The Effects of Aqueous Extract of Anacyclus Pyrethrum on Sperm Count and Reproductive Organs in Adult Male Rats

authors:

avatar Mohammad Reza Shahraki 1 , avatar Sarieh Shahraki 1 , * , avatar Mohammad Reza Arab 2 , avatar Mahnaz Shahrakipour 3

Department of Physiology, Zahedan University of Medical Sciences, Zahedan, Iran
Department of Anatomy, Zahedan University of Medical Sciences, Zahedan, Iran
Department of Epidemiology and Statistics, Faculty of Health, Zahedan University of Medical Sciences, Zahedan, Iran
Zahedan Journal of Research in Medical Sciences: Vol.17, issue 2; e1984
published online: January 16, 2013
article type: Short Communication
received: April 29, 2013
accepted: May 15, 2013

How To Cite Shahraki M R, Shahraki S, Arab M R, Shahrakipour M. The Effects of Aqueous Extract of Anacyclus Pyrethrum on Sperm Count and Reproductive Organs in Adult Male Rats. Zahedan J Res Med Sci. 2015;17(2):e1984. 

Abstract

Background:

More than 80 million individuals suffer from infertility globally. Various factors such as some drugs and toxins have harmful effects on fertility. Anacyclus pyrethrum plant in Indian traditional medicine is used for treatment of many diseases including infertility.

Materials and Methods:

In this experimental study 48 male adult rats were divided randomly into four groups (N=12) including one control group (A) and three test groups (B, C and D). Test groups (B, C and D) received root aqueous extract of A. pyrethrum intraperitoneally with doses of 50, 100 and 150 mg/kg for 28 days, respectively. At the end of the treatment period, the reproduction variables such as weight of body and sex organs, the sperm count in epididymis and right and left vas deferens and percent of abnormal spermatozoids were determined. The test groups were compared to the controls using analysis of variance following Tukey.

Results:

Data analysis of body and sex organs’ weight, sperm count of epididymis and right and left vas deferens and percent of abnormal spermatozoids showed a significant difference between the tests and control groups (p=0.02, p=0.0001); however, no significant difference was found between two groups regarding vas deferens weight.

Conclusion:

The results of the present study showed that root aqueous extract of A. pyrethrum increased the weights of body and sex organs, increase of sperm count of epididymis and right and left vas deferens, and reduction of percent of abnormal spermatozoids in treated rats.

Introduction

Infertility is a complex disorder affecting more than 80 million people worldwide [1]. Approximately 30 % of infertilities are due to male factors [2]. In only 40% of all cases the infertility is detectable, and in the remaining 60% it is not pathologically detectable [3]. Many factors including drug treatment, chemotherapy, toxins, air pollutions and insufficient vitamins intake have destructive effects on a normal spermatogenesis [4]. Some toxic compounds such as 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) can increase the production of abnormal sperms [5]. Besides, study of Saba et al. [6] showed that extract of Lagenaria breviflora augments the production of abnormal sperms [7].

Although androgens are primarily used in the treatment of sexual disorders, in both men and women [8], the use of medicinal herbs have always been under attention [9]. The World Health Organization (WHO) in its 31th summit in developed countries has recommended administration of medicinal herbs, because of their standards, advantages and healthiness [10]. Experimental reports suggest that compounds with androgenic effects could increase the weight of reproductive organs and glands [11].

Various studies have shown that compounds such as flavonoids in medicinal herbs have androgenic effects and stimulates the spermatogenesis [12]. Anacyclus pyrethrum is a medical herb which derives from the root Pellitory and in Indian language is called Akarkara. In Indian traditional medicine this plant has been shown to strengthen the immune system of human [13].

Medicinally, A. pyrethrum root has a pungent efficacy in promoting a free flow of saliva, in relieving toothache, in alleviating chronic catarrh and acne [14]. Pellitory root in Indian traditional medicine has been widely used as an aphrodisiac and as a medicine for rejuvenation and vitality [15].

It has been reported that oral administration of the root powder has aphrodisiac actions [16], so that in male rats the administration of alkylamide-rich extracts of A. pyrethrum improved their sexual behaviors [17]. Therefore, the present study was aimed to evaluate the effects of intraperitoneal injection of aqueous extract of A. pyrethrum root on weight of reproductive organs and glands and on sperm count in epididymis and vas deferens of male adult rats.

