bad impact of exposure to cigarette smoke on men’s reproductive health can be reduced, one way is by using plant extracts as herbal medicine therapy. One of the plants that has benefits for improving the health of the reproductive organs is the flower part of the telang plant (Clitoria ternatea). Butterfly pea flowers have long been used as a medicinal ingredient in various countries. The compounds in it help protect against cell damage due to free radicals and inflammation, such as anthocyanin compounds which are believed to be antioxidants (Pramesemara, 2017).

Flowers of the telang plant (Clitoria ternatea) are cultivated throughout the world to meet the demands of the herbal agro-industry medicinal industry and aesthetic purposes (Oguis et al., 2019). Butterfly pea flowers contain several secondary metabolites such as tannins, triterpenoids, saponins, flavonoids and alkaloids (Aziza et al., 2021). Butterfly pea flower extract weighed 59.65 g. and yield value 11.93% has very good antioxidant activity with IC50 value amounting to 20.787 ppm (Kuswandari et al., 2022).

Research using butterfly pea flower extract (Clitoria ternatea L.) to observe Sertoli cells and seminiferous tubule epithelium in male white rats (Rattus norvegicus) has never been done before. The content of butterfly pea flowers is quite complex. Therefore, for the purpose of knowing the effect, research was carried out using male white rats (Rattus norvegicus) exposed to cigarette smoke and given treatment with an extract from the butterfly pea flower (Clitoria ternatea L.).

However, the protective effect of Clitoria ternatea extract on the testicular architecture specifically the seminiferous tubule epithelium and Sertoli cells in the context of cigarette smoke-induced damage remains unexplored. Therefore, this study aimed to investigate the role of C. ternatea extract in mitigating such damage. This study is aligned with the global health agenda outlined in the Sustainable Development Goals (SDGs), particularly SDG 3 (Good Health and Well-Being). More specifically, this research addresses SDG Target 3.9, which emphasizes reducing illnesses and health risks caused by exposure to hazardous chemicals and environmental pollution. Cigarette smoke represents a major source of toxic environmental exposure that adversely affects male reproductive health. By investigating the protective effects of Clitoria ternatea extract against cigarette-smoke-induced testicular damage, this study contributes to the development of safe, accessible, and plant-based strategies to mitigate reproductive toxicity and promote male reproductive well-being.

MATERIALS AND METHODS

Study period and location

The research was performed from May to July 2023. The extraction of Clitoria ternatea flowers was carried out at the Pharmacology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya. Animal maintenance, treatment, and sample collection were conducted at the Pharmacology Laboratory, Faculty of Medicine, Universitas Airlangga. Histopathological preparation was performed at the Veterinary Pathology Division Laboratory, while measurements and data analysis were conducted at the Embryology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga.

Plant material and extraction

The flowers of Clitoria ternatea were shade-dried and powdered. Extraction was performed using 70% ethanol in a 1:10 (w/v) ratio. The mixture was macerated for 3 × 24 hours at room temperature with intermittent stirring. The extract was filtered, evaporated under reduced pressure using a rotary evaporator at 40–45°C, and stored at 4°C until use. The yield was approximately 11.93% (Kuswandari et al., 2022).

Experimental design

This study was a true experimental study using a post-test only control group design with a simple random treatment design. A total of 25 Wistar male rats aged 2–3 months (250 g body weight) were used. The rats were divided randomly into five groups: C− (no exposure to cigarette smoke or extract), C + (exposed to cigarette smoke only), T1, T2, and T3 (exposed to cigarette smoke and administered Clitoria ternatea extract at doses of 150, 300, and 600 mg/kg BW/day, respectively). Cigarette smoke exposure was given to C +, T1, T2, and T3 groups, using two cigarettes per day per group for 35 days. Doses were based on previous research by (Intisari, 2022).

Cigarette smoke exposure

Rats in groups C +, T1, T2, and T3 were exposed daily to the smoke of two unfiltered commercial cigarettes (each containing 2.5 mg nicotine and 30 mg tar) for 35 days. Exposure was performed in an acrylic chamber (60 × 40 × 30 cm), with smoke introduced using a peristaltic pump for 10 minutes per cigarette. Group C− rats were not exposed.

