Medicinal plants as a potential source of Phosphodiesterase-5 inhibitors: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence and distress caused by erectile dysfunction (ED) to both male and female partners are increasing at a steady rate. ED has now become the most treated sexual disorder for men among young and old age groups due to varying physical and psychological factors. The treatment with synthetic Phosphodiesterase-5 (PDE5) inhibitors are cost-effective but due to adverse effects such as priapism, loss of vision, heart attack and syncope, the daily life patterns of these patients are distressed and hence the need for alternative medicaments or sources are of utmost important. Therefore, the exploration of medicinal plants as PDE5 inhibitors will be worthwhile in tackling the problems as many plant extracts and fractions have been long used as aphrodisiacs and sexual stimulants which may be found to be active against PDE5 enzyme. AIM OF THE STUDY: To provide a review on the different medicinal herbs traditionally used as natural aphrodisiacs, libido or sexual enhancers which are proven for their PDE5 inhibitory effect. MATERIALS AND METHODS: Ethnobotanical and scientific information was procured, reviewed and compiled from the literature search of electronic databases and search engines. RESULTS: A total of 97 medicinal plants exhibiting PDE5 inhibitory effect are reviewed in this paper which is supported by preclinical experimental evidence. Among them, 77 plants have been selected according to their traditional and ethnobotanical uses as aphrodisiacs and the rest are screened according to their effectiveness against predisposing factors responsible for ED and sexual dysfunction such as diabetes and hypertension or due to the presence of phytochemicals having structural similarity towards the identified natural PDE5 inhibitors. In addition, sixteen alkaloids, sixty-one phenolics and eight polycyclic aromatic hydrocarbons have been isolated or identified from active extracts or fractions that are exhibiting PDE5 inhibitory activity. Among them, isoflavones and biflavones are the major active constituents responsible for action, where the presence of prenyl group for isoflavones; and the methoxy group at C-5 position of flavones are considered essential for the inhibitory effect. However, the prenylated flavonol glycoside, Icariin and Icariside II isolated from Epimedium brevicornum Maxim (hory goat weed) are the most effective inhibitor, till date from natural sources. Traditional medicines or formulations containing extracts of Ginkgo biloba L., Kaempferia parviflora Wall. ex Baker, Clerodendrum colebrookianum Walp., Eurycoma longifolia Jack and Vitis vinifera L. are also found to be inhibitors of PDE5 enzyme. CONCLUSION: The review suggests and supports the rational use of traditional medicines that can be further studied for the development of potential PDE5 inhibitors. Many traditional medicines are still used in various regions of Africa, Asia and South America that are poorly characterized and experimented. Despite the availability of a vast majority of traditional formulations as aphrodisiacs or sexual stimulants, there exists a need for systemic evaluation on the efficacy as well as the mechanism of action of the herbal constituents for the identification of novel chemical moieties that can be further developed for maximum efficacy.

Isolation and characterisation of N-benzyl tadalafil as a novel adulterant in a coffee-based dietary supplement.

A new tadalafil analogue was detected via high-performance liquid chromatography (HPLC)-diode array detection (DAD) during routine screening of health foods suspected of adulteration with erectile dysfunction drugs. The UV absorption spectrum of the unknown was almost identical to that of tadalafil. The analogue was purified by preparative HPLC and structural elucidation carried out by mass spectrometric and NMR spectroscopic experiments. The spectral data revealed that this tadalafil analogue bears a benzyl group instead of the methyl group. The isolated compound was identified as N-benzyl tadalafil. Considering the risk it poses to public health, this new PDE-5 analogues for ED should be included on the inspection list for illegal products.

Depsidones and a dihydroxanthenone from the endophytic fungi Simplicillium lanosoniveum (J.F.H. Beyma) Zare & W. Gams PSU-H168 and PSU-H261.

Three new compounds including two depsidones (simplicildones J and K) and one dihydroxanthenone (globosuxanthone E) together with nine known compounds were obtained from the crude extracts of two endophytic fungi Simplicillium lanosoniveum (J.F.H. Beyma) Zare & W. Gams PSU-H168 and PSU-H261 which were isolated from the leaves of Hevea brasiliensis. The structures were elucidated by spectroscopic evidence. The absolute configuration of globosuxanthone E was established by means of experimental and calculated TDDFT ECD data. Simplicildone K exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with equal MIC values of 128 µg/mL. Simplicildone K and globosuxanthone E displayed antifungal activity against Cryptococcus neoformans ATCC90113 with the same MIC values of 32 µg/mL. In addition, known botryohordine C and simplicildone A showed phosphodiesterase 5 inhibitory activity with the IC50 values of 5.69 and 9.96 µM, respectively, and were noncytotoxic toward noncancerous Vero cells.

