International Journal of Impotence Research (2013) 25, 212–216 & 2013 Macmillan Publishers Limited All rights reserved 0955-9930/13 www.nature.com/ijir ORIGINAL ARTICLE A novel aphrodisiac compound from an orchid that activates nitric oxide synthases A Subramoniam1, A Gangaprasad2, PK Sureshkumar1, J Radhika1 and BK Arun1 Nitric oxide (NO) is known to have roles in several crucial biological functions including vasodilation and penile erection. There are neuronal, endothelial and inducible NO synthases that influence the levels of NO in tissues and blood. NO activates guanylate cyclase and thereby increases the levels of cyclic GMP (cGMP). Viagra (sildenafil), a top selling drug in the world for erectile dysfunction, inhibits phosphodiesterase-5, which hydrolyses cGMP to GMP. Thus, it fosters an NO-mediated increase in the levels of cGMP, which mediates erectile function. Here, we show the aphrodisiac activity of a novel chemical isolate from the flowers of an epiphytic orchid, Vanda tessellata (Roxb.) ex Don, which activates neuronal and endothelial, but not inducible, NO synthases. The aphrodisiac activity is caused by an increase in the level of NO in corpus cavernosum. The drug increases blood levels of NO as early as 30 min after oral administration. The active compound was isolated by column chromatography. Based on the spectral data, the active compound is found to be a new compound, 2,7,7-tri methyl bicyclo [2.2.1] heptane. We anticipate that our findings could lead to the development of a commercially viable and valuable drug for erectile dysfunction. International Journal of Impotence Research (2013) 25, 212–216; doi:10.1038/ijir.2013.18; published online 18 April 2013 Keywords: aphrodisiac compound; mounting behavior; nitric oxide synthase; orchid INTRODUCTION Enhancement of mounting and mating behavior by the alcohol 5 It is of interest to develop a novel herbal drug for treating sexual extract of V. tessellata flower was observed earlier by us. The dysfunction, particularly erectile dysfunction.1–4 The commercially major aim of this study was to determine the utility of the plant available top selling drug for sexual dysfunction is a synthetic flower to use as an aphrodisiac medicine, which includes isolation drug, Viagra (sildenafil).3 The common side effects of Viagra are of the active compound (AC) and determination of its mechanism headaches, facial flushing, dyspepsia and upset stomach.3 Other of action. problems with Viagra can include bluish vision, blurred vision or sensitivity to light.3,4 Many aphrodisiac formulations are available in the market from various sources to improve sex drive in men. MATERIALS AND METHODS However, the safety and efficacy of the drugs are not proved Animals beyond doubt. Hardly any herbal drug other than yohimbine has Swiss albino mice (26–30 g body weight) were used for the experiments, been cleared by Food and Drug Administration, USA as a drug for which were reared in Tropical Botanic Garden and Research male sexual dysfunction. Institute (TBGRI) animal house and fed with standard pellet diet and Screening traditional orchids for various pharmacological water ad libitum. Animal experiments were approved by the Institute Animal Ethics Committee (IAEC) and the animals were maintained under properties led to the discovery of aphrodisiac activity in the 5 6 standard laboratory conditions as per the guidelines of the Committee for flowers of Vanda tessellata and leaves of Trichopus zylanicus by the Purpose of Control and Supervision of Experiments on Animals the investigators’ group at Tropical Botanic Garden and Research (CPCSEA). Institute. V. tessellata (Roxb.) Hook. ex. Don (Syn: Vanda roxburghii R. Br.) is an epiphytic orchid found in India, Sri Lanka and many Preparation of alcohol extract of V. tessellata flower other tropical regions in Asia. The flower and, to some extent, the root is known to stimulate mounting behavior of male mice. This V. tessellata (Roxb.) ex Don was collected from Thiruvananthapuram District, Kerala State and identified by the Taxonomists of TBGRI. A voucher activity is present in the alcohol extract, and the extract À 1 5 specimen, No. 50080, was deposited in the Herbarium of TBGRI. The (200 mg kg ) also increases mating performance in mice. flowers were cut into small pieces and air dried at room temperature. It is well known in traditional medicine that this plant has Ethanol extract of the dried flower was prepared as described.5 therapeutic powers.5,7 A paste made from the leaves of this orchid is used for external application in fevers. It is an ingredient of Isolation of active fraction and assay of mounting behavior Rasna Panchaka Quatha, an Ayurvedic formulation used in the treatment of arthritis and rheumatism. The root is used as an The alcohol extract of V. tessellata (flower) was suspended in water (1 g/50 ml) and sequentially extracted with (two times with 50 ml each) antidote against scorpion sting and a remedy for bronchitis. 