Effect of Elettaria cardamomum L. on hormonal changes and spermatogenesis in the propylthiouracil‐induced hypothyroidism male BALB/c mice

Abstract Introduction Spermatogenesis is significantly influenced by the thyroid gland. Thyroid disorders can be caused by a variety of factors. Traditionally, Ellettaria cardamomum has been used to treat a variety of ailments. The effects of E. cardamomum extract (ECE) on spermatogenesis in hypothyroid mice were investigated in this study. Methods In this study 42 male mice, weighing (25–35 g) were randomly divided in six groups: control group (taking normal saline, 0.5 mL/day, by oral gavage [P.O.]), hypothyroid group (taking 0.1% propylthiouracil in drinking water for 2 weeks), hypothyroid groups treated by levothyroxine (15 mg/kg/day, P.O.) and hypothyroid groups treated by ECE (100, 200 and 400 mg/kg/day, P.O.). After the end of experiments the mice were anaesthetised and blood samples were collected for hormonal analysis. Results The sperm count and microscopic studies of testes were done also. Our results showed that the T3, T4, testosterone levels and spermatogenesis in hypothyroid animals decreased and thyroid‐stimulating hormone, follicle‐stimulating hormone and luteinizing hormone increased compared with control group. Treatment by ECE reverse these effects in comparison with hypothyroid group. Conclusions According to our findings, the ECE may stimulates thyroid gland function and increases testosterone and spermatogenesis.


| INTRODUC TI ON
Infertility and related problems are known as one of the important issues in the life of couples. 1 According to the available statistics, 35% of couples' infertility cases are related to men and 25% of infertility cases are related to both couples. The most common cause of male infertility is their inability to produce a sufficient number of healthy and active sperms and disturbances in the spermatogenesis process. 2,3 The binding of follicle-stimulating hormone (FSH) to Sertoli cells causes the synthesis of androgen-binding protein (APB). 4 Testosterone is secreted by Leydig cells. These cells are located in the interstitial tissue between the spermatogenic tubes. The amount of testosterone synthesis and secretion is regulated by the amount of luteinizing hormone (LH) in the blood. The level of testosterone secretion is directly related to the level of LH. 5 Thyroid gland function disorders and related hormonal fluctuations can each affect the body's chemical interaction mechanism.
As long as the disturbances resulting from these fluctuations lead to irreparable damage that endangers natural life. Thyroid hormones, which were previously thought to have no effect on spermatogenesis, are now recognized as an important factor influencing this process. The identification of thyroid hormone receptors on Sertoli cells in the testis initiated further studies to investigate the role of these hormones in male reproduction. 6 The effects of changing thyroid hormones on reproductive systems have been widely studied in humans and animal models, and the results have generally shown that changing thyroid function from the normal state leads to a decrease in fertility. 7 Medicinal plants are of special interest as a potential source of new chemotherapeutic drugs due to having a wide spectrum of bioactive compounds; because herbal medicines, due to their natural origin, are more compatible with living organisms, including the human body, than chemical medicines and cause fewer side effects. 8 The green cardamom plant with the scientific name Elettaria cardamomum is an herbaceous, perennial and evergreen plant. The origin of this plant is reported in South India and Sri Lanka. 9 E. cardamomum fruit is rich in volatile oils, which mainly include phenolic and flavonoid compounds. Starch, protein, waxes and sterols are other compounds found in green cardamom extract. 10 Phenolic compounds extracted from cardamom fruit have been proven to have antibacterial activity against Staphylococcus aureus and Escherichia coli extracted from prostate glands. 11 Kumari and Dutta 12 stated that the use of cardamom with a dose of 2% effectively reduces physiological changes and improves parenchymal levels in the lungs. In another study, it was shown that the use of green cardamom extract improves lung congestion, asthma, heart disease, eyelid inflammation and abdominal insufficiency. 13 Considering that the oral consumption of this plant has been common among people for a long time, and its therapeutic use in traditional medicine, and so far, there are no studies on the therapeutic effects of this plant. The effect of the plant on the process of spermatogenesis in patients with hypothyroidism has not been done, this study design to investigate the protective effect of E. cardamomum on spermatogenesis process in the hypothyroid male mice.

