A Novel Aphrodisiac Compound from an Orchid That Activates Nitric Oxide Synthases

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. herbal drug (AF). We show that the AF in a concentration- and

Nitric oxide (micro mole/L) 20

0 0 1530456075 Time (in min) Figure 1. Effect of active fraction from Vanda tesselatta ﬂower on blood nitric oxide (NO) levels in mice. (a) High performance thin layer chromatography (HPTLC) proﬁle of the active chloroform fraction; arrow indicates active compound. HPTLC was carried out using silica gel-60 À 1 À 1 F253 (Merck) plates; solvent system: chloroform:hexane (4:1(v v )). (b) Effect of a single dose (50 mg kg , per os) of the active fraction (AF) on blood NO levels in mice as a function of time after AF administration. Experimental design: to determine time-dependent effect of AF on blood NO levels, male mice were divided into control and three experimental groups. Experimental mice received the AF (50 mg kg À 1, per os) in 2% Tween-80 and the control animals received the vehicle. Blood was collected from the three experimental groups by cardiac puncture under ether anesthesia at 15, 45 and 75 min after drug administration, respectively; n ¼ 3 in each group.

& 2013 Macmillan Publishers Limited International Journal of Impotence Research (2013), 212 – 216 A novel aphrodisiac compound from an orchid A Subramoniam et al 214 a 400 Table 2. Effect of aphrodisiac compound (AC) administration for 14 days (daily) to mice on serum biochemical parameters

300 Control Aphrodisiac compound treated (mg kg À 1)

200 51020

Glucose (mg dl À 1)106±4 107±3 110±5 113±6 À 1 ± ± ± ± 100 GOT (IU l ) 73.1 6.1 76.1 5.1 77.2 5.3 80.1 5.2 GPT (IU l À 1) 38.0±2.9 39.3±2.5 40.0±2.6 42.8±2.4 % increase in NOS activity AP (KAU) 9.4±0.9 9.9±0.8 9.8±0.7 10.2±0.8 Total protein (g dl À 1) 6.3±0.6 6.0±0.5 6.4±0.5 7.1±0.7* 0 Albumin (g dl À 1) 3.1±0.4 3.0±0.3 3.3±0.4 3.6±0.4* 0 50 100 150 200 HDL (mg dl À 1) 19.1±1.2 20.2±1.3 20.6±1.6 22.2±1.6* À 1 ± ± ± ± Aphrodisic compound (µg/ml) LDL (mg dl ) 35.2 2.8 38.2 3.1 40.2 4.6 42.2 3.1* Triglyceride (mg dl À 1)143±8 147±9 146±8 144±9 À 1 ± ± ± ± b 120 Cholesterol (mg dl )1427 146 5 149 8 185 11* Urea (mg dl À 1) 34.1±1.6 33.6±1.8 36.9±2.5 41.2±1.9* Creatine (mg dl À 1) 2.22±0.11 2.41±0.14 2.40±0.13 2.61±0.16* 100 Abbreviations: AC, active compound; AP, alkaline phosphatase; GOT, 80 glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Values are mean±s.d., n ¼ 6 in each case. Values marked with asterisk are 60 signiﬁcantly different from control values, *Po0.05. The drug was administered orally in 2% Tween-80 using a feeding canulla. Control 40 group of mice received the vehicle in an identical manner.

