International Journal of Impotence Research (2012) 24, 31–37 & 2012 Macmillan Publishers Limited All rights reserved 0955-9930/12 www.nature.com/ijir ORIGINAL ARTICLE Effect of Curculigo orchioides on hyperglycemia-induced oligospermia and sexual dysfunction in male rats M Thakur1,2, NS Chauhan2, V Sharma2, VK Dixit2 and S Bhargava2 1Central institute for Laboratory Medicine, Charite, Campus Benjamin Franklin, Hindenburgdamm, Berlin, Germany and 2Department of Pharmaceutical Sciences, Dr H S Gour University, Sagar, MP, India Sustained hyperglycemia is considered as a major cause of sexual and erectile dysfunction in human population. Curculigo orchioides (CO) is considered as a sexual tonic in Ayurvedic system of medicine with potent antioxidant and adaptogenic properties. The aqueous extract of the herb was evaluated for its effectiveness against streptozotocin-induced hyperglycemic stress and subsequent sexual dysfunction due to hyperglycemia in male rats. Six groups with eight male rats in each group were used for this study and the study was carried out for 28 days. The body and organ weights of the animals were recorded. Behavioral analysis of rats was undertaken to observe the effect on mount, ejaculation and intromission (latencies and frequencies) and hesitation time. Blood glucose and serum testosterone levels were determined 28 days past treatment with CO at 100 and 200 mg kgÀ1 doses. Glibenclamide and sildenafil citrate were used as positive controls. This deleterious effect of sustained hyperglycemia and associated stress was prominently ameliorated in animals treated with aqueous extract of CO. CO treatment was helpful in ameliorating the damage caused by sustained hyperglycemia evidenced in the principle parameters viz. male sexual behavior, sperm count, penile erection index and seminal fructose content Antioxidant and anabolic activities of the extract under investigation could be a major attribute in preserving the sexual functions in hyperglycemic male rats. The study validates the use of CO in traditional medicine for curing diabetes-induced sexual dysfunction and compromised sexual potency. International Journal of Impotence Research (2012) 24, 31–37; doi:10.1038/ijir.2011.43; published online 15 September 2011 Keywords: Curculigo orchioides; penile erection; sexual behavior; sexual dysfunction; sildenafil citrate Introduction ability to cure sexual dysfunction in males, and the plants having this kind of activity are defined as Sexual dysfunction including erectile dysfunction rasayana. A rasayan may be defined as smooth pro- (ED) is common in men with diabetes. Diabetic men stressor, which reduces reactivity of host defense are three times as likely to develop ED as non- systems and decreases damaging effects of various diabetic men.1,2 The causes of diabetes-induced stressors due to increased basal level of mediators sexual dysfunction are multi-factoral, but most involved in the stress response.4,5 commonly reflect endothelial dysfunction and auto- Curculigo orchioides (CO) Gaertn. (family Amarylli- nomic neuropathy. Increased oxidative stress is also daceae) is a well-known herb in traditional Indian considered as an important factor causing diabetes- system of medicine and has been designated as a induced sexual dysfunction.3 Vajikaran Rasayan.6,7 It is claimed to be useful in Ayurveda the traditional system of medicine in treatment of piles, asthma, jaundice, diarrhea, colic, India is endowed with numerous plants having the gonorrhea and sexual dysfunction.8 It is also a constituent of ‘Chywanprash’ an age-old Ayurvedic formulation reported in ancient texts for invigorating and rejuvenating the physiological functions. CO has Correspondence: Dr M Thakur, Central Institute for been used in several metabolism enhancing and Laboratory Medicine, Charite, Campus Benjamin Franklin, Hindenburgdamm, Berlin 12200, Germany. aphrodisiac formulation in the Indian system of medicine. Rhizomes possess immunostimulant7 aph- E-mails: [email protected] or 5,6 9 10 [email protected] rodisiac, antidiabetic and hepatoprotective activity. Received 28 August 2010; revised 7 April 2011; accepted Diabetes and sustained hyperglycemia results in 10 August 2011; published online 15 September 2011 reduced libido.11 Ayurveda and traditional Chinese Effect of Curculigo orchioides M Thakur et al 32 medicine recognizes CO as an important aphrodisiac female rats were prepared for the experimentation as herb. Ayurveda the traditional system of medicine in per the methodology reported by Agmo.13 After 15 Indian subcontinent also recommends the use of this days of preparation the female rats were used for herb as a ‘Virya Rasayan’. Virya Rasayanas are herbs experimentation. useful in improving the sperm count. Sustained Blood samples were collected by retro-orbital hyperglycemia is reported to be major cause of ED, puncture from rats fasted overnight. First sample lower sperm count and low serum testosterone (T) was collected 96 h after STZ injection. On day 28 