Stesura Seveso Archivio Italiano di Urologia e Andrologia 2014; 86, 3188 ORIGINAL PAPER A study on the effects of the hydroalcholic extract of the aerial parts of Alhagi camelorum on prolactin and pituitary-gonadal activity in rats with hypercholesterolemia Ali Zarei 1, Saeed Changizi Ashtiyani 2, Gholam Hassan Vaezi 3 1 Young Researchers Club, Abadeh Branch, Islamic Azad University, Abadeh, Iran; 2 Department of Physiology, Arak University of Medical Sciences, Arak, Iran; 3 Department of biology, Islamic Azad University, Damghan Branch, Semnan, Iran. Background: Although endocrine disor- ders are not a common cause of infer- tility, in some cases, testing thyroid function, and hypo- thalamus - pituitary - gonadal axis can determine the cause of infertility. We aimed to investigate the effect of the aerial parts of Alhagi camelorum extract on pro- lactin, cortisol and pituitary - gonadal axis activities in rats with hypercholesterolemia. Materials and methods: In this study, 35 male wistar rats in 5 groups (n = 7) were assigned as: control group with normal diet, the sham group with fat diet and three experimental groups of hypercholesterolaemic animals which received Alhagi camelorum extract at a minimum dose of 100 mg/kg, average dose of 200 ml/kg and maximum dose of 300 mg/kg over a period of 21 days. At the end of the period, blood samples were collected from all groups and blood factors were then measured and analyzed. Results: In the sham group compared to the control, cholesterol levels increased and FSH levels decreased, whereas cholesterol levels reduced in the experimental groups. Alhagi camelorum extract also reduced testos- terone level and increased prolactin and gonadotropins. Conclusion: Alhagi camelorum extract at low and aver- age doses reduced cortisol, testosterone and cholesterol and increased gonadotropins. So it can cause reproduc- tive disorders in male rats. The extract at maximum dose can increase cortisol and prolactin. As these two hormones work together to produce milk, this plant can help to boost breastfeeding. KEY WORDS: Alhagi camelorum; Prolactin; Testosterone; Cholesterol; Gonadotropin. Submitted 8 April 2014; Accepted 30 January 2014 Summary No conflict of interest declared. state in which no pregnancy occurs after a year of sex activity without using birth control methods. When dis- cussing infertility, people generally believe that most problems are related to women. In fact, nearly 30% of infertility problems are related to men and 20% are com- mon problems between women and men. So, 50% of men are involved in problems related to infertility. However, this rate is different in different countries and in different studies. Obesity is associated with various hormonal changes that can be responsible for changes in sperm motility and abnormal sexual function. Evidence suggests that in obese men, more androgen changes into estrogen in fat tissue and serum testosterone level decreases. However, by increasing the negative feedback of estrogen on pituitary gland, gonadotropin levels decrease. Pulse obesity also affects GnRH-FSH-LH which may affect sex hormones secretion and sperm maturation by disrupting Leydig and Sertoli cells (1, 2). Endocrine disorders are not among the common causes of infertili- ty; however, in some cases, the cause of infertility can be determined by testing thyroid, gonadotropins, prolactin and testosterone. The level of FSH rises with germinal cell aplasia and the level of testosterone in men with hypogonadotropic hypogonadism decreases (1). Leydig cells are the main source of testosterone and have recep- tors for prolactin which at normal levels increases testos- terone secretion. These studies suggest a synergy between prolactin, LH and testosterone. However, high levels of prolactin reduces testosterone and leads to frigidity (1, 2). Prolactin is regulated by dopamine and some other factors such as TRH. Dopamine is a neuro- transmitter that has an inhibitory effect on the hypothal- amic-pituitary-gonadal axis (2). TRH, secreted from the hypothalamus gland, stimulates prolactin secretion. Cholesterol is the precursor of steroid hormones and cholesterol changes into pregnenolone by P450 in mito- chondria (1, 3, 4). On the other hand, increased level of blood cholesterol is associated with coronary artery dis- ease, fat liver and infertility. Excessive fat causes the male hormone of testosterone to be converted to estrogen which reduces the production of sex cells. DOI: 10.4081/aiua.2014.3.188 INTRODUCTION Today, with advances in science and technology, it is rec- ognized that infertility is not just a problem for women. Male factors are also involved. Infertility is defined as a Zarei_Stesura Seveso 09/10/14 10:38 Pagina 188 189Archivio Italiano di Urologia e Andrologia 2014; 86, 3 Effects of the hydroalcholic extract of the aerial parts of Alhagi camelorum on prolactin and pituitary-gonadal activity in rats with hypercholesterolemia Cholesterol levels can be lowered by diet or drugs (1, 4). Many plants and compounds can be effective in reducing cholesterol. Alhagi camelorum is one of the plants in tra- ditional medicine which is used to treat metabolic, gas- trointestinal