% increase in NOS activity 20

0 significantly influenced by AC (macrophage is known to contain Corpus Brain Macrophage inducible NOS), whereas the enzymes from neuronal (brain) and Cavernosum CC were activated by AC almost to the same level (Figure 2b). Enzyme source The activity of NOS from CC was almost completely inhibited by 100 mM non-selective inhibitor of NOS, N-omega-nitro-L-arginine c 180 methyl ester hydrochloride (Sigma: N-5751), whereas eNOS 160 inhibitor, N-methyl-L-arginine acetate (Sigma: M7033), and neuronal NOS inhibitor, N-omega-nitro-L-arginine (Sigma: N-5501), 140 inhibited the enzyme activity by 45% and 58%, respectively, 120 indicating the presence of both types of NOS in the CC. It is already known that CC contains both types of the enzyme, which 100 are involved in erection. AC (aphrodisiac compound), in the 80 presence of non-selective inhibitor, did not show any influence on 60 the CC enzyme activity, while in the presence of eNOS inhibitor, the activity was increased by AC from 45% (55% was inhibited by 40 the inhibitor) to 94%, and in the presence of neuronal NOS Enzyme activity (% of control) 20 inhibitor, the activity was increased by AC from 58 to 110% (Figure 2c). These observations indicate that AC activates both 0 eNOS and nNOS. CACEIEI + NI NI + NI + NI + AC AC EI EI + AC AC did not exhibit any conspicuous toxicity in the short-term (14 days) limited toxicity evaluation in male mice at 5 mg kg À 1 Figure 2. Activation of nitric oxide synthase (NOS) by the (therapeutic dose) and 10 mg kg À 1 doses. Food and water intake aphrodisiac compound (AC) from Vanda tessellata flower. (a) as well as the weight of body and organs were not influenced by Activation of corpus cavernosum NOS by different concentrations of the AC. (b) Effect of the AC (50 mgmlÀ 1) on the activity of NOS the treatment (data not shown). As given in Table 2, serum biochemical parameters were also not altered by the treatment from different tissues. (c) Effect of inhibitors of neuronal NOS and À 1 À 1 endothelial NOS on the activation of corpus cavernosum NOS by the (5 or 10 mg kg AC). However, at 20 mg kg , there were AC. C, control; AC, aphrodisiac compound; EI, endothelial NOS marginal increases in serum glutamate pyruvate transaminase, inhibitor; NI, neuronal NOS inhibitor. Each assay was carried out in glutamate oxaloacetate transaminase, high-density lipoprotein, triplicate. In all, 100 mM (final concentration in the assay system) low-density lipoprotein, protein and albumin. These increased inhibitor was used in all cases.The inhibitor was added in the assay values were within the normal range. Thus, even at this high dose system before the addition of the enzyme (tissue homogenate). (20 mg kg À 1), deleterious changes were not observed. Spectral data of the AC was obtained to elucidate its structure. IR (KBr): 2923 and 2984 (v C-H aliphatic, sym. and asym. str.), 1461 system) effect of the drug on corpus cavernosum (CC) tissue NOS (v C-alkyl group) and 1377 (v C-CH3 symmetric) Cm-1. MS: m/z 136 þ activity. We show that the drug in a concentration-dependent (M , À 2) of C10 H18. À 1 1 manner activated NOS. At 100 mgml level, the drug enhanced H NMR (CDCl3, 500 MHz): d 2.05–1.98 (m, 2H, C1 and 4-H), 1.68 the enzyme activity more than 150% (Figure 2a). (S, 1H, C2-H, 1.56 (S, 6H, C7-(CH3)2), 1.42 (S, 3H, C2-CH3), 1.25 (S, 2H, To get some clue regarding the influence of the aphrodisiac C3-H) and 0.86–0.85 (m, 4H, C5 and 6-H) p.p.m. 13 compound (chemical isolate) on different types of NOS (inducible C NMR (CDCl3, 500 MHz): d 37 (C-7), 32 (C-4), 31 (C-1), 30 (C-3), NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS)), the 29.71 (C-5), 29.37 (C-6), 27 (C-2), 22(C7-(CH3)2 and 14 C2-CH3) p.p.m. effect of the aphrodisiac compound (AC) on the NOS of different Based on the spectral data, the isolated compound is 2, 7, 7-tri tissues was studied. The activity of NOS from macrophage was not methyl bicyclo [2.2.1] heptane. Figure 3 shows the structure

