A novel aphrodisiac compound from an orchid that activates nitric oxide synthases

A Corrigendum to this article was published on 16 July 2014

Abstract

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.

Introduction

It is of interest to develop a novel herbal drug for treating sexual dysfunction, particularly erectile dysfunction.1, 2, 3, 4 The commercially available top selling drug for sexual dysfunction is a synthetic drug, Viagra (sildenafil).3 The common side effects of Viagra are headaches, facial flushing, dyspepsia and upset stomach.3 Other 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. However, the safety and efficacy of the drugs are not proved beyond doubt. Hardly any herbal drug other than yohimbine has been cleared by Food and Drug Administration, USA as a drug for male sexual dysfunction.

Screening traditional orchids for various pharmacological properties led to the discovery of aphrodisiac activity in the flowers of Vanda tessellata5 and leaves of Trichopus zylanicus6 by the investigators’ group at Tropical Botanic Garden and Research Institute. V. tessellata (Roxb.) Hook. ex. Don (Syn: Vanda roxburghii R. Br.) is an epiphytic orchid found in India, Sri Lanka and many other tropical regions in Asia. The flower and, to some extent, the root is known to stimulate mounting behavior of male mice. This activity is present in the alcohol extract, and the extract (200 mg kg−1) also increases mating performance in mice.5

It is well known in traditional medicine that this plant has 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 Rasna Panchaka Quatha, an Ayurvedic formulation used in the treatment of arthritis and rheumatism. The root is used as an antidote against scorpion sting and a remedy for bronchitis. Investigators have reported that the plant has anti-inflammatory,7 antiarthritic,7, 8 antimicrobial,9 antipyretic9 and wound-healing10 properties.

Enhancement of mounting and mating behavior by the alcohol extract of V. tessellata flower was observed earlier by us.5 The major aim of this study was to determine the utility of the plant flower to use as an aphrodisiac medicine, which includes isolation of the active compound (AC) and determination of its mechanism of action.

Materials and methods

Animals

Swiss albino mice (26–30 g body weight) were used for the experiments, which were reared in Tropical Botanic Garden and Research Institute (TBGRI) animal house and fed with standard pellet diet and water ad libitum. Animal experiments were approved by the Institute Animal Ethics Committee (IAEC) and the animals were maintained under standard laboratory conditions as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Preparation of alcohol extract of V. tessellata flower

V. tessellata (Roxb.) ex Don was collected from Thiruvananthapuram District, Kerala State and identified by the Taxonomists of TBGRI. A voucher specimen, No. 50080, was deposited in the Herbarium of TBGRI. The flowers were cut into small pieces and air dried at room temperature. Ethanol extract of the dried flower was prepared as described.5

Isolation of active fraction and assay of mounting behavior

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) hexane, chloroform, ethyl acetate and butanol; each fraction was tested for aphrodisiac activity using mounting behavior in mice.6 (The chloroform fraction was found to be active. Ethyl acetate, butanol and water fractions did not exhibit any activity at 50 mg kg−1 level.)

Isolation of AC (aphrodisiac compound) by column chromatography

In all, 1 g of the fraction was chromatographed on 35 g silica gel (60–120 mesh); eluted with hexane:chloroform (4:1 (v v−1)) (200 ml of 10 ml fractions were collected); and each fraction was monitored for activity by measuring blood nitric oxide (NO) level after administration to mice. The 3rd and 4th fractions contained the pure AC, which moved as a single spot on silica gel high performance thin layer chromatography (HPTLC) (Rf value: 0.94). The yield of alcohol extract was about 38% of dried flower; the yield of chloroform fraction was about 18% of alcohol extract; and that of AC was about 15% of chloroform fraction and 1.2% of the dry flower.

NO measurement

NO in the serum was determined by measuring its stable non-volatile breakdown product, sodium nitrate using Griess assay system with copper–cadmium alloy.11

Assay of NO synthase

Calbiochem NO synthase (NOS) assay kit (EMD Chemicals Inc., Darmstadt, Germany) was used to assess the enzyme activity. In this method, the nitrate formed from NO is converted into nitrite using nitrate reductase enzyme.12 Lactate dehydrogenase is used to destroy excess NADPH, which interferes with the estimation of nitrite by Griess reagent. The nitrite content is measured using Griess reagents. The nitrite level shows the amount of NO formed (by NOS activity).

Results

Based on enhancement of mounting behavior, in this study, an active chloroform fraction was isolated from the alcohol extract by solvent fractionation. The chloroform fraction (active fraction (AF)) was resolved into several components on silica gel HPTLC (Figure 1a).

