
==== Front
J Adv Pharm Technol Res
J Adv Pharm Technol Res
JAPTR
J Adv Pharm Technol Res
Journal of Advanced Pharmaceutical Technology & Research
2231-4040
0976-2094
Wolters Kluwer - Medknow India

JAPTR-15-194
10.4103/JAPTR.JAPTR_458_23
Original Article
Effect of hesperidin in the prevention of aluminum chloride-induced testicular dysfunction in rats
Parasuraman Subramani 12
Huey Loh Yee 1
Jee Yap Shen 1
1 Unit of Pharmacology, Toxicology and Basic Health Sciences, Faculty of Pharmacy, AIMST University, Bedong, Kedah, Malaysia
2 Center for Global Health Research, Saveetha Medical College and Hospital, Chennai, Tamil Nadu, India
Address for correspondence: Dr. Subramani Parasuraman, Unit of Pharmacology, Toxicology and Basic Health Sciences, Faculty of Pharmacy, AIMST University, Bedong, Kedah, Malaysia. E-mail: parasuraman@aimst.edu.my/parasuphd@gmail.com
Jul-Sep 2024
22 7 2024
15 3 194199
03 10 2023
21 4 2024
11 4 2024
Copyright: © 2024 Journal of Advanced Pharmaceutical Technology & Research
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Hesperidin is a citrus bioflavonoid and has numerous pharmacological actions. Hesperidin’s effect on testicular dysfunction has not been well researched. Hence, the present study is aims to investigate the effect of hesperidin on aluminium chloride (AlCl3)-induced testicular dysfunction in rats. Both vitamin C (200 mg/kg) and hesperidin (50, 100, and 200 mg/kg) were administered orally for 21 days. At the end of the study, the blood samples were obtained from all animals for investigation of biochemical and hematological parameters. Then, bilateral orchiectomy was carried out to remove testicles from the animals, and sperm was collected and examined under a microscope. Finally, organs such as the liver, kidney, and testicles were also collected and utilized for histopathological analysis. Part of the liver sample was used for determination of antioxidant enzymes such as reduced glutathione (GSH) and catalase (CAT) levels. The rats administered with AlCl3 showed elevated levels of biochemical and hematological parameters and a reduction in levels of sperm count, sperm motility, and oxidative stress parameters, whereas the rats administered with vitamin C/hesperidin (200 and 400 mg/kg) were able to ameliorate AlCl3-induced testicular dysfunction by attenuating AlCl3-induced changes in biochemical and hematological parameters, sperm motility, sperm count, and oxidative stress. Both vitamin C and hesperidin had significant ameliorative effects against AlCl3-induced testicular dysfunction.

Bioflavonoids
reactive oxygen species
testicular dysfunction
vitamin C
==== Body
pmcINTRODUCTION

Male infertility factors account for almost half of all occurrences of infertility in 10%–15% of infertile couples. Exposure to heavy metals may have an adverse impact on male fertility, either directly or indirectly. Heavy metals and metal ions can cause oxidative stress and impair antioxidant capability. As a result, testicular tissue is damaged by oxidative stress, which can lead to infertility and poor semen quality.[1] Aluminum (Al) is the most abundant metal in the Earth’s crust, and it is commonly used in a wide range of human activities. Al is extensively utilized in several industries, including the packaging of food.[2] Human exposure to Al may be a significant cause of decreasing sperm counts and male fertility.