| Plant material and extraction
The E. cardamomum plant was purchased from Avicenna's Medicinal Plants Center of Hamedan Province Agricultural Jihad Organization (herbarium code: 36586) in the early summer of 2014. Since the prepared plant was fresh, it was spread on a paper for a few days and completely dried. Then the dried seeds were powdered with an electric mill. To prepare the extract 200 g of the seed powder was poured into 1000 mL of ethanol and place in the dark room for 1 week; then, the resulting mixture was filtered and concentrated in vacuum at 45°C using a rotary apparatus (EYELA). The resultant extract was dried and stored in the refrigerator at 4°C until the experiment. The ECE prepared as 100, 200 and 400 mg/kg of body weight of animal and were given by oral rout (P.O.). The selected dose based on earlier report. 14

| Preparation of a solution containing 0.1% propylthiouracil
In order to induce hypothyroidism in mice, the method of feeding propylthiouracil (PTU) in drinking water was used. To prepare the above solution, 0.1 mg of each tablet was dissolved in 100 mL of drinking water and given to the animals. Daily, the above solution was freshly prepared and freely provided to the mice. After 2 weeks, rats consuming this solution will develop hypothyroidism. 15

| Preparation of levothyroxine solution
Levothyroxine drug was obtained in the form of oral tablets containing 0.1 mg of the active ingredient (Iran Hormone Pharmaceutical Company). Then each tablet was dissolved in 20 mL of distilled water.
The resulting solution will have a concentration of 5 μg/mL. From the resulting solution, each animal was fed with a dose of 15 μg/kg and with a certain volume of 0.2 mL by insulin syringe to each mouse daily and by gavage. 16

| Animals' maintenance and ethical considerations
In this experimental study, 42 male BALB/c mice weighing 25-35 g and six-weeks-old were purchased from Pasteur Institute in Tehran. The animals were kept in a suitable place in terms of humidity and temperature, and their cages were cleaned regularly.
Animals had free access to food and water. All mice were kept in standard cages with a temperature of 22-25°C and suitable humid-

| Study design and grouping
The animals were randomly divided into six groups as follows:

| Blood and tissue sampling
The animals were anaesthetised by ketamine (Alfasan Co., 80 mg/ kg) and xylazine (Alfasan Co., 10 mg/kg). Blood sampling was done directly from the heart at 10:00 AM using a sterile 5 cc syringe and immediately after the end of the blood collection, the tubes were placed in the centrifuge. The device set to 10 min with 1700 g and afterward serum transfer into the special microtubes. Serum used to determine the concentration of T 3 , T 4 , thyroid-stimulating hormone (TSH), testosterone, LH and FSH hormones.
In addition, the testicular tissue sample was separated from the animals and after weighing and examining the morphological characteristics, it was immediately placed in 10% formalin solution to fix and prepare histological sections. Also, epididymal tissue samples of the left testicle were separated from each animal and separated in physiological serum at 37°C for sperm count. Also, the gonadosomatic index (GSI) was expressed as the percentage of the total body weight in relation to the organ weight (GSI = [organ weight/ total body weight] × 100%).

| Hormone measurement methods
One of the hormone measurement methods is the enzyme-linked immunosorbent assay (ELISA) method, which consists of a two-step enzymatic reaction with the sandwich method, and at the end of the test, instead of a coloured product, a fluorescent product is created.

| Histological studies
Immediately after blood sampling, by making a longitudinal incision in the scrotum, the testicles were removed with tweezers and its extra tissues were removed, and the tissue sections were carefully prepared and were studied using an optical microscope, first with a 10 and then with a 40 eyepiece. In this study, direct observation by light microscope and comparison of cell density in experimental groups with the control group was done. For morphometric analysis, 10 fields of view from each slide were randomly selected under the microscope and photographed by a digital camera. The photos were transferred to the computer and the diameter of the seminiferous tubules was measured using the Image Tool computer software, and the cells were counted visually under a light microscope with a magnification of 400. To count these cells, after preparing slides, the samples were observed by a microscope equipped with a camera.
10 sections of seminiferous tubules were selected from each slide sample corresponding to each mouse in different places of the field of view and counted, then the average of each mouse was taken.

| Statistical analysis
The normality of the obtained data was evaluated using the Shapiro-Wilk test. Then the average data were presented as mean ± standard deviation (SD). Using the mean data, one-way ANOVA and Tukey's post hoc test were performed to compare two by two between groups regarding each group and the differences between them were investigated. The criterion of significant difference between the data was considered as p < .05.