Materials and Methods

In this experimental study was performed on 48 Wistar strain male albino rats, aging 5-7 month, weighting 225±50 g, which were kept at the Animal laboratory of Zahedan University of Medical Sciences. In this experimental study, the animals were housed at room temperature (20±20oC), and light was set at 12 h light–dark cycle. They were maintained in plastic cages separately and had free access to the food and water. The study protocol was approved by the Institutional Animal Ethics Committee. After weighting of animals with a digital balance (EK-b10, Japan) the rats were randomly divided into four groups each with 12 animals; group A were given only standard dry rat pellet diet and water ad libitum. The test groups, (B, C and D) received doses of 50, 100 and 150 mg/kg of aqueous extract of A. pyrethrum root for 28 days by intraperitoneal route, respectively [18]. In the present study, a Sham group also was used for comparison, but since the results for the Sham and control group were similar, we eliminated the Sham group from the study to facilitate analyzes and interpretation of our results. A. pyrethrum roots purchased from a local market in Zahedan, and were approved at the Herbarium center of Biology department of University of Sistan and Balouchestan.

For the preparation of aqueous extract of A. pyrethrum, the plant roots were dried, powdered and then subjected to soxhelet apparatus for extraction with distilled water for 24 h. The extract obtained was filtered through a 30×100 mm filter paper and then dried at temperature of 37ºC to get the powder form. At the time of experiments, the dried powder was dissolved in physiologic serum in determined doses and then was injected intraperitoneally to the experimental groups [19]. At the end of the treatment period, the animals were anaesthetized with ether, killed by cervical dislocation and subjected for the various analyses. Body, testes, vas deferens and right and left epididymides were weighed and removed for sperm analysis after an inguinal dissection.

Sperm counting was performed according to the method described by Sharma et al. [20]. In brief, a hemocytometer with improved Neubauer ruling was employed for counting the spermatozoa. A 20-fold dilution was made by mixing the sperm suspension with normal saline (0.9% NaCl). The preparation was then thoroughly mixed, and one drop was added to both sides of the hemocytometer. Spermatozoa on both sides of the hemocytometer were counted, and the average number was recorded. The average number of spermatozoa counted was multiplied to volume, surface and dilution coefficients. Since a number of sperms are damaged during mincing of epididymis, only intact spermatozoa were counted. To evaluate the sperm abnormalities, one drop of sperm suspension was stained with 1% eosin (alcoholic) smeared on slides, fixed and made permanent slides. The slides were examined under the microscope (Olympus) using ×400 objectives and abnormal sperm cells were counted and the percentage was calculated [21-22]. Results are expressed as mean±SEM. The test groups were compared to the controls using analysis of variance following tukey tests. All the statistical analyses were carried out using SPSS-19 software. A p-value below 0.05 was considered statistically significant.

Results

Analysis of data by the Tukey test revealed no significant difference of vas deferens weight in A. pyrethrum treated rats and the control. However, a significant difference was observed between the groups in the weights of body, testes and epididymis (Table 1 and 2). The effects of A. pyrethrum extract on weight of body, testes, right and left epididymis, right and left vas deferens and sperm count in left and right epididymis have been fully described in the table 1 and 2.

Table 1.

Effects of A. pyrethrum extract on weight of body, testes and right and left epididymis (N=12)

Variables GroupsBody weight (g)Right testis (g)Left testis (g)Right epididymis (g)Left epididymis (g)
A (mean±SEM)239.50±10.410.880±0.0060.882±0.0080.485±0.0660.490±0.067
B (mean±SEM)256.16±18.310.910±0.0040.911±0.0070.485±0.0660.490±0.067
p-Value0.02<0.00010.0010.0010.004
C (mean±SEM)291.58±11.820.947±0.0030.951±0.0070.675±0.0100.667±0.025
p-Value<0.0001<0.0001<0.0001<0.0001<0.0001
D (mean±SEM)296.00±12.031.012±0.0311.005±0.0100.698±0.0110.699±0.011
p-Value<0.0001<0.0001<0.0001<0.0001<0.0001
Table 2.