The provisional tolerable weekly intake of Al for food additives is 2 mg/kg.[2] Al migration occurs when food contacts with Al foil during food packing. As a result, most adults ingest 1–10 mg/day of Al from natural sources.[3] The most common way that humans are exposed to Al is through their digestive systems, and the rate of absorption in this area is approximately 0.2%. Through receptor-mediated transferrin endocytosis, Al (or around 0.005% of Al-protein complexes) can reach the nervous system, accumulate in the brain, and cause neurodegeneration.[4] Al also exhibits prooxidant effects and increases the risk of neurodegenerative disorders.[5] Chronic Al exposure reduces the antioxidant enzymes and influences prooxidant systems.[6] The toxicological properties of Al include hepatotoxicity, neurodegenerative disorders, microcytic hypochromic anemia, reproductive toxicity, and genotoxicity. These effects are due to the oxidative degradation of deoxyribonucleic acid and the production of free radicals.[7]

Plants are a major source of medicine for early drug discovery, and they have been used for centuries to treat a wide range of illnesses. Plants synthesize secondary metabolites and these substances play significant roles in the defense, growth, and development of the plant but are not necessary for the essential growth and development of the plant. The biological effects of these secondary metabolites on plants and other living things are diverse. Nearly 50,000 secondary metabolites have been found in plants including hesperidin.[8]

Hesperidin is a bioflavonoid and is abundantly found in citrus fruits (Rutaceae family). Hesperidin is known for its antioxidant activity and exhibits several pharmacological actions against systemic diseases.[9] Hesperidin’s effect on testicular dysfunction has not been extensively studied. Hence, the present study is aims to investigate the effect of hesperidin on aluminium chloride (AlCl3)-induced testicular dysfunction in rats.

METHODS

Effect of hesperidin on aluminum chloride-induced testicular dysfunction

Healthy, adult, male Sprague-Dawley (SD) with a weight of 210 ± 15 g were used in the study. The animals were divided into six groups, namely vehicle (normal control), AlCl3, AlCl3 + vitamin C (200 mg/kg), AlCl3 + hesperidin 50 mg/kg, AlCl3 + hesperidin 100 mg/kg, and AlCl3 + hesperidin 200 mg/kg administered groups. The animal doses of hesperidin and vitamin C were based on the scientific literature.[1011] Testicular dysfunction was induced by administering an intraperitoneal injection of AlCl3 (20 mg/kg) (dissolved in water).[8] Both vitamin C and hesperidin were suspended in carboxymethyl cellulose and administered orally once per day for 21 days. AlCl3 was administered after 1 h of vitamin C/hesperidin administration. Body weight changes were monitored regularly.

At the end of the study, the animals were anesthetized using diethyl ether, and the blood sample was collected through retro-orbital plexus puncture and used for the analysis of biochemical (using a Reflotron Plus biochemical analyzer [Roche Diagnostics, Germany]) and hematological (performed by Gribbles Pathology Lab Sdn. Bhd., Malaysia) parameters. Then, bilateral orchiectomy was carried out to remove both testicles from the animals. Semen was extracted from the right cauda epididymis and used for the determination of sperm count and motility.[12] Finally, part of the liver sample was collected and stored at −20°C until used for determination of antioxidant enzymes such as reduced glutathione (GSH) and catalase (CAT) levels using Ellman’s reagent method and hydrogen peroxide method, respectively.[13] Later, organs including the liver, kidney, and testicles were also collected, preserved in 10% neutral formalin, and utilized for histopathological analysis.[14]

Statistical analysis

Data were presented as mean ± standard error of the mean (SEM). Analysis was carried out using one-way ANOVA followed by Turkey’s multiple comparisons post-hoc test. P ≤0.05 was considered statistically significant.

RESULTS

In the present study, reduction in body weight was observed in the AlCl3-administered group, but the results were not significant [Figure 1]. The effect of hesperidin on biochemical and hematological parameters in rats administered with AlCl3 is summarized in Tables 1 and 2. The rats administered with AlCl3 showed a significant increase in the levels of aspartate transaminase, alanine aminotransferase, alkaline phosphatase, total bilirubin, urea, creatinine, neutrophils, and lymphocytes and a significant decrease in the level of albumin and platelets when compared to the control group, whereas both vitamin C and hesperidin 100 and 200 mg/kg prevented the AlCl3-induced changes in the biochemical and hematological parameters.