| Histopathological examination of testis from the different experimental groups
The cross-section of spermatogenic tubules have shown in the

| Effect of E. cardamomum extract on the thyroid hormones and thyroid-stimulating hormone
The test of between-subject effects for T 3 , T 4 , and TSH were (F According to the results listed in Table 1, in the examination of the data obtained from the T 3 , T 4 , and TSH, the comparison between the control group and the PTU group shows a significant difference between them, and treatment with PTU significantly decreased the serum level of T 3 and T 4 (p < .05), and significantly increased the serum level of TSH (p < .05). Treatment with ECE reverse the effect of PTU and has led to a significant increase in the serum level of T 3 and T 4 (p < .05), and significant decrease in the serum level of TSH (p < .05) compared to the PTU group.

| Effect of E. cardamomum extract on the sexual hormones
The test of between-subject effects for testosterone, FSH, and LH were (F (5,36) = 40.67 and p-value < .001; F (5,36) = 153.70 and pvalue < .001; F (5,36) = 216.40 and p-value < .001, respectively), the main variable effect of ECE on testosterone, FSH, and LH level in the blood of male mice with 99% confidence is significant.
As shown in the Table 2, in the examination of the data obtained from the testosterone, FSH, and LH, the comparison between the control group and the PTU group shows a significant difference between them, and treatment with PTU significantly decreased the serum level of testosterone (p < .05), and significantly increased the serum level of FSH and LH (p < .05). Treatment with ECE reverse the effect of PTU and has led to a significant increase in the serum level of testosterone (p < .05), and significant decrease in the serum level of FSH and LH (p < .05) compared to the PTU group.

F I G U R E 1
Histopathological examination of testis from the different experimental groups. Section from control group shown normal microscopic structure of spermatogenic tubules and spermatogonial cells (asterisk) (A); the cross-section of 6-n-propylthiouracil (PTU) group showed that spermatogenic tube has an abnormal shape and atrophy and severe reduction of cell population as well as separation of spermatogonia (arrow) and Sertoli (arrowhead) cells are observed (B); the cross-section from levothyroxine (LEVO) group has shown the normal microscopic state of the tissue (C); the cross-section from Elettaria cardamomum extract (ECE) 100 group, treatment with this dose of ECE had a negligible effect on the microscopic structure of the spermatogenic tube tissue (D); the cross-section from ECE 200 group, treatment with this dose of ECE had little effect on the microscopic structure of the spermatogenic tube tissue (E), the cross-section from ECE 400 group, treatment with this dose of ECE has a significant effect on the improvement of the microscopic structure of the spermatogenic tube tissue, so that the damaged tube lining tissue (star) is regenerating (F). Haematoxylin and eosin ×400 magnification, scale bar = 50 μm.

| Effect of E. cardamomum extract on the sperm count and gonadosomatic index
The test of between-subject effects for sperm counts and GSI were (F (5,36) = 25.90 and p-value < .001; F (5,36) = 24.81 and pvalue < .001, respectively), the main variable effect of ECE on sperm counts and GSI in male mice with 99% confidence is significant. Figure 2A,B show the sperm counts and GSI factors, in the examination of the data obtained from the sperm counts and GSI, the comparison between the control group and the PTU group shows a significant difference between them, and treatment with PTU significantly decreased the sperm counts and GSI (p < .05). Treatment with ECE reverse the effect of PTU and has led to a significant increase in the sperm counts and GSI (p < .05) compared to the PTU group.

| DISCUSS ION
This study was conducted in order to determine the effect of ECE on  Therefore, hypothyroidism has a negative effect on the process of spermatogenesis. 21 Research shows that phytoestrogens in plant extracts have the ability to bind to oestrogen receptors 22 and by creating a negative feedback on LH, it reduces the amount of testosterone. 23,24 Phytochemical studies show that cardamom seeds have chemical compounds such as alpha-terpineone, diterpenes, menthone, limonene, sabinene, heptane, myrcene, beta-nerolidol, linalool, beta-pinene, alpha pinene, eugenyl acetate, gamma-sitosterol, citronellol, terpinene and pinene. Chemical analysis shows that cardamom contains alkaloid, flavonoid, saponin, sterol and tannin.
The citronellol composition of this plant is known as a sedative and anti-depressant. 25 In the study of Taheri

| CON CLUS ION
According to the results of the present research, the ECE decreased the hypothyroid inhibitory effects on testicular tissue, increased the number of sex cells, and increased testosterone secretion.

FU N D I N G I N FO R M ATI O N
This study did not receive any external funding.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.