Tissue collection and histological preparation

On day 36, rats were anesthetized intraperitoneally with ketamine (0.1 mL/kg BW) and xylazine (0.8 mL/kg BW). The testes were excised, fixed in 10% neutral buffered formalin (NBF) for 24–48 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H and E).

Histological evaluation

• Seminiferous tubule epithelium thickness: Measured using Image Raster by drawing a perpendicular line from the basement membrane to the lumen in five fields per tubule, across five tubules per animal. Results were expressed in µm (Altoe et al., 2014).
• Sertoli cell count: Counted under 400× magnification with Optilab software. Five tubules were selected per animal, and five fields examined per tubule (Nukman, 2018).

Research procedure

The procedure began with a 7-day adaptation period. Following this, cigarette smoke exposure and extract treatments were conducted for 35 days. On day 36, rats were anesthetized, dissected, and testes collected for histopathological analysis to assess seminiferous epithelium thickness and Sertoli cell counts.

Data collection

Epithelial thickness was measured from the basement membrane to the lumen surface in each seminiferous tubule, averaged from five different locations (Altoe et al., 2014; Pramesemara, 2017). Sertoli cells were counted in five tubules per group, with five fields per tubule observed via the spiral field-sweeping technique was performed as described by (Nukman, 2018).

Data analysis

Statistical analysis was performed using ANOVA in SPSS software. Significant differences (p < 0.05) were further analyzed using Duncan’s multiple range test and Pearson correlation analysis to assess relationships among variables.

RESULTS

Seminiferous tubule epithelium thickness

The mean thickness of the seminiferous tubule epithelium in all groups is presented in Table 1. A significant reduction was observed in the C + group (48.81 ± 6.13 µm) compared to the C group (92.39 ± 6.86 µm) (p < 0.05), indicating the impact of cigarette smoke exposure. Groups treated with Clitoria ternatea extract exhibited a dose-dependent increase in epithelial thickness. The mean values for T1, T2, and T3 were 61.29 ± 7.41 µm, 71.43 ± 6.92 µm, and 78.72 ± 6.61 µm, respectively. The T3 group showed no statistically significant difference compared to C− (p > 0.05), but remained significantly different from the C + group (p < 0.05). The comparison of group means is illustrated in Figure 2.

 

Table 1: Mean and standard deviation of Seminiferous Tubule Epithelial Thickness exposed cigarette smoke and given butterfly pea flower extract.

Treatment	N	Seminiferous tubule epithelial thickness (mm) (Mean ± SD)
C -	5	92.39 ± 7.22c
C +	5	48.81 ± 1.59a
T1	5	68.34 ± 16.07b
T2	5	73.46 ± 11.98b
T3	5	78.72bc ± 6.61bc

 

Different superscripts on the same table indicate significant significant differences (p<0.05). N = Number of test animals frequencies. Treatment = Groups consisting of C-, C+, T1, T2, and T3. Mean ± SD = Mean value and samples distribution for each group. C + = Exposure 2 (two) cigarettes/day and given 0.5% CMC Na 1 ml for 35 days. C - = Given 0.5% CMC Na 1 ml for 35 days only. T1 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 150 mg/kg BW/day in 2 ml for 35 days. T2 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 300 mg/kg BW/day in 2 ml for 35 days. T3 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 600 mg/kg BW/day in 2 ml for 35 days.

 

 

Table 2: Mean and standard deviation of total of sertoli cell count exposed cigarette smoke and given butterfly pea flower extract.

Treatment	N	Sertoli Cell Count (Mean ± SD)
C-	5	22.52 ± 1.97b
C+	5	17.28 ± 2.92a
T1	5	21.84 ± 1.26b
T2	5	22.88 ± 4.35b
T3	5	23.05 ± 1.61b

 

Different superscripts on the same table indicate significant significant differences (p<0.05). N = Number of test animals frequencies. Treatment = Groups consisting of C-, C+, T1, T2, and T3. Mean ± SD = Mean value and samples distribution for each group. C+ = Exposure 2 (two) cigarettes/day and given 0.5% CMC Na 1 ml for 35 days. C- = Given 0.5% CMC Na 1 ml for 35 days only. T1 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 150 mg/kgBW/day in 2 ml for 35 days. T2 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 300 mg/kgBW/day in 2 ml for 35 days. T3 = Exposure 2 (two) cigarettes/day and given butterfly pea flower extract at a dose of 600 mg/kgBW/day in 2 ml for 35 days.