Figure 1 Effect of hesperidin on body weight of experimental rats. All the values are mean ± standard error of the mean (n = 6). AlCl3: Aluminum chloride

Table 1: Effect of hesperidin on biochemical parameter

Group	Total protein (g/L)	AST (U/L)	ALT (U/L)	ALP (U/L)	Albumin (g/L)	Globulin (g/L)	Total bilirubin (mg/dL)	Urea (mg/dL)	Creatinine (mg/dL)	
Control	75.50±2.35	88.17±3.52	45.17±2.65	103.83±4.67	44.50±2.45	29.50±2.93	0.93±0.08	22.83±1.40	0.25±0.02	
AlCl3 20 mg/kg	57.00±5.80	121.33±6.13**	82.83±5.28***	136.67±5.53**	30.17±3.03**	25.33±4.30	1.53±0.16***	42.83±4.15***	0.55±0.03***	
Vitamin C 200 mg/kg	70.17±3.86	94.50±2.25#	51.83±3.11###	109.50±9.38#	44.17±2.52##	25.33±1.54	0.92±0.06###	24.50±2.20###	0.29±0.01###	
Hesperidin 50 mg/kg	62.83±2.34	113.17±8.83*	68.00±7.80*	124.33±4.70	35.67±1.76	24.17±1.45	1.13±0.08#	33.00±3.97	0.35±0.02###	
Hesperidin 100 mg/kg	65.33±5.00	95.50±5.34#	55.50±4.88##	111.17±3.15	42.83±3.47#	22.17±2.09	0.97±0.05##	27.50±2.26##	0.35±0.02###	
Hesperidin 200 mg/kg	69.83±4.96	95.33±3.37#	52.83±2.91##	110.00±6.29#	43.33±1.28#	25.50±2.93	0.97±0.08##	25.17±1.76##	0.31±0.04###	
*P<0.05, **P<0.01, and ***P<0.001 compared with that of the control group, #P<0.05, ##P<0.01 and ###P<0.001 compared with that of AlCl3 20 mg/kg administered group. All the values are mean±SEM (n=6). SEM: Standard error of the mean, ALT: Alanine aminotransferase, AST: Aspartate transaminase, ALP: Alkaline phosphatase, AlCl3: Aluminum chloride

Table 2: Effect of hesperidin on hematological parameter

Parameter	Control	AlCl320 mg/kg	Vitamin C 200 mg/kg	Hesperidin 50 mg/kg	Hesperidin 100 mg/kg	Hesperidin 200 mg/kg	
Hemoglobin (g/L)	131.83±7.47	99.50±6.79	126.50±10.48	112.17±12.63	119.83±4.09	126.17±6.83	
Red blood cell count (×1012/L)	4.85±0.32	4.17±0.22	4.75±0.17	4.37±0.23	4.73±0.17	4.80±0.15	
Platelet count (×109/L)	985.83±45.30	818.17±48.86*	985.33±24.75#	899.00±40.37	962.00±35.69	982.00±28.29	
Total white blood cell (×109/L)	9.35±0.40	10.68±0.52	9.13±0.41#	9.32±0.27	9.13±0.18#	9.28±0.26	
Neutrophils (×109/L)	0.85±0.03	1.17±0.09***	0.89±0.04##	0.98±0.03	0.91±0.03##	0.86±0.02###	
Lymphocytes (×109/L)	10.57±0.22	11.70±0.25*	10.38±0.27##	10.38±0.24##	10.32±0.21##	10.83±0.11	
Monocytes (×109/L)	1.31±0.06	1.44±0.06	1.27±0.06	1.28±0.07	1.34±0.03	1.29±0.02	
Eosinophils (×109/L)	0.11±0.01	0.12±0.01	0.11±0.01	0.12±0.01	0.11±0.01	0.11±0.01	
*P<0.05 and ***P<0.001 compared with that of the control group, #P<0.05, ##P<0.01, and ###P<0.001 compared with that of AlCl3 20 mg/kg administered group. All the values are mean±SEM (n=6). AlCl3: Aluminum chloride, SEM: Standard error of the mean

In sperm analysis, AlCl3-administered animals showed a significant reduction in motility and number of sperm cells when compared to control animals, whereas vitamin C/hesperidin restored the motility of sperm cells and sperm count [Figures 2 and 3].