and liver diseases, rheumatic disorder, migraines and warts. Laboratory studies indicate that Alhagi camelorum extract reduces body temperature and heart rate. The extract also inhibits the action of acetyl- choline to relax the muscles and is helpful in opening the urinary tract and disposal of kidney stones (5). This plant, commonly called camel thorn (Figure 1), with the scientific name of Alhagi maurorum belongs to the plant family of leguminosae (papilionaceous). The fam- ily has about 550 genera and more than 13000 species (6). Other chemical researches on this plant indicate that it contains sterols and fatty acids (7, 8), flavonoids (9, 10), coumarins (8), alkaloids (6, 8), and vitamins. About 12 types of flavonoids have been isolated from this plant (1). Studies have shown that flavonoids have anti- andro- genic and antifertility effects on the reproductive system of dogs (12, 13). Studies on the bioactive compounds which have the potential to inhibit or stop cancer cells can pave the way to discover more effective drugs (14). Nowadays, people are increasingly using fruits and vegetables due to their protective effects against illnesses like cancer, cardiovas- cular and liver diseases (15, 16). This is due to the antiox- idant compounds present in plants, including vitamins B and C, carotenoids, lycopene and flavonoids, which pre- vent the damages caused by free radicals (17, 19). As infertility and lipid disorders are increasing and most of the anti-fat drugs and contraceptives available in the pharmaceutical market of Iran and the world have mul- tiple side effects, and also thanks to the increasing ten- dency towards herbal remedies because of their fewer side effects, any study on medicinal plants is of great value (4). With this in mind, the present study aimed to investigate the effects of the extract of the aerial parts of Alhagi camelorum on prolactin and pituitary-gonadal axis activities in rats with hypercholesterolemia. METHODS This is an experimental study .All animals were taken from Razi Institute in Fars Province and were kept in standard conditions of temperature and light.This study is based on observing all moral codes of working with laboratory animals established by the Ministry of Health and Medical Training (Iran). Before the research, All the animal were weighed to be within a certain weight range. Initially, 35 male wistar rats with the average weight of 5 ± 170 g were randomly divided into 5 groups (n = 7) as follows: controls - the animals in this group did not receive any drug or solvent during the experiment and their diet was normal. Sham group consisted of hyperc- holesterolemic rats which received 0.2 ml of solvent (nor- mal saline) for 21 days as gavage; (2% cholesterol was added to their food to make them hypercholesterolemic); experimental group 1, hypercholesterolaemic rats which received a minimum dose of 100 mg/kg of Alhagi camelo- rum extract for 21 days as gavage; experimental group 2: hypercholesterolemic rats that were gavaged for 21 days with an average dose of 200 mg/kg of the extract; and experimental group 3 were hypercholesterolemic rats receiving maximum dose of 300 mg/kg of the extract for the same period as gavage feeding. PREPARATION METHOD FOR HIGH CHOLESTEROL FOOD To obtain a 2% high-cholesterol diet, 20 grams of Merck pure cholesterol powder (Fluke Chemika) was solved in 5 ml of olive oil and the solution was well mixed with a kilogram of rat diet. To avoid deterioration of the food it was kept in the refrigerator for only two days (20, 21). EXTRACTION Alhagi camelorum plants were collected from the suburb of Abadeh (Fars Province/Iran) and were identified and con- firmed by the PNU (Payame Noor University) Department of Botany (herbarium code was 002/040/073). To prepare the Alhagi camelorum alcoholic extract, after providing the aerial parts and removing impurities, 800 grams of the col- lected plant samples were crushed and mixed with ethyl alcohol 98% by the ratio of 1 to 5. The content obtained was kept in a package for 48 hours in vitro and it was carefully filtered by passing it through different small and big filters. Then it was placed in a water bath to concentrate. Finally, different concentrations of the obtained extract (about 15 g per 100 g of crushed plant) was prepared by adding different amounts of nor- mal saline. During the experimental period all experimental groups were fed with high fat diet. During the test period (21 days) the animals were daily injected at 9 am. After com- pleting this course and in order to measure plasma bio- chemical factors of the animals, they were mildly anes- thetized with ether and their blood was collected and then centrifuged at 3000 rpm. The serum was separated and transferred to the laboratory for measurement of fac- tors. To measure cholesterol, prolactin, testosterone, cor- tisol, and gonadotropin RIA (RIA), Pars Azmoon kits and RIA 1000 machine (made in USA) were used. For statis- tical analysis the mean obtained (mean ± SEM), one way Figure 1. The aerial parts of camel thorn (Alhagi Camelorum). Zarei_Stesura Seveso 09/10/14 10:38 Pagina 189 Archivio Italiano di Urologia e Andrologia 2014; 86, 3 A. Zarei, S. Changizi