Effect of A. pyrethrum extract on weight of right and left vas deferens and sperm count in left and right epididymis (N=12)

Variables GroupsRight vas deferensi (g)Left vas deferens (g)Sperm count in right epididymis (106/mL)Sperm count in left epididymis (106/mL)
A (mean±SEM)0.105±0.0100.100±0.01044.27±30.8944.44±26.12
B (mean±SEM)0.099±0.0270.097±0.02751.28±26.7451.41±27.45
p-Value0.90.8<0.0001<0.0001
C (mean±SEM)0.110±0.0170.0108±0.01760.90±23.4961.25±23.83
p-Value0.80.9<0.0001<0.0001
D (mean±SEM)0.106±0.0130.103±0.01364.42±24.3964.24±23.72
p-Value0.90.9<0.0001<0.0001
Table 3.

Effect of A. pyrethrum extract on sperm count in right and left vas deferens and percentage of abnormal spermatozoa (N=12) *

Variables GroupsSperm count in right deferens (106/mL)Sperm count in left deferens (106/mL)Abnormal Spermatozoa (%)
A (mean±SEM)5.18±2.955.24±2.821.91±0.66
B (mean±SEM)5.88±1.845.73±2.251.75±0.62
p-Value<0.0001<0.00010.004
C (mean±SEM)6.57±1.856.63±1.221.66±0.65
p-Value<0.0001<0.00010.002
D (mean±SEM)7.31±3.767.28±2.380.75±0.62
p-Value<0.0001<0.0001<0.0001

* p<0.05 Comparisons with the Control group (A)

Moreover, analysis of data using the Tukey showed that there is a significant difference between the test and control groups in sperm count in epididymis and vas deferens and also in percentage of spermatozoa with abnormal morphology (Tables 2, 3). The effects of A. pyrethrum extract on sperm count in right and left vas deferens and percentage of abnormal spermatozoa is presented in table 3.

4. Discussion

The results of present study showed that the root aqueous extract of A. pyrethrum increased the body weight in the treated group compared to the control group. Several experiments including the Sharma et al. [19] study have demonstrated that aqueous or alcoholic extracts of this plant can enhance body weights of the test group in comparison with the control group. Additionally, the present study showed that testes and epididymis average weight were elevated significantly in the defined doses, which is in accordance with previous studies on other types of herbs [19, 23, 24]; however, vas deferens average weight was not affected. Our study showed that the plant extract leads to considerable elevation of average sperm count in epididymis and vas deferens, which fully supports the results of Sharma et al. [19, 23, 24]. To the best of our knowledge, our study for the first time revealed that the aqueous extract of A. pyrethrum reduces the percentage of spermatozoa with abnormal morphology.

Gaining weight is usually associated with steroid production, and is a biological indicator for effectiveness of medicinal herbs in production of steroid hormones [15]. Production of testosterone can result from gonadotropin activity or from increase of testosterone steroid precursors [25]. It is also presumed that steroidogenic components in the plant extract may improve the function of gonadotropin [26].

With respect to structure and performance, a male reproductive organ is dependant to testosterone and other androgens, two male sex hormones which are involved in growth and secretory action of reproductive glands [27-29]. Androgens are essential for development, growth and normal activity of male testes and sex glands, and previous studies have shown that there is a strong correlation between testosterone levels in blood and weight of testes, epididymis, seminal vesicle and prostate [30]. The mechanism of the testosterone activity in increasing the weight of sex organs is through stimulation of protein expression in target cells. After a short time which testosterone is converted to dihydro testosterone (DHT) by α-5 reductase enzyme the DHT binds to cytoplasmic protein receptors and enters the nucleus to stimulate the transcription of DNA to RNA which through translation of RNA to proteins, eventually lead to increase of the weight of body and reproductive organs [31, 32]. Administration of this plant orally to male rats has been reported to increase of serum levels of testosterone compared to the control group [18]. Since androgenic effects are associated with the blood testosterone levels, probably A. pyrethrum extract has a role in testosterone secretion from gonads [33]. These effects may be correlated to neurotransmitters level or their activity in the cell [34, 35].