Optimization of Extraction Technology of Majun Mupakhi Ela and its Effect on Hydrocortisone-induced Kidney Yang Deficiency in Mice.

We used Box-Behnken design-based (BBD) response surface methodology (RSM) in this research to optimize the extraction process of Traditional medicine Majun Mupakhi Ela (MME) and evaluate its effect on hydrocortisone-induced kidney yang deficiency. Three independent parameters were applied to evaluate the maximum phosphodiesterase type 5 (PDE5) inhibition activity of MME extracts in vitro. The optimal processing conditions (extraction time 2 h, solid-liquid ratio 1:16, extraction once) gave a maximum PDE5 inhibition rate of 84.10%, flavonoid content of 0.49 mg/ml, icariin content of 0.028 mg/ml and targeted extraction yield of 26.50%. In animal experiments, MME extracts significantly increased the adrenal mass index, semen weight index, preputial gland weight index, and penis weight index in mice; in the middle and high dose group, the level of serum testosterone increased by 7664.29% and 14207.14% respectively, compared with the model group, and the level of PDE5 decreased by 67.22% and 74.69% respectively compared with the control group. These results indicate that MME has a significant positive effect on the hypothalamus-pituitary-gonadal axis, improve mating ability and not only has inhibits PDE5 activity but also significantly inhibits the expression of PDE5 in penile tissues, potential to become erectile dysfunction (ED) therapies for the clinical management of patients with kidney yang deficiency.

In silico screening of anthraquinones from Prismatomeris memecyloides as novel phosphodiesterase type-5 inhibitors (PDE-5Is).

OBJECTIVE: Prismatomeris memecyloides Craib (Rubiaceae) is a medicinal plant traditionally used by ethnic minorities in Vietnam for the treatment of erectile dysfunction (ED). The aim of this study was to investigate the chemical compositions and screen in silico its possible inhibitory effect against PDE-5 which reduced cyclic guanosine-3',5'-monophosphate (cGMP) levels and indirectly caused the male ED. METHODS: Separation of natural compounds were carried out on chromatographic column with silica gel or reversed phase materials, eluting with different solvent gradients. The structures of all isolated compounds were elucidated on the basis of spectroscopic data (HR-MS, 1D/2D-NMR). Docking simulation study of compound (1-7) was performed by using flexible side chains protocol based on Iterated Local Search Global Optimizer Algorithm of AutoDock/Vina v.1.1.2. Pharmacokinetic parameters and toxicity prediction were also calculated by appropriate softwares. RESULTS: From the methanol extract of roots of P. memecyloides collected in Vietnam, seven compounds including four anthraquinone/one anthraquinone glycoside namely damnacanthal (1), lucidin-ω-methyl ether (2), 3-methylalizarin (3), rubiadin-3-methyl ether (4), and 1-O-methylrubiadin 3-O-primeveroside (5) along with two iridoid glucosides, asperulosidic acid (6) and aitchisonide A (7) were isolated. The molecular modeling results showed that 5 anthraquinone compounds possess the lowest binding energies to PDE-5. The anthraquinone glucoside 1-O-methylrubiadin 3-O-primeveroside (5) potentially inhibited PDE-5 similarly to commercial PDE-5Is sildenafil (SLD) and tadalafil (TLD). Calculated pharmacokinetic results like pIC50,pred; miLogP, TPSA, enzyme inhibitory of anthraquinone glucoside (5) were similar and even higher to those of the commercial PDE-5 inhibitors. Especially the predictive toxicity of 1-O-methylrubiadin 3-O-primeveroside (5) was even lower than those of SLD and TLD. CONCLUSION: This is the first study to find a scientific-based evidence for the ethnic use of P. memecyloides as medicinal plant for the treatment of ED. The result indicates that the anthraquinones (damnacanthal (1), lucidin-ω-methyl ether (2), 3-methylalizarin (3) and rubiadin-3-methyl ether (4)), especially anthraquinone glycoside (1-O-methylrubiadin 3-O-primeveroside (5)) are compounds of potential novel drug class for the ED treatment.

Phosphodiesterase 5 Inhibitors from Derris scandens.