Ashtiyani, G. Hassan Vaezi 190 ANOVA test and Tukey and Duncan tests were used. All statistical analyses were done using SPSS software ver- sion 17 (P < 0.05). RESULTS As shown in Table 1 the amount of cholesterol in the sham group increased significantly compared to the con- trol group and in the group receiving the minimum dose of the extract the cholesterol level significantly decreased compared to the sham group (P = 0.02). The differences between experimental groups are not significant. In the case of FSH, mean value in the sham group shows a significant decrease than in the control group. And the group receiving an average dose of Alhagi camelorum extract shows a significant increase compared to the sham group and the groups receiving the minimum and maxi- mum doses of the extract (P = 0.001). LH level in the sham group did not show significant changes than in the control group. However, in groups with minimum and average doses there is a significant increase compared to both the sham group and the group receiving the highest dose of the extract (P = 0.01). In the case of testosterone in the sham group no significant change was seen if weighed against the control group, however, the amount of it in groups receiving minimum and average doses a significant decrease was seen com- pared to the sham group (P = 0.02). None of the changes in experimental groups were significant. Prolactin level increased in the sham group compared to the control group, but these changes were not signifi- cant. Its level in the group receiving the maximum dose showed a significant increase as compared to the sham group and the groups which received minimum or aver- age doses of the extract (P = 0.007). However, no signif- icant difference was observed between the average and minimum groups. Cortisol levels in the sham group did not show any sig- nificant changes as compared to the control group, but in the groups receiving minimum and average doses of the extract it was significantly lower than in the sham group. Cortisol levels in the group receiving the highest dose of the extract compared to sham group as well as the group receiving the least and average doses of the extract showed a significant increase (P = 0.000). DISCUSSION Test results showed that by increasing the amount of cholesterol in the sham group, FSH levels decreased. The administration of the extract to hypercholesterolaemic rats increased gonadotropin and prolactin levels and decreased the level of testosterone. The level of cortisol in the group receiving the highest dose of the extract reduced, but it decreased in groups with the minimum and average doses. Nowadays, with the prevalence of obesity, it has become an important issue that how fertility in men is affected by obesity and fat. In this study, the relationship between increased cholesterol level and functions of pituitary- gonadal axis in the sham group was measured. It is important because if obesity is the cause of male infertil- ity, it can be treated. Obesity affects fertility in men by various mechanisms, among which we can refer to changes in gonadotropin secretion from the pituitary gland, changes in sex hormone-binding globulins, decreased libido, sperm DNA damage ,and so on. Another important effect of obesity on fertility is the reduced testicular activity. In adipose tissue ten percent of testosterone which is male hormone turns into estra- diol which is a female hormone. Increased estradiol makes the breasts grow larger in men and obese men typically have larger breasts (24-22). However, studies in this field are controversial. For example, Pauli et al. (25) and Relvany et al. (26) showed that weight gain does not affect the fer- tility of the semen quality parame- ters while Wagner et al. (2010) and Paasch et al. (2010) stated that increased BMI has a negative impact on sperm quality and fertil- ity indices (27-28), which is con- sistent with the results of the pres- ent study as it was seen that in the control group by increasing choles- terol, the level of FSH decreased. On the effect of the extract on pitu- itary - gonadal axis activities it can be said that on one hand, the extract reduced cholesterol, testos- terone and on the other, it in - creased prolactin and gona do tro - pin levels. One of the most impor- tant ways to adjust LH and FSH Groups Control Sham AC AC AC (100 mg/kg) (200 mg/kg) (300 mg/kg) Parameters Cholesterol 69.8 ± 2.9 86.57 ± 1.9 66.33 ± 3.3 70.83 ± 7.24 76.16 ± 5.2 * ! LH 1.07 ± 0.2 0.71 ± 0.03 1.40 ± 0.3 1.54 ± 0.2 0.85 ± 0.03 " ! # ! FSH 2.8 ± 0.4 1.03 ± 0.07 1.64 ± 0.45 3.34 ± 0.5 1.60 ± 0.2 * $ ! # Testosterone 6.9 ± 2.7 5.94 ± 1.8 1.65 ± 0.3 0.55 ± 0.1 2.67 ± 0.6 ! ! Prolactin 3.45 ± 0.25 5.16 ± 0.55 6.49 ± 0.7 6.28 ± 1.4 9.84 ± 1.8 " # ! Cortisol 20.2 ± 0.8 20.77 ± 4.8 10.27 ± 0.2 10.33 ± 1.40 27.15 ± 2.4 ! ! " # ! * marks a significant change compared with the control group, ! represents a significant change compared with the sham group. " represents a significant change between the minimum and maximum doses of Alhagi Camelorum extract. % indicates significant change between average and maximum doses of Alhagi Camelorum extract. ! represents a significant change in minimum