Spermatogenesis is a complex process among structural epithelial elements of testes and hormonal system [36]. FSH is a stimulator of spermatogenesis and LH accelerates the release of testosterone. Testosterone augments the blood flow, improves the growth of target tissues, and directly motivates spermatogenesis [37]. Accordingly, our results possibly are associated with chemical constitutes of A. pyrethrum. The root of this plant has been analyzed and it was determined that the root contains a brown spicy resin substance named Pyrethrin. Additionally, the root of this plant involves vaporizing oil, resin and various Tannic acids [14]. Botanists have reported that this plant contains N-isobutyldienediynamide and warm water soluble polysaccharides [38, 39]. Phytochemical studies on this plant have demonstrated the presence of alkylamide and polymeric polysaccharides both of which has potent androgenic effects and also stimulates secretion of the testosterone [40]. Additionally, Sharma et al. in their previous studies have shown that isolated alkylamides from extract of A. pyrethrum root have positive effects on reproduction variables such as weight of body and sex organs as well as the sperm count in epididymis [39].

Similarly, Cicero et al. reported that alkyl amide from the A. pyrethrum has positive effects on reproduction [38]. Tail domain of spermatozoa is prone to oxidation due to the presence of unsaturated fatty acids in the plasma membrane. Thakur et al. showed that date seed oil with antioxidant properties suppresses lipids peroxidation, hence reduces the number of abnormal permatozoids [22]. In the present study, antioxidant effects of A. pyrethrum plant have not been examined, but Kalim et al. in their study have evaluated the antioxidant effects of the A. pyrethrum plant [41]. Therefore, it is probable that A. pyrethrum plant contributes to the reduction of abnormal spermatozoids through a similar mechanism.

Therefore, with respect to the noted evidences, the significant increase of some examined variables could be explained through the fact that increasing the weights of reproductive organs is associated with androgenic and anabolic factors. Androgens can stimulate the growth of reproductive organs and then increase their weight [9]. Thus it is probable that natural components have androgenic properties [11]. It is presumed that the existence of Plitorin, an alkyl amide in the A. pyrethrum extract, might be related to the observed effects. The main reason for this hypothesis is fortification of sex characteristics by alkyl amides that are extracted from Lepidium meyenii root [40]. It seems that the alkyl amid has a testosterone-like activity or stimulates the testosterone secretion [41]. In conclusion, this study indicated that intraperitoneal injection of aqueous extract of A. pyrethrum root in male adult rats increased the weight of body, epididymides and vas deferens and augment of sperm count in these organs; however it reduces the number of abnormal spermatozoids, and in this way improves the quality of spermatozoids. Therefore, the positive effects of A. pyrethrum plant on different reproductive parameters might improve the production of forthcoming drugs and dietetic components for prognosis or treatment of infertility

Acknowledgements

References

  • 1.

    Zegers F, Hochschild JE, Schwarze V, Alam F. Infertility international encyclopedia of public health. USA: Academic Press; 2008.

  • 2.

    Bhasin S, de Kretser DM, Baker HW. Clinical review 64: Pathophysiology and natural history of male infertility. J Clin Endocrinol Metab. 1994;79(6):1525-9. [PubMed ID: 7989450]. https://doi.org/10.1210/jcem.79.6.7989450.

  • 3.

    Yang WM, Kim HY, Park SY, Kim HM, Chang MS, Park SK. Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes. J Ethnopharmacol. 2010;128(3):693-6. [PubMed ID: 20219665]. https://doi.org/10.1016/j.jep.2010.02.020.

  • 4.

    Purvis K, Christiansen E. Infection in the male reproductive tract. Impact, diagnosis and treatment in relation to male infertility. Int J Androl. 1993;16(1):1-13. https://doi.org/10.1111/j.1365-2605.1993.tb01146.x.

  • 5.

    Etuk EU, Muhammad AA. Fertility enhancing effects of aqueous stem bark extract of Lophiralanceolata in male sparguedawley rats. Int J Plant Phys Biochem. 2009;1(1):1-4.

  • 6.

    Saba AB, Oridupa OA, Oyeyemi MO, Osanyigbe OD. Spermatozoa morphology and characterictics of male Wistar rats administerated with ethanolic extract of Lagenaria Breviflora Roberts. Afr J Biotechnol. 2009;8(7):1170-5.

  • 7.

    Drug policies and managment medical plant. Switzerland: World Health Organization. WHO. 1978:31-3.

  • 8.