7 hexane, chloroform, ethyl acetate and butanol; each fraction was tested for Investigators have reported that the plant has anti-inflammatory, aphrodisiac activity using mounting behavior in mice.6 (The chloroform 7,8 9 9 10 antiarthritic, antimicrobial, antipyretic and wound-healing fraction was found to be active. Ethyl acetate, butanol and water fractions properties. did not exhibit any activity at 50 mg kg À 1 level.) 1Department of Phytopharmacology, Tropical Botanic Garden and Research Institute (TBGRI), Palode, Thiruvananthapuram, India and 2Department of Botany, University of Kerala, Thiruvananthapuram, India. Correspondence: Dr A Subramoniam, T.C. 14–80, Anayara, Thiruvananthapuram, Kerala 695029, India. E-mail: [email protected] Received 19 July 2012; revised 12 February 2013; accepted 13 March 2013; published online 18 April 2013 A novel aphrodisiac compound from an orchid A Subramoniam et al 213 Isolation of AC (aphrodisiac compound) by column time-dependent manner increased the levels of NO in the serum chromatography (Figure 1b and Table 1). The effect on NO levels was observed as In all, 1 g of the fraction was chromatographed on 35 g silica gel (60–120 early as 15 min after the AF administration and the maximum mesh); eluted with hexane:chloroform (4:1 (v v À 1)) (200 ml of 10 ml effect was observed about 45 min after the AF administration fractions were collected); and each fraction was monitored for activity by (Figure 1b). measuring blood nitric oxide (NO) level after administration to mice. The The AC (aphrodisiac compound) was isolated in a pure form 3rd and 4th fractions contained the pure AC, which moved as a single spot from the AF by activity (increase in blood NO levels in mice) - on silica gel high performance thin layer chromatography (HPTLC) (Rf value: 0.94). The yield of alcohol extract was about 38% of dried flower; guided column chromatography. A fast-moving compound with the yield of chloroform fraction was about 18% of alcohol extract; and that of an Rf value of 0.94 on HPTLC was identified as the aphrodisiac AC was about 15% of chloroform fraction and 1.2% of the dry flower. compound (AC). It is about 15% of chloroform fraction (1.2% of dried flower). There was a positive correlation between the drug NO measurement concentration, mounting behavior and blood NO levels (Table 1). As NO levels in the blood could be enhanced by an increase in NO in the serum was determined by measuring its stable non-volatile breakdown product, sodium nitrate using Griess assay system with the activity of NOS, we studied the in vitro (in cell-free biochemical copper–cadmium alloy.11 Table 1. Effect of Vanda tessellata on mounting behavior and blood Assay of NO synthase NO levels of male mice Calbiochem NO synthase (NOS) assay kit (EMD Chemicals Inc., Darmstadt, Germany) was used to assess the enzyme activity. In this method, the Treatment NO levels Number of mounts nitrate formed from NO is converted into nitrite using nitrate reductase (mmol l À 1) per 15 min enzyme.12 Lactate dehydrogenase is used to destroy excess NADPH, which interferes with the estimation of nitrite by Griess reagent. The nitrite Control 36.8±2.7 2.4±0.2 content is measured using Griess reagents. The nitrite level shows the Alcohol extract 72.0±5.1* 13.8±1.8* amount of NO formed (by NOS activity). (200 mg kg À 1) Chloroform fraction (AF) from alcohol extract RESULTS 25 mg kg À 1 48.1±3.0* 5.8±0.3* Based on enhancement of mounting behavior, in this study, an 50 mg kg À 1 75.2±4.9* 14.5±1.2* 100 mg kg À 1 92.4±8.1* 17.9±2.02* active chloroform fraction was isolated from the alcohol extract by À 1 solvent fractionation. The chloroform fraction (active fraction (AF)) AC from AF (5 mg kg ) 69.1±5.0* 12.1±2.1* was resolved into several components on silica gel HPTLC Abbreviations: AC, Active compound; AF, active fraction; ANOVA, analysis (Figure 1a). of variance; NO, nitric oxide. Although the herbal drug (alcohol extract) reproducibly Inbred Swiss albino mice were used for the studies. Blood was collected for increases the mounting behavior in a dose-dependent manner, NO determination on the 46th min after drug administration. Serum NO the biochemical events influenced by the herbal drug were not was determined as given under the Materials and methods section. known initially. Our first objective was development of a suitable Mounting behavior was observed for 15 min (30 min after herbal drug 5 ± non-invasive biochemical assay based on the physiology administration) as described. Values are mean s.d., n ¼ 6, *Po0.001 (compared with respective control value). Differences between groups of erection to quantify easily the aphrodisiac action of the were examined by ANOVA followed by Dunnett’s test.