Figure 1
figure1

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 F253 (Merck) plates; solvent system: chloroform:hexane (4:1(v v−1)). (b) Effect of a single dose (50 mg kg−1, 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−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=3 in each group.

Although the herbal drug (alcohol extract) reproducibly increases the mounting behavior in a dose-dependent manner, the biochemical events influenced by the herbal drug were not known initially. Our first objective was development of a suitable non-invasive biochemical assay based on the physiology of erection to quantify easily the aphrodisiac action of the herbal drug (AF). We show that the AF in a concentration- and time-dependent manner increased the levels of NO in the serum (Figure 1b and Table 1). The effect on NO levels was observed as early as 15 min after the AF administration and the maximum effect was observed about 45 min after the AF administration (Figure 1b).

Table 1 Effect of Vanda tessellata on mounting behavior and blood NO levels of male mice

The AC (aphrodisiac compound) was isolated in a pure form from the AF by activity (increase in blood NO levels in mice) -guided column chromatography. A fast-moving compound with an Rf value of 0.94 on HPTLC was identified as the aphrodisiac compound (AC). It is about 15% of chloroform fraction (1.2% of dried flower). There was a positive correlation between the drug concentration, mounting behavior and blood NO levels (Table 1).

As NO levels in the blood could be enhanced by an increase in the activity of NOS, we studied the in vitro (in cell-free biochemical system) effect of the drug on corpus cavernosum (CC) tissue NOS activity. We show that the drug in a concentration-dependent manner activated NOS. At 100 μg ml−1 level, the drug enhanced the enzyme activity more than 150% (Figure 2a).

Figure 2
figure2

Activation of nitric oxide synthase (NOS) by the aphrodisiac compound (AC) from Vanda tessellata flower. (a) Activation of corpus cavernosum NOS by different concentrations of the AC. (b) Effect of the AC (50 μg ml−1) on the activity of NOS from different tissues. (c) Effect of inhibitors of neuronal NOS and endothelial NOS on the activation of corpus cavernosum NOS by the AC. C, control; AC, aphrodisiac compound; EI, endothelial NOS inhibitor; NI, neuronal NOS inhibitor. Each assay was carried out in triplicate. In all, 100 μM (final concentration in the assay system) inhibitor was used in all cases.The inhibitor was added in the assay system before the addition of the enzyme (tissue homogenate).

To get some clue regarding the influence of the aphrodisiac compound (chemical isolate) on different types of NOS (inducible NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS)), the effect of the aphrodisiac compound (AC) on the NOS of different tissues was studied. The activity of NOS from macrophage was not significantly influenced by AC (macrophage is known to contain inducible NOS), whereas the enzymes from neuronal (brain) and CC were activated by AC almost to the same level (Figure 2b).

The activity of NOS from CC was almost completely inhibited by 100 μM non-selective inhibitor of NOS, N-omega-nitro-L-arginine methyl ester hydrochloride (Sigma: N-5751), whereas eNOS inhibitor, N-methyl-L-arginine acetate (Sigma: M7033), and neuronal NOS inhibitor, N-omega-nitro-L-arginine (Sigma: N-5501), inhibited the enzyme activity by 45% and 58%, respectively, indicating the presence of both types of NOS in the CC. It is already known that CC contains both types of the enzyme, which are involved in erection. AC (aphrodisiac compound), in the presence of non-selective inhibitor, did not show any influence on the CC enzyme activity, while in the presence of eNOS inhibitor, the activity was increased by AC from 45% (55% was inhibited by the inhibitor) to 94%, and in the presence of neuronal NOS inhibitor, the activity was increased by AC from 58 to 110% (Figure 2c). These observations indicate that AC activates both eNOS and nNOS.

AC did not exhibit any conspicuous toxicity in the short-term (14 days) limited toxicity evaluation in male mice at 5 mg kg−1 (therapeutic dose) and 10 mg kg−1 doses. Food and water intake as well as the weight of body and organs were not influenced by the treatment (data not shown). As given in Table 2, serum biochemical parameters were also not altered by the treatment (5 or 10 mg kg−1 AC). However, at 20 mg kg−1, there were marginal increases in serum glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, high-density lipoprotein, low-density lipoprotein, protein and albumin. These increased values were within the normal range. Thus, even at this high dose (20 mg kg−1), deleterious changes were not observed.