    Saxena S. Role of total alkaloids of Mucunapruviens Bakerin spermatogenesis in male rats. Indian J Nat Prod. 1987;3:3-7.

  • 9.

    Chauhan NS, Sharma V, Dixit VK. Effect of Asteracantha longifolia seeds on the sexual behaviour of male rats. Nat Prod Res. 2011;25(15):1423-31. [PubMed ID: 19753500]. https://doi.org/10.1080/14786410802588493.

  • 10.

    Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspects Med. 2006;27(1):1-93. [PubMed ID: 16105678]. https://doi.org/10.1016/j.mam.2005.07.008.

  • 11.

    Sharma V, Thakur M, Chauhan NS, Dixit VK. Immunomodulatory activity of petroleum ether extract of Anacyclus pyrethrum. Pharm Biol. 2010;48(11):1247-54. [PubMed ID: 20843161]. https://doi.org/10.3109/13880201003730642.

  • 12.

    Bentley R, Trimen H. Mediclinical plants. Delhi: Asiatic Publishing House; 2004.

  • 13.

    India: Ministry of health and family welfare. 2007.

  • 14.

    Rassayana PH. Ayurvedic herbs for longevity and rejuvenation. London: Taylor & Francis; 2003.

  • 15.

    Singh J, O'Neill C, Handelsman DJ. Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology. 1995;136(12):5311-21. [PubMed ID: 7588276]. https://doi.org/10.1210/endo.136.12.7588276.

  • 16.

    Luo Q, Li Z, Huang X, Yan J, Zhang S, Cai YZ. Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H2O2-induced DNA damage in mouse testicular cells and beneficial effect on sexual behavior and reproductive function of hemicastrated rats. Life Sci. 2006;79(7):613-21. [PubMed ID: 16563441]. https://doi.org/10.1016/j.lfs.2006.02.012.

  • 17.

    Khaki F, Fathiazad F, Nouri M. The effect of Ginger on spermatogenesis and sperm parameters of rats. Iran J Reprod Med. 2009;7(1):7-12.

  • 18.

    Parandin R, Yousofvand N, Ghorbani R. The enhancing effects of alcoholic extract of Nigella sativa seed on fertility potential, plasma gonadotropins and testosterone in male rats. Iran J Reprod Med. 2012;10(4):355-62. [PubMed ID: 25246898].

  • 19.

    Sharma V, Boonen J, Spiegeleer BD, Dixit VK. Androgenic and spermatogenic activity of alkylamide-rich ethanol solution extract of Anacyclus pyrethrum DC. Phytother Res. 2013;27(1):99-106. [PubMed ID: 22473789]. https://doi.org/10.1002/ptr.4697.

  • 20.

    Sharma VTM, Chauhan Singh N, Dixit VK. Evaluation of the anabolic, aphrodisiac and reproductive activity of anacyclus pyrethrum DC in male rats. Sci Pharm. 2009;77:97-110. https://doi.org/10.3797/scipharm.0808-14.

  • 21.

    Sharma V, Thakur M, Chauhan NS, Dixit VK. Effects of petroleum ether extract of Anacyclus pyrethrum DC. on sexual behavior in male rats. Zhong Xi Yi Jie He Xue Bao. 2010;8(8):767-73. [PubMed ID: 20727332].

  • 22.

    Thakur M, Chauhan NS, Bhargava S, Dixit VK. A comparative study on aphrodisiac activity of some ayurvedic herbs in male albino rats. Arch Sex Behav. 2009;38(6):1009-15. [PubMed ID: 19139984]. https://doi.org/10.1007/s10508-008-9444-8.

  • 23.

    Haren MT, Morley JE, Chapman IM, O'Loughlin PD, Wittert GA. Defining 'relative' androgen deficiency in aging men: how should testosterone be measured and what are the relationships between androgen levels and physical, sexual and emotional health? Climacteric. 2002;5(1):15-25. [PubMed ID: 11974555].

  • 24.

    Gray JM, Nunez AA, Siegel LI, Wade GN. Effects of testosterone on body weight and adipose tissue: role of aromatization. Physiol Behav. 1979;23(3):465-9. [PubMed ID: 504438].

  • 25.