and average doses of Alhagi Camelorum extract. Table 1. Effects of different doses of the extract of the aerial parts of Alhagi Camelorum (AC) on cholesterol, cortisol, testosterone, prolactin and gonadotropin. Zarei_Stesura Seveso 09/10/14 10:38 Pagina 190 191Archivio Italiano di Urologia e Andrologia 2014; 86, 3 Effects of the hydroalcholic extract of the aerial parts of Alhagi camelorum on prolactin and pituitary-gonadal activity in rats with hypercholesterolemia gonadotropin levels is through negative feedback effect of testosterone. That is when the level of this hormone increases, gonadotropin levels decrease and vice versa (29). In this study it seems reasonable that by increasing testosterone gonadotropins decreased. Leydig cells are the main source of testosterone. Leydig cells have receptors for prolactin that at normal levels increases testosterone. This suggests a collaboration between PRL and LH and testosterone. However, high levels of prolactin, reduce testosterone (1). The results of this study showed that the Alhagi camelorum extract reduced cholesterol and testosterone, but it increased gonadotropins and prolactin. LHRH hormone can be another possibility for the reduc- tion of testosterone level. This hormone increases LH and FSH hormones and at the same time inhibits testic- ular testosterone synthesis and secretion by reducing LH receptors (30, 31). Therefore, in this study, despite the increase in FSH and LH, lower testosterone seems to be reasonable and the results of this study is consistent with previous studies on effect of blue plate extract (Centella asiatica) on spermatogenesis, as studies show the chemi- cal compounds in both plants are similar (32). Testosterone is one of the hormones needed for sper- matogenesis. Lower level of this hormone could possibly reduce the number of spermatogonial and spermatocytes cells. These cells produce growth factors such as activin and in the presence of calcium ions, cause karyokinesis, cytokinesis and sperm differentiation (33-34). Studies on Alhagi camelorum plant indicates that on one hand this herb may inhibit calcium channels and on the other active components of the plant including flavonoids have contraceptives and anti-androgenic effects on reproductive system (12, 13). Alkaloids are key ingredients in this plant. Alkaloids by reducing androgens lead to atrophy of epithelial cells and subsequently prohibits androgenic effects on tissues and thus cancer is treated (4, 35). Alkaloids also easily cross the cell membrane and thus they destroy the cytoskele- ton, help a variety of free radicals to release and ulti- mately cause detrimental changes in cellular structure which in turn causes higher activity of white blood cells (anti-inflammatory), while some studies suggest that they have also antioxidant effects (35, 36-38) In addition, alkaloids inhibit cholesterol synthesis (39). Similar studies on Berberis indicates that the alkaloid compounds in this plant such as berberine and berbamine can be effective in the prevention of coronary artery diseases and could possibly reduce total choles- terol levels . Blocking calcium channel is the major effect of berbamine. Berberine increases the production of a receptor in the liver that binds with cholesterol and facil- itates its disposal (20, 21). Since cholesterol is the pre- cursor of steroid hormones, the extract probably lowers testosterone by reducing cholesterol. Bashtiny et al. in a study on feeding animals with Alhagi camelorum showed that it increased their milk produc- tion. This is consistent with our findings as increased prolactin level is one of the most important factors for increasing milk (40). Injecting prolactin and hydrocorti- sone acetate lactogenic hormone in a variety of lactating mammals induces the synthesis and accumulation of beta-carotene in the mammary glands of rats. Research shows that glucocorticoids can be effective only when they are accompanied by prolactin. Studies also showed that milk secretion is the result of several hormones, and herbal extracts directly stimulate endogenous prolactin secretion. As a result, it works effectively on mammary glands. 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Asian-Aust J Anim Sci. 2007; 20:563-568. 39. Zarei A, Changizi Ashtiyani S, Rasekh F, Mohammadi A. The effect of Physalis Alkekengi extracts on lipids concentrations in rats. Arak university of Medical Journal. 2011; 14:36-42 [in Persian]. 40. Bashtini J, Fazaeli H, Mirhadi A, Malekkhahi M. Effect of feed- ing Alhaji browse to lactating ewes on milk yield and performance of lambs. Animal science researches. 2013; 23:39-48. 41. Sepehri H. La synthese de Beta casein par lacide pectique obtenu des extraits des plantes lactogenes. Journal of science, University of Tehran. 1991; 20:61-72. Correspondence Ali Zarei, PhD Young Researchers Club, Abadeh Branch, Islamic Azad University, Abadeh, Iran Saeed Changizi Ashtiyani, PhD (Correspondig Author) dr.ashtiyani@arakmu.ac.ir Department of Physiology, Arak University of Medical Sciences, Arak, Iran Gholam Hassan Vaezi, PhD Department of biology, Islamic Azad University, Damghan Branch, Semnan, Iran Zarei_Stesura Seveso 09/10/14 10:38 Pagina 192