 

Sertoli cell count

The mean Sertoli cell counts are shown in Table 2. A significant decrease was found in the C+ group (29.2 ± 3.0 cells/tubule) compared to the C− group (54.4 ± 4.2 cells/tubule) (p < 0.05). In the treatment groups, Sertoli cell counts increased progressively with dose: T1 (36.6 ± 3.4 cells/tubule), T2 (46.2 ± 4.0 cells/tubule), and T3 (51.6 ± 3.8 cells/tubule). The T3 group was not significantly different from the C − group (p > 0.05), and was significantly higher than the C + group (p < 0.05). The distribution of Sertoli cell numbers across groups is depicted in Figure 3. Representative histological sections showing Sertoli cells at 400× magnification are presented in Figure 5.

 

Correlation analysis

Pearson correlation analysis revealed a strong positive correlation between Sertoli cell count and seminiferous tubule epithelium thickness (r = 0.661, p = 0.000), as shown in Table 3. This correlation was statistically significant at the 0.01 level (two-tailed), indicating that higher Sertoli cell numbers were associated with greater epithelial thickness.

 

Table 3: Correlation analysis of seminiferous tubules epithelium thickness and total sertoli cell count exposed cigarette smoke and given butterfly pea flower extract.

Variable	Sertoli cells	Seminiferous tubules ephitelium thickness
Sertoli cells		0.661**
Seminiferous tubules epithelium thickness	0.661**

 

**. Correlation is significant at the 0.01 level (2-tailed).

 

DISCUSSION

Cigarette smoke exposure is a known potent inducer of oxidative stress due to its high levels of free radicals and toxic components. The imbalance between reactive oxygen species (ROS) and endogenous antioxidant defenses contributes to lipid peroxidation, germ cell apoptosis, and structural testicular damage (Kelainy et al., 2019; Aitken, 2018). In this study, histological alterations such as reduced seminiferous tubule epithelium thickness and decreased Sertoli cell numbers in smoke-exposed rats confirm these mechanisms of oxidative injury.

 

Sertoli cells play an essential role in maintaining testicular function by supporting spermatogenesis and synthesizing androgen-binding protein (ABP), which facilitates testosterone action within the seminiferous tubules (Alves et al., 2013). Damage to Sertoli cells caused by ROS disrupts these processes, leading to impaired spermatogenic cell differentiation, epithelial thinning, and potential infertility (Kovacevic et al., 2006). The susceptibility of Sertoli cells to oxidative damage is further enhanced by their polyunsaturated fatty acid content, which is highly prone to lipid peroxidation, producing malondialdehyde (MDA) as a biomarker of oxidative stress (Vijayprasad et al., 2014).

Treatment with Clitoria ternatea extract showed a dose-dependent protective effect on both Sertoli cell counts and seminiferous epithelium thickness. This protective action can be attributed to bioactive compounds such as flavonoids, anthocyanins, tannins, and alkaloids (Kuswandari et al., 2022; Aziza et al., 2021). Flavonoids and anthocyanins possess potent antioxidant activity, functioning as free radical scavengers, regulators of antioxidant enzyme expression (e.g., superoxide dismutase, glutathione peroxidase), and inhibitors of ROS regeneration (Ashidi et al., 2019). Tannins stabilize cellular membranes and chelate metal ions, while alkaloids contribute to DNA and membrane protection under oxidative stress (Derbak et al., 2021).

 

The antioxidant constituents of C. ternatea may also influence the hypothalamic–pituitary gonadal (HPG) axis. Flavonoids and alkaloids are known to stimulate anterior pituitary function, increasing the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby promoting Sertoli cell proliferation and enhancing spermatogenesis (Fadhilah et al., 2022). The observed improvement in seminiferous epithelium structure and Sertoli cell counts at higher doses (600 mg/kg BW) suggests a dual mechanism involving both direct antioxidative effects and hormonal modulation.