    Bhasin S, Fielder T, Peacock N, Sod-Moriah UA, Swerdloff RS. Dissociating antifertility effects of GnRH-antagonist from its adverse effects on mating behavior in male rats. Am J Physiol. 1988;254(1 Pt 1):E84-91. [PubMed ID: 3276216].

  • 26.

    O'Donnell L, McLachlan RI, Wreford NG, Robertson DM. Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology. 1994;135(6):2608-14. [PubMed ID: 7988449]. https://doi.org/10.1210/endo.135.6.7988449.

  • 27.

    Glina S. Testosterone and erectile dysfunction. J Med Health Gonads. 2009;23:465-9.

  • 28.

    Setty BS, Riar SS, Kar AB. Androgenic control of epididymal function in rhesus monkey and rabbit. Fertil Steril. 1977;28(6):674-81. [PubMed ID: 405258].

  • 29.

    Damassa DA, Smith ER, Tennent B, Davidson JM. The relationship between circulating testosterone levels and male sexual behavior in rats. Horm Behav. 1977;8(3):275-86. [PubMed ID: 881168].

  • 30.

    Thakur DV. Effects of chlorophytom borivilianum on androgenic and sexual behavior of male rats. Indian Drugs. 2006;43:300-6.

  • 31.

    Subramoniam A, Madhavachandran V, Rajasekharan S. Aphrodisiac property of Trichopus zeylanicus extract in male mice. J Ethnopharmacol. 1997;57(1):21-7. https://doi.org/10.1016/S0378-8741(97)00040-8.

  • 32.

    Gonzales GF, Cordova A, Vega K, Chung A, Villena A, Gonez C. Effect of Lepidium meyenii (Maca), a root with aphrodisiac and fertility-enhancing properties, on serum reproductive hormone levels in adult healthy men. J Endocrinol. 2003;176(1):163-8. [PubMed ID: 12525260].

  • 33.

    Chauhan N. Spematogenenic activity of rizhumes of Curculigoorchiodes Gaertn on male rats. Int J Appl Res Nat Prod. 2008;2(2):26-31.

  • 34.

    Zarrow M, Mccarthy JL. Expremenatal endocrinology. A sourcebook of basic technique. New York: Academic Press; 1964.

  • 35.

    Crombie L. Isolation and Structure of an N-isoButyldienediynamide from Pellitory (Anacyclus pyrethrum DC.). Nature. 1954;174(4435):832-3. https://doi.org/10.1038/174832a0.

  • 36.

    Bendjeddou D, Lalaoui K, Satta D. Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis. J Ethnopharmacol. 2003;88(2-3):155-60. [PubMed ID: 12963136].

  • 37.

    Thakur M, Thompson D, Connellan P, Deseo MA, Morris C, Dixit VK. Improvement of penile erection, sperm count and seminal fructose levels in vivo and nitric oxide release in vitro by ayurvedic herbs. Andrologia. 2011;43(4):273-7. [PubMed ID: 21486409]. https://doi.org/10.1111/j.1439-0272.2010.01068.x.

  • 38.

    Cicero AF, Bandieri E, Arletti R. Lepidium meyenii Walp. improves sexual behaviour in male rats independently from its action on spontaneous locomotor activity. J Ethnopharmacol. 2001;75(2-3):225-9. [PubMed ID: 11297856].

  • 39.

    Sharma V, Boonen J, Chauhan NS, Thakur M, De Spiegeleer B, Dixit VK. Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 2011;18(13):1161-9. [PubMed ID: 21757328]. https://doi.org/10.1016/j.phymed.2011.06.001.

  • 40.

    Zheng BL, He K, Kim CH, Rogers L, Shao Y, Huang ZY, et al. Effect of a lipidic extract from lepidium meyenii on sexual behavior in mice and rats. Urology. 2000;55(4):598-602. [PubMed ID: 10736519].

  • 41.

    Kalim MD, Bhattacharyya D, Banerjee A, Chattopadhyay S. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complement Altern Med. 2010;10:77. [PubMed ID: 21159207]. https://doi.org/10.1186/1472-6882-10-77.

  • 42.

    Bivalacqua TJ, Liu T, Musicki B, Champion HC, Burnett AL. Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. Eur Urol. 2007;51(6):1732-40. [PubMed ID: 17113219]. https://doi.org/10.1016/j.eururo.2006.10.061.