Table 2 Effect of aphrodisiac compound (AC) administration for 14 days (daily) to mice on serum biochemical parameters

Spectral data of the AC was obtained to elucidate its structure. IR (KBr): 2923 and 2984 ( C-H aliphatic, sym. and asym. str.), 1461 ( C-alkyl group) and 1377 ( C-CH3 symmetric) Cm-1. MS: m/z 136 (M+, −2) of C10 H18.

1H NMR (CDCl3, 500 MHz): δ 2.05–1.98 (m, 2H, C1 and 4-H), 1.68 (S, 1H, C2-H, 1.56 (S, 6H, C7-(CH3)2), 1.42 (S, 3H, C2-CH3), 1.25 (S, 2H, C3-H) and 0.86–0.85 (m, 4H, C5 and 6-H) p.p.m.

13C NMR (CDCl3, 500 MHz): δ 37 (C-7), 32 (C-4), 31 (C-1), 30 (C-3), 29.71 (C-5), 29.37 (C-6), 27 (C-2), 22(C7-(CH3)2 and 14 C2-CH3) p.p.m.

Based on the spectral data, the isolated compound is 2, 7, 7-tri methyl bicyclo [2.2.1] heptane. Figure 3 shows the structure assigned to the compound. (Scanned images of spectra of the compound and the equipments used for the same are given under Supplementary Information.) This plant has been reported to contain heptacosane (C27H56) and octacosanol (C26H58) as anti-inflammatory molecules.8

Figure 3
figure3

Structure of the active compound.

Discussion

In this study, an important activator (AC) of eNOS and nNOS has been discovered. The mechanism of activation of the enzyme by AC remains to be studied. Fluid shear-stress-mediated Akt-dependent phosphorylation of eNOS has been shown in the physiological activation of eNOS.13, 14 It has been suggested that shear stress activates a pathway including phosphatidylinositol 3′-kinase and the serine/threonine kinases (Akt), which phosphorylates and activates eNOS at resting levels of intracellular calcium.14 The extracellular purine nucleotide-mediated eNOS phosphorylation and activation is calcium and protein kinase Cδ-dependent.15 Nitro-oleic acid stimulates the phosphorylation of eNOS at Ser(1179), resulting in an increase in eNO production.16 The activation of NOS by AC may involve phosphorylation of NOS.

The drug 2,7,7-trimethyl bicyclo[2.2.1] heptane activates CC nNOS and eNOS. This results in increased production of NO, which 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 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.

Figure 4
figure4

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. (+) The increase in activity or quantity and (—) the decrease.

The aphrodisiac drug from V. tessellata could increase the levels of cGMP by increasing its formation from GTP, whereas it is known that Viagra (sildenafil citrate) inhibits phosphodiesterase-5, which specifically degrades cGMP. It is of interest to note that both the present aphrodisiac drug and Viagra can increase the levels of cGMP, but the herbal drug increases its formation from GTP, whereas Viagra inhibits its degradation. The herbal drug appears to be useful for developing as an attractive alternative to Viagra. If required, the orchid can be cultivated in large scales for drug production. An Indian patent application has been filed on these findings.19

In conclusion, in this study, a novel aphrodisiac compound that activates eNOS and nNOS was discovered from an orchid, V. tessellata. The finding has the potential to develop a valuable medicine for sexual dysfunction or erectile dysfunction. Further, the discovery is a significant addition to the knowledge in the field of sexual dysfunction.

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Acknowledgements

The research was supported by the Department of Science and Technology (DST), Government of India. We gratefully acknowledge Mangalam S Nair for her help in obtaining some of the spectral data and the interpretation of the spectra.

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Correspondence to A Subramoniam.

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AS (Principal Investigator of the Department of Science and Technology, Government of India, funded Project) wrote the project with assistance from AG and PKS, designed the work, analyzed the experimental data and wrote the paper. AG (co-investigator of the project) helped in designing the experiments and in writing the manuscript. PKS (co-investigator of the project) helped in writing the project proposal and the collection of Vanda tessellata from the field. JR and KBR (Research Fellows in the project) carried out animal experiments, biochemical assays and isolation of active principles under the direct supervision of AS.

Supplementary Information accompanies the paper on International Journal of Impotence Research website

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Subramoniam, A., Gangaprasad, A., Sureshkumar, P. et al. A novel aphrodisiac compound from an orchid that activates nitric oxide synthases. Int J Impot Res 25, 212–216 (2013). https://doi.org/10.1038/ijir.2013.18

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Keywords

  • aphrodisiac compound
  • mounting behavior
  • nitric oxide synthase
  • orchid

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