A strong positive correlation was found between Sertoli cell counts and seminiferous epithelium thickness (r = 0.661, p < 0.01), confirming the close association between cellular support and tissue structure integrity. This finding aligns with previous morphometric studies showing correlations between Sertoli cell characteristics (volume occupancy, nuclear size, numerical density) and spermatogenic capacity (França et al., 2016). Comparable findings were also observed in studies by Ghosh et al. (1992) and França et al. (2016), supporting that Sertoli cell dimensions and density influence germ cell populations and testosterone regulation.

Antioxidant plant extracts have demonstrated similar reproductive benefits in earlier studies. Punica granatum extract preserved testicular histology and improved sperm quality in smoke-exposed rats (Angelia et al., 2022). Likewise, Mucuna pruriens supplementation restored spermatogenic arrest and normalized antioxidant enzyme activities (Ashidi et al., 2019) and Peganum harmala alkaloids mitigated sperm damage and oxidative stress in ram semen (Derbak et al., 2021). These reports reinforce the current study’s findings that C. ternatea extract mitigates smoke-induced gonadotoxicity through its phytochemical constituents.

Prior investigations have shown cigarette smoke to cause severe damage to Sertoli cells, marked by increased HSCORE values, reduced cell counts, and disrupted seminiferous structures (Yüksel et al., 2014; Sobinoff et al., 2014). Passive smoke exposure has been reported to reduce epithelial thickness and elevate oxidative stress markers, corroborating the present results. The administration of C. ternatea extract likely restored the testicular microenvironment necessary for spermatogenesis by attenuating oxidative stress and supporting Sertoli cell function.

Although this study confirms the protective and dose-dependent efficacy of C. ternatea extract, limitations persist. The lack of molecular assays for antioxidant enzymes (e.g., SOD, catalase, MDA) and hormonal profiling limits detailed mechanistic insights. Further investigations integrating biochemical and reproductive endpoints, as well as toxicity and long-term safety analyses, are warranted. Nonetheless, the current findings provide strong evidence that Clitoria ternatea extract confers protection against cigarette smoke-induced testicular damage through combined antioxidant and endocrine-modulatory mechanisms (Mohamed et al., 2012).

CONCLUSIONS

This study demonstrated that Clitoria ternatea flower extract provides a dose-dependent protective effect against cigarette-smoke-induced testicular damage in male rats (Rattus norvegicus). Administration of 600 mg/kg BW of C. ternatea extract (T3 group) was the most effective in maintaining seminiferous tubule epithelium thickness and preserving Sertoli cell count. These findings indicate that the protective effect is likely mediated through the antioxidant and anti-apoptotic properties of the plant’s phytochemical constituents.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to the Faculty of Veterinary Medicine, Airlangga University, for providing the necessary facilities and support throughout this research. Special thanks to the Ethics Commission of the Faculty of Veterinary Medicine for approving the study protocol. We also extend our appreciation to the laboratory staff and all individuals who contributed to the successful completion of this study.

Novelty Statement

This study is the first to demonstrate the dose-dependent protective effect of Clitoria ternatea L. extract on both seminiferous tubule epithelial thickness and Sertoli cell count in male rats exposed to cigarette smoke. The novelty lies in the combined evaluation of histopathological parameters and their correlation, highlighting the potential of Clitoria ternatea as a natural antioxidant-based intervention for male reproductive health impaired by environmental toxins.

AUTHOR’s CONTRIBUTION

W contributed to the conception and design of the study, supervised the research process, and critically revised the manuscript. PAF was responsible for experimental work, data collection, and drafting the initial version of the manuscript. WT and SS assisted in conducting laboratory experiments, data analysis, and interpretation of results. NH and SPM contributed to histopathological examination, evaluation, and preparation of figures and tables. ISH assisted in data analysis and provided critical feedback on the manuscript draft.

Ethical approval

This research has received an ethical clearance certificate from the Ethics Commission of the Faculty of Veterinary Medicine, Airlangga University, Surabaya (No.1.KEH.074.05.2023).

Generative AI and AI-assisted technology statement

The authors declare that no generative AI or AI-assisted technologies were used in the writing, editing, data analysis, or figure generation processes for this manuscript. All content was solely produced and reviewed by the authors.

Conflict of interest

The authors have declared no conflict of interest.

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