International Journal of Impotence Research (2013), 212 – 216 & 2013 Macmillan Publishers Limited A novel aphrodisiac compound from an orchid A Subramoniam et al 215 assigned to the compound. (Scanned images of spectra of the physiological activation of eNOS.13,14 It has been suggested that compound and the equipments used for the same are given shear stress activates a pathway including phosphatidylinositol under Supplementary Information.) This plant has been reported 30-kinase and the serine/threonine kinases (Akt), which phos- to contain heptacosane (C27H56) and octacosanol (C26H58)as phorylates and activates eNOS at resting levels of intracellular anti-inflammatory molecules.8 calcium.14 The extracellular purine nucleotide-mediated eNOS phosphorylation and activation is calcium and protein kinase Cd-dependent.15 Nitro-oleic acid stimulates the phosphorylation of eNOS at Ser(1179), resulting in an increase in eNO DISCUSSION production.16 The activation of NOS by AC may involve In this study, an important activator (AC) of eNOS and nNOS has phosphorylation of NOS. been discovered. The mechanism of activation of the enzyme by The drug 2,7,7-trimethyl bicyclo[2.2.1] heptane activates CC AC remains to be studied. Fluid shear-stress-mediated Akt- nNOS and eNOS. This results in increased production of NO, which dependent phosphorylation of eNOS has been shown in the activates guanylate cyclase that catalysis the conversion of GTP into cyclic GMP. The role of cyclic GMP (cGMP) in the regulation of CC smooth muscle tone and penile erection are elucidated.3,4,17 Structure of the aphrodisiac compound The relaxation is mainly caused by a decrease in the levels of intracellular calcium. This is brought about by cGMP- and cAMP- mediated pathways.17,18 Thus, AC could activate eNOS and nNOS and bring about aphrodisiac action through cGMP-mediated pathway. AC is likely to stimulate erectile function in CC as shown in Figure 4. AC could concomitantly influence both CC and CNS signaling. Neuronal NOS has many functions including sexual arousal in men and women. The aphrodisiac drug from V. tessellata could increase the levels of cGMP by increasing its formation from GTP, whereas it is known 2,7,7-trimethyl bicyclo[2.2.1] heptane that Viagra (sildenafil citrate) inhibits phosphodiesterase-5, which Figure 3. Structure of the active compound. specifically degrades cGMP. It is of interest to note that both the

Suggested action of aphrodisiac compound (2,7,7, tri methyl bicyclo [2.2.1] heptane from Vanda tessellata on the pathway principally responsible for sexually stimulated erection

L-arginine Aphrodisiac compound

NO synthase (e-NOS & n-NOS) (+)

(+) Sildenafil (Viagra) Soluble Guanyl Cyclase GTP

(--) Phospho diesterase (+) GMP cGMP

(+) i calcium (--) PKG Potassium (+)

(+) Myosin light chain phosphatase

Dephosphorylation of myosin light chain

Corpus cavernosal smooth muscle relaxation and erection

Figure 4. Suggested action of the aphrodisiac compound from Vanda tessellata on the pathway principally responsible for sexually stimulated erection. NO, nitric oxide; e-NOS, endothelial nitric oxide synthase; n-NOS, neuronal nitric oxide synthase; cGMP, cyclic GMP; i calcium, intracellular calcium. ( þ ) The increase in activity or quantity and (--) the decrease.