Figure 2 Effect on sperm motility. All the values are mean ± standard error of the mean (n = 6). aP < 0.05, bP < 0.01, and cP < 0.001 compared with that of the control group; eP < 0.01 and fP < 0.001 compared with that of AlCl3 20 mg/kg administered group. AlCl3: Aluminum chloride

Figure 3 Effect of hesperidin on sperm count. All the values are mean ± standard error of the mean (n = 6). cP < 0.001 compared with that of the control group; eP < 0.01 and fP < 0.001 compared with that of AlCl3 20 mg/kg administered group. AlCl3: Aluminum chloride

In the antioxidant assay, the animal administered with AlCl3 showed significant decreases in the levels of GSH and CAT when compared to the control group [Figures 4 and 5], whereas vitamin C/hesperidin restored the levels of GSH and CAT [Figures 4 and 5].

Figure 4 Effect of hesperidin on reduced glutathione enzyme. All the values are mean ± standard error of the mean (n = 6). cP < 0.001 and aP < 0.05 compared with that of the control group; fP < 0.001 compared with that of AlCl3 20 mg/kg administered group. AlCl3: Aluminum chloride

Figure 5 Effect of hesperidin on catalase enzyme. All the values are mean ± standard error of the mean (n = 6). cP < 0.001 compared with that of the control group; fP < 0.001 compared with that of AlCl3 20 mg/kg administered group. AlCl3: Aluminum chloride

In histopathological analysis, the liver of the control group and AlCl3 + Vitamin C/hesperidin 200 mg/kg administered animals showed normal configuration of hepatocytes. The liver of AlCl3-administered animals showed binucleated hepatocytes, mild sinusoidal congestion, and mild centrilobular necrosis [Figure 6]. The liver of animals co-administered with hesperidin 50 or 100 mg/kg administered animals showed binucleated hepatocytes and mild sinusoidal congestion [Figure 6]. AlCl3-administered showed mild vacuolar degeneration of tubules in the kidney [Figure 7]. The kidneys of the control group and AlCl3 + vitamin C or hesperidin 50/100/200 mg/kg showed normal renal cells [Figure 7]. These findings indicated hesperidin possesses a nephroprotective effect at concentrations of 50 mg/kg and above. The testis of the control group and AlCl3 + Vitamin C/hesperidin 200 mg/kg administered animals showed normal seminiferous tubule morphology (the testis is surrounded by a thick tunic of connective tissue) [Figure 8]. The testis of the AlCl3-administered animals shows oligospermia and increased spaces between tubules. The testis of the AlCl3 + hesperidin 50/100 mg/kg administered animals show increased spaces between tubules [Figure 8].

Figure 6 Histopathology of liver in rats (photomicrograph of hematoxylin and eosin; x400) (a) Control; (b) AlCl3 20 mg/kg; (c) Vitamin C 200 mg/kg; (d) Hesperidin 50 mg/kg; (e) Hesperidin 100 mg/kg; (f) Hesperidin 200 mg/kg

Figure 7 Histopathology of kidney in rats (photomicrograph of haematoxylin and eosin; x400) (a) Control; (b) AlCl3 20 mg/kg; (c) Vitamin C 200 mg/kg; (d) Hesperidin 50 mg/kg; (e) Hesperidin 100 mg/kg; (f) Hesperidin 200 mg/kg

Figure 8 Histopathology of testis in rats (photomicrograph of hematoxylin and eosin; x100) (a) Control; (b) AlCl3 20 mg/kg; (c) Vitamin C 200 mg/kg; (d) Hesperidin 50 mg/kg; (e) Hesperidin 100 mg/kg; (f) Hesperidin 200 mg/kg