Phosphodiesterase 5 inhibitors have been used as a first-line medicine for the treatment of erectile dysfunction. In the search for new phosphodiesterase 5 inhibitors from natural sources, we found that the 95% ethanol extract of Derris scandens stem showed phosphodiesterase 5 inhibitory activity with an IC50 value of about 7 µg/mL. Seven isoflavones and a coumarin constituent isolated from this plant were investigated for phosphodiesterase 5 inhibitory activity. The results showed that osajin (8: ), 4',5,7-trihydroxybiprenylisoflavone (4: ), and derrisisoflavone A (2: ) had the ability to inhibit phosphodiesterase 5 with IC50 values of 4, 8, and 9 µM, respectively. These compounds exhibited selectivity on phosphodiesterase 5 over phosphodiesterase 1, however, the selectivity on phosphodiesterase 5 over phosphodiesterase 6 was low. In order to quantitatively determine these bioactive constituents in D. scandens extract, LC-QTOF-MS method has been developed and validated. The limit of quantitation values in the range of 0.1 - 5 µg/mL were obtained. The assay showed satisfactory precision and accuracy. The results from our method showed that the 95% ethanol extract of D. scandens stem was comprised of all eight compounds, with derrisisoflavone A (2: ) and lupalbigenin (3: ) presenting as the major constituents.

Determination of synthetic phosphodiesterase-5 inhibitors by LC-MS2 in waters and human urine submitted to dispersive liquid-liquid microextraction.

High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS2) with a triple quadrupole is proposed for determining the synthetic phosphodiesterase-5 inhibitors, sildenafil, tadalafil and vardenafil, and the active metabolite N-desmethyl sildenafil. The method was successfully applied to the analysis of waters of different origins and human urine samples. Dispersive liquid-liquid microextraction (DLLME) was applied in the classical way for water analysis, whereas a previous extraction into an organic solvent was necessary for urine samples, the acetonitrile extract being used as dispersant solvent in the DLLME step. The analytes were determined by LC-ESI-MS2 in the multiple reaction monitoring mode. Detection limits were in the 5-50 and 15-250ngL-1 ranges for water and urine samples, respectively. The repeatability was calculated using the relative standard deviation, obtaining values of between 3.6% and 10.1%. The enrichment factors were between 75 and 81. Accuracy of the procedure was calculated through recovery assays and average recoveries ± SD (n = 48) of 93.6 ± 3.5 and 91.1 ± 3.5 were obtained for water and urine samples, respectively. None of the samples analyzed contained the target compounds, at least above the corresponding detection limits.

Simultaneous Detection of Three Phosphodiesterase Type 5 Inhibitors and Eight of Their Analogs in Lifestyle Products and Screening for Adulterants by High-Performance Thin-Layer Chromatography.

An HPTLC method is proposed to permit effective screening for the presence of three phosphodiesterase type 5 inhibitors (PDE5-Is; sildenafil, vardenafil, and tadalafil) and eight of their analogs (hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil) in finished products, including tablets, capsules, chocolate, instant coffee, syrup, and chewing gum. For all the finished products, the same simple sample preparation may be applied: ultrasound-assisted extraction in 10 mL methanol for 30 min followed by centrifugation. The Rf values of individual HPTLC bands afford preliminary identification of potential PDE5-Is. Scanning densitometry capabilities enable comparison of the unknown UV spectra with those of known standard compounds and allow further structural insight. Mass spectrometric analysis of the material derived from individual zones supplies an additional degree of confidence. Significantly, the proposed screening technique allows focus on the already known PDE5 Is and provides a platform for isolation and chemical categorization of the newly-synthesized analogs. Furthermore, the scope could be expanded to other therapeutic categories (e.g., analgesics, antidiabetics, and anorexiants) that are occasionally coadulterated along with the PDE5-Is. The method was successfully applied to screening of 45 commercial lifestyle products. Of those, 31 products tested positive for at least one illegal component (sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, or dimethylsildenafil).

Semi-synthetic derivatives of natural isoflavones from Maclura pomifera as a novel class of PDE-5A inhibitors.