levels, thus this study was designed to evaluate the the blood samples were collected by retro-orbital role of this herb on erectile function, and low values puncture 3 h after pellet diet and administration for seminal parameters (that is, seminal fructose of extract. Blood glucose levels were determined content, sperm count) in male rats.12 using the glucose oxidase kit as per instructions (Invitrogen, Darmstadt, Germany). Sexual behavior of male rat was assessed on day 14 Materials and methods to make the rats sexually experienced (data not shown) and day 28 of experimentation. The experi- Animal stock ment was carried under dim-red light and the A total of 66 Wistar strain albino rats (48 males and 18 behavioral aspects were video recorded for the females) weighing 220–250 g were fed on standard duration of 60 min for each rat using a digital camera diet and water ad libitum. The animals were housed (Olympus, Hamburg, Germany, EX120). Observa- at room temperature (24±2 1C) on a reversed light– tional and behavioral analysis was performed in a dark cycle (06:00 hours to 18:00 hours). Animal wooden chamber with a glass wall (70 Â 40 Â 60 cm3) experimentation was carried out after prior permis- under diffused red light in the dark phase of the sion from the Institutional Ethical Committee of light–dark cycle. The chamber had a special small Dr HS Gour University, Sagar (MP), India. The opening at the side for introducing the female as guidelines of Committee for the Purpose of Control stimulus. The video recorded data were subjected to and Supervision of Experiments on Animals, India analysis using freeware version of Etholog v 2.2.5r were adhered to during the whole experimentation. EB Ottoni,14 (Sao Paulo) run on Windows XP. Male animals in group of eight each were taken for the studies and dosing protocol for different groups were as follows. The experiment started 2 weeks Collection, extraction and characterization after the confirmation of diabetes in all the groups, Rhizomes of CO were collected in and around the the experiment lasted 28 days thereafter. regions of Sagar MP (India), and identified at the Group I: was administered vehicle only (normo- Department of Botany Dr H S Gour University, Sagar. glycemic control). A voucher specimen of the same has been deposited Group II: 50 mg kgÀ1 body weight (b.w.) streptozo- (no. MKNS-Pharma-2008). Rhizomes were dried in tocin (STZ) intraperitoneally (i.p.) once (hypergly- sunlight and coarsely powdered. Powdered drug cemic control). passing through 60 mm mesh size and retained on Group III: 50 mg kgÀ1 b.w. STZ, i.p. once and 80 mm mesh size was fed in a Soxhlet extractor 100 mg kgÀ1 b.w. of CO extract p.o., daily (CO100). (Mahendra Scientific, Kanpur, India) and defatted Group IV: 50 mg kgÀ1 b.w. STZ, i.p. once and with petroleum ether 60–80 1C. Defatted drug was 200 mg kgÀ1 b.w. of p.o., daily (CO200). further extracted with deionized water and the Group V: 50 mg kgÀ1 b.w. STZ i.p. once and aqueous extract was subjected to lyophilization. The 600 mgkgÀ1 b.w. of glibenclamide i.p. (positive % yield of aqueous extract was found to be nearly 29% of crude material used for extraction. CO extract control for hyperglycemia) daily (STZ50 þ Glib600). Group VI: 50 mg kgÀ1 b.w. STZ i.p. once and was characterized by high-performance thin-layer 5mgkgÀ1 b.w. of sildenafil citrate i.p. (positive chromatography and high-performance liquid chro- matography with mass detection. High-performance control) daily (STZ50 þ Sil5). thin-layer chromatography chromatogram against gal- Glibenclamide is an orally administered anti- 15 diabetic agent and was used as a positive control lic acid as standard was developed. Standardization for evaluating the role of better management of of the extract was carried out to ensure the composi- glucose metabolism on sexual behavior and erectile tion and uniformity of the extract for future reprodu- function if any. Similarly, sildenafil citrate that is a cibility of phytochemical parameters. CO extract phosphodiesterase inhibitor is useful in treatment of administered to rats was also found to contain appre- ED. Because sustained hyperglycemia may cause ciable amounts of phenolic compounds, quercetin, ED therefore, to evaluate whether the treatment epigallocatechin and a small amount of saponins. with phosphodiesterase inhibitor may be useful in diabetes-associated ED, STZ50 þ Sil5 group was included in the study as a positive control.1,3 In vivo studies Female rats were also divided in three groups Effect on androgenic activity and sexual behavior. comprising of six animals in each group. Receptive Two rats from each group were killed on day 0 and International Journal of Impotence Research Effect of Curculigo orchioides M Thakur et al 33 the rest were killed on day 28 of experimentation. observed for a period of 30 min. The PE was The weight of body and the secondary sexual organs recorded when the rats bent down to lick their erect (testis, seminal vesicles and epididymis) of all the penis.