DISCUSSION

In the present study, hesperidin prevented the AlCl3-induced testicular dysfunction by attenuating AlCl3-induced changes in biochemical and hematological parameters, sperm motility, sperm count, and oxidative stress. Al has been indicated to induce male reproductive toxicity by various mechanisms including an increase in the oxidative stress enzyme levels, affecting the endocrine system, impairment of the blood–testis barrier, alteration in membrane function, changes to spermatogenesis, and changes in steroidogenesis.[15] Al also causes a dose-dependent reduction in sperm viability, motility, and increased malondialdehyde (MDA) level.[16]

In the present study, a decrease in body weight (results were not significant) was observed in the AlCl3-administered group, and this may be due to the anorectic effect of Al.[17] In the biochemical analysis, AlCl3-administered rats showed elevated levels of liver and renal markers which indicate that animals are at risk of liver and kidney damage. In experimental animals, AlCl3 disturbs the mitochondrial Deoxyribonucleic acid (DNA) transcript and mitochondrial energy metabolism and causes an increase in liver enzymes.[18] AlCl3-induced nephrotoxicity is due to the accumulation of Al in the renal system.[19] Chronic Al exposure causes oxidative stress and suppresses liver and kidney functions.

In the present study, vitamin C and hesperidin prevented AlCl3-induced changes in sperm count and motility. The ameliorative of hesperidin is based on its ability to scavenge free radicals. The excessive reactive oxygen species may disrupt mitochondrial energy production which leads to a significant increase in total abnormal sperm rate and decreases sperm motility.[20] Al can also cross the blood–testis barrier and cause oxidative stress, which affects the testes’ cellular membranes and interferes with spermatogenesis.[21]

AlCl3-administered rats also showed decreases in the levels of GSH and CAT. Ogunlade et al. also found decreased antioxidant enzymes and increased MDA levels in AlCl3-administered animals.[22] The reduction in the antioxidant enzyme activities demonstrated an impaired antioxidant system, leading to oxidative stress-induced testicular dysfunction/toxicity. However, vitamin C/hesperidin ameliorates AlCl3-induced oxidative stress by increasing antioxidant enzyme levels. It has been inferred that vitamin C/hesperidin prevents testicular dysfunction by scavenging free radicals and lowering their levels. In a clinical study, hesperidin is able to reduce the level of oxidative stress markers by increasing antioxidant enzyme levels.[23] During the cryopreservation-thawing process, hesperetin (a metabolite of hesperidin) improves the quality of human sperm (viability and motility) and also protects sperm from reactive oxygen species, lipid peroxidation, and apoptosis.[24]

The findings of histopathological analysis indicate hesperidin possesses hepatoprotective, nephroprotective, and testicular protective effects against AlCl3-induced tissue damage. In histopathological analysis, AlCl3-administered animals showed mild-to-moderate tissue damage. The histopathology of the liver, kidney, and testis of animals in the control group, AlCl3 + vitamin C, and AlCl3 + hesperidin 100/200 mg/kg showed minimal histological changes or normal cellular arrangement. Al is known to exhibit hepatotoxicity, nephrotoxicity, and testicular damage.[182526]

The present study data indicated that AlCl3-inducing testicular dysfunction by increasing oxidative stress enzymes and co-administration of hesperidin prevents the AlCl3-induced testicular dysfunction.

CONCLUSION

AlCl3 caused testicular dysfunctions in experimental rats. The rats co-administered with vitamin C/hesperidin (200 and 400 mg/kg) were able to ameliorate AlCl3-induced changes in biochemical and hematological parameters and prevented AlCl3-induced testicular dysfunction in experimental animals. Both vitamin C and hesperidin exhibited antioxidant activity, and inhibition of AlCl3-induced changes may be mediated through their antioxidant effect.

Ethics approval

Prior to the study, permission was obtained from the AIMST University Human and Animal Ethics Committee to carry out animal experiments (AUAEC/FOP/2021/05).

Financial support and sponsorship

The authors received financial support for publication of this article from AIMST University.

Conflicts of interest

There are no conflicts of interest.
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