& 2013 Macmillan Publishers Limited International Journal of Impotence Research (2013), 212 – 216 A novel aphrodisiac compound from an orchid A Subramoniam et al 216 present aphrodisiac drug and Viagra can increase the levels of 3 Bivalacqua TJ, Champion HC, Hellstrom WJG, Kadowitz PJ. Pharmacotheraphy for cGMP, but the herbal drug increases its formation from GTP, erectile dysfunction. Trends Pharm Sci 2000; 21: 484–489. whereas Viagra inhibits its degradation. The herbal drug appears 4 Dean RC, Lue TF. Physiology of penile erection and pathophysiology of erectile to be useful for developing as an attractive alternative to Viagra. dysfunction. Urol Clin N Am 2005; 32: 379–403. If required, the orchid can be cultivated in large scales for 5 Suresh PK, Subramoniam A, Pushpangadan P. Aphrodisiac activity of Vanda drug production. An Indian patent application has been filed on tessellata (Roxb) Hook extract in male mice. Indian J Pharmacol 2000; 32: 38–41. 19 6 Subramoniam A, Madhavachandran V, Rajasekharan S, Pushpangadan P. Aphro- these findings. disiac property of Trichopus zeylanicus extract in male mice. J Ethnopharmacol In conclusion, in this study, a novel aphrodisiac compound that 1997; 57: 21–27. activates eNOS and nNOS was discovered from an orchid, 7 Basu K, Das Gupta B, Bhattacharya SK, Lal R, Das PK. Anti-inflammatory principles V. tessellata. The finding has the potential to develop a valuable of Vanda roxburghii. Curr Sci 1972; 40: 86–87. medicine for sexual dysfunction or erectile dysfunction. Further, 8 Chawala AS, Sharma AK, Handa SS, Dhar KL. Chemical studies and anti-inflam- the discovery is a significant addition to the knowledge in the field matory activity of Vanda roxburghii roots. Indian J Pharm Sci 1992; 54: 159–161. of sexual dysfunction. 9 Ahmed F, Sayeed A, Islam A, Salam SMA, Sadik G, Sattar MA et al. Antimicrobial activity of extracts and a glycoside from Vanda roxburghii R. Br. Pak J Biol Sci 2002; 5: 189–191. CONFLICT OF INTEREST 10 Nayak BS, Suresh R, Rao AV, Pillai GK, Davis EM, Ramkissoon V et al. Evaluation of The authors declare no conflict of interest. wound healing activity of Vanda roxburghii R.Br. (Orchidacea): a preclinical study in a rat model. Int J Low Extrem Wounds 2005; 4: 200–204. 11 Sastry KVM, Moudgal RM, Mohan RJ, Tyagi JS, Rao GS. Spectrophotometric ACKNOWLEDGEMENTS determination of serum nitrite and nitrate by copper–cadmium alloy. Anal Bio- The research was supported by the Department of Science and Technology (DST), chem 2002; 306: 79–82. Government of India. We gratefully acknowledge Mangalam S Nair for her help in 12 Dawson J, Knowles RG. A microtiter-plate assay of nitric oxide synthase activity. obtaining some of the spectral data and the interpretation of the spectra. Mol Biotechnol 1999; 12: 275–279. 13 Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. AUTHOR CONTRIBUTIONS Nature 1999; 399: 601–605. 14 Fisslthaler B, Dimmeler S, Hermann C, Busse R, Fleming I. Phosphorylation and AS (Principal Investigator of the Department of Science and Technology, activation of the endothelial nitric oxide synthase by fluid shear stress. Acta Government of India, funded Project) wrote the project with assistance from AG Physiol Scand 2000; 168: 81–88. and PKS, designed the work, analyzed the experimental data and wrote the 15 da Silva CG, Specht A, Wegiel B, Ferran C, Kaczmarek E. Mechanism of purinergic paper. AG (co-investigator of the project) helped in designing the experiments activation of endothelial nitric oxide synthase in endothelial cells. Circulation and in writing the manuscript. 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The pharmacology of nitric oxide in the peripheral nervous J Sex Med 2005; 16: 459–469. system of blood vessels. Pharmacol Rev 2003; 55: 271–324. 19 Subramoniam A, Sureshkumar PK, Gangaprasad A, Radhika J, Arun KB. (inventors). 2 Knowles GR, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994; 298: A novel aphrodisiac drug prepared using Vanda tessellata flower. Indian patent 249–258. application, File No: 2299/CHE/2010.

Supplementary Information accompanies the paper on International Journal of Impotence Research website (http://www.nature.com/ijir)

International Journal of Impotence Research (2013), 212 – 216 & 2013 Macmillan Publishers Limited