Natural (iso)flavonoids have been recently reported to inhibit cyclic nucleotide phosphodiesterases (PDEs) and induce vasorelaxation, albeit the results described in the literature are discordant. The cGMP-selective isoform PDE-5A, in particular, represents the target of sildenafil and its analogues in the treatment of erectile dysfunction (ED) and pulmonary hypertension by promoting relaxation in vascular smooth muscle through the activation of the NO/cGMP pathway. We undertook this study to verify if osajin and pomiferin, two natural prenylated isoflavones and major constituents of Maclura pomifera extracts previously investigated for their anticancer, antibacterial and antidiabetic properties, show inhibitory activity on PDE-5A. These two isoflavones were isolated from the plant extracts and then synthetically modified to obtain a set of semi-synthetic derivatives with slight and focused modifications on the natural scaffold. The compounds were at first screened against PDE-5A in vitro and, based on the encouraging results, further tested for their relaxant effect on isolated rat artery rings. Computational docking studies were also carried out to explore the mode of interaction with the target protein. The obtained data were compared to the behaviour of the well-known PDE-5A inhibitor sildenafil. Our results demonstrate that semi-synthetic derivatives of osajin and pomiferin show an inhibitory effect on the isolated enzyme that, for some of the compounds, is accompanied by a vasorelaxant activity. Based on our findings, we propose the here described isoflavones as potential lead compounds for the development, starting from natural scaffolds, of a new class of PDE-5A inhibitors with vasorelaxant properties.

Application of Orbitrap-mass spectrometry to differentiate isomeric sildenafil- and thiosildenafil-like analogues used for the adulteration of dietary supplements.

Two groups of isomeric phosphodiestrase-type 5 inhibitors (PDE-5), consisting of four sildenafil- and three thiosildenafil-like analogues, have been successfully differentiated using high-resolution MS/MS. The optimised MS/MS data obtained from each compound were used to build a database with the aid of mass processing software. Isomeric compounds with very close chromatographic separation like dimethylsildenafil and homosildenafil could be distinguished by their unique fingerprint fragment ions in the MS/MS database. All fragment ions were within the mass tolerance of 5 ppm. One case study using an adulterated dietary supplement is included to provide more insights into this application.

Moringa oleifera extract enhances sexual performance in stressed rats.

Aphrodisiacs are required to improve male sexual function under stressful conditions. Due to the effects of oxidative stress and dopamine on male sexual function, we hypothesized that Moringa oleifera leaves might improve male sexual dysfunction induced by stress. Therefore, the effects on various factors playing important roles in male sexual behavior, such as antioxidant effects, the suppression of monoamine and phosphodiesterase type 5 (PDE-5) activities, serum testosterone and corticosterone levels, and histomorphological changes in the testes, of a hydroethanolic extract of M. oleifera leaves were investigated. Various doses of extract including 10, 50, and 250 mg/kg body weight (BW) were given orally to male Wistar rats before exposure to 12 h-immobilization stress for 7 d. The results demonstrated that the extract showed both antioxidant and monoamine oxidase type B (MAO-B) suppression activities. At 7 d of treatment, the low dose of extract improved sexual performance in stress-exposed rats by decreasing intromission latency and increasing intromission frequency. It also suppressed PDE-5 activity, decreased serum corticosterone level, but increased serum testosterone, numbers of interstitial cells of Leydig and spermatozoa. The increased numbers of interstitial cells of Leydig and spermatozoa might have been due to the antioxidant effect of the extract. The increased sexual performance during the intromission phase might have been due to the suppression of MAO-B and PDE-5 activities and increased testosterone. Therefore, M. oleifera is a potential aphrodisiac, but further research concerning the precise underlying mechanisms is still needed.

Pyrazolopyrimidines in 'all-natural' products for erectile dysfunction treatment: the unreliable quality of dietary supplements.

A herbal food supplement advertised as a potency pill was screened for the presence of PDE5 inhibitors. The resulting signals were characterised by UV, LC-MS in ESI-negative mode, and NMR spectroscopy using 1D and 2D experiments. Several substances were identified, bearing the basic chemical structure of sildenafil, but were not supposed to exhibit PDE5 inhibition. These compounds may be process-related impurities or by-products of different reaction steps in the synthesis of PDE5 analogues. As they were found to be present in different capsules at different concentrations, this is an example of the unreliable quality of dietary supplements.

Isolation and structural characterization of a new tadalafil analog (2-hydroxyethylnortadalafil) found in a dietary supplement.

A screen for known PDE-5 inhibitors in a dietary supplement product marketed for "enhanced sexual performance" detected a compound that structurally resembled tadalafil. The compound was isolated from the supplement matrix using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and a fraction collector, and was further characterized using nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), as well as high-resolution accurate mass mass spectrometry (HRAM-MS). The analog had an accurate mass of m/z 420.15614 (error is 1.77235ppm) for the protonated species [M+H](+), corresponding to a molecular formula of C23H22N3O5. Mass spectral fragmentation data suggested that the modification occurred in place of the CH3 located on the pyrazinopyridoindole-1,4-dione of tadalafil. NMR was utilized to further elucidate the configuration of the substitution. The analysis indicated that the moiety is a CH2CH2OH, hydroxyethyl group. The new analog has been named 2-hydroxyethylnortadalafil.

Novel mechanisms of anticancer activities of green tea component epigallocatechin- 3-gallate.

After water, tea is the most widely consumed beverage. The major active constituents in green tea are catechins, of which epigallocatechin-3-gallate (EGCG) is the most abundant and active compound. Animal experimental studies using EGCG alone or green tea catechins with EGCG being a major component have generated a mounting body of evidence suggesting that EGCG as a naturally occurring compound and commonly consumed beverage ingredient is a promising cancer preventive agent. However, the relationship between green tea consumption and reduced cancer risk seen from epidemiologic studies is not as encouraging as that observed in animal studies and remains inconclusive. In the present article, the achievements using EGCG or green tea catechins for cancer prevention were reviewed, the latest identified anticancer mechanisms of EGCG and the emerging mechanism-based cancer therapies of EGCG were outlined, and the potential reasons for the discrepancy in animal studies and epidemiological studies were tentatively analysed. On the basis of these analyses, it could be anticipated that future intervention trials in humans would be able to achieve consistent cancer prevention effects provided that the timely intervention of EGCG or green tea catechins at appropriate high-dose levels presumably approaching their upper safety limits have had been fully considered.

Identification of new synthetic PDE-5 inhibitors analogues found as minor components in a dietary supplement.

A dietary supplement sold in erotic shops was analysed. It contains dithiodesmethylcarbodenafil as the major component, which was already reported as an adulterant in dietary supplements. Additionally three more compounds were found and their structures were elucidated after isolation using NMR and mass spectroscopy. They were designated as isonitrosoprodenafil, dithiodesethylcarbodenafil and norcarbodenafil.

Simultaneous identification of 18 illegal adulterants in dietary supplements by using high-performance liquid chromatography-mass spectrometry.

We developed a method for the identification of 18 illegal adulterants in dietary supplements for erectile dysfunction by using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a Cosmosil 3C18-EB column. The mobile phase consisted of 0.1% formic acid solution and 0.1% formic acid in acetonitrile, with gradient elution at a flow rate of 0.15 mL/min. The proposed method may be useful for the identification of illegal adulterants and for quality control of dietary supplements.

Isolation and identification of novel propoxyphenyl thiosildenafil found in natural health food product.

A novel phosphodiesterase-5 (PDE-5) inhibitor was found in a natural health food product. The previously unknown sildenafil analogue was isolated using preparative HPLC. The structure of the compound was elucidated using HPLC with diode array detection (DAD), time-of-flight mass spectrometry (TOF/MS), and nuclear magnetic resonance spectroscopy (NMR). An [M + H](+) ion was detected at m/z 505.2077 by LC-TOF/MS that was consistent with C23H32N6O3S2 (-0.98 ppm). By NMR analysis, the analogue was identified as 1-methyl-5-(5-(4-methylpiperazin-1-ylsulfonyl)-2-propoxyphenyl)-3-propyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-thione. In this structure, the ethoxy group of thiosildenafil was substituted by a propoxy group of the unknown compound. Therefore, this novel thiosildenafil analogue was named propoxyphenyl thiosildenafil.

Magnetic molecularly imprinted polymer for the selective extraction of sildenafil, vardenafil and their analogs from herbal medicines.

The successfully developed magnetic molecularly imprinted polymers (MMIPs) toward six synthetic phosphodiesterase type-5 (PDE-5) inhibitors were described. Sildenafil was used as template for the preparation of MMIPs using superparamagnetic core-shell nanoparticle as supporter. The obtained MMIPs were characterized using transmission electron microscope, Fourier transform infrared, X-ray diffraction, and vibrating sample magnetometer. High performance liquid chromatography (HPLC) with diode array detector (DAD) was used for the analysis of target analytes. The application of MMIPs as selective sorbent in the cleanup of herbal medicine samples prior to HPLC offered simple sample preparation. The adsorption capacity and selectivity of prepared MMIPs and magnetic non-molecularly imprinted polymers were investigated. The binding isotherms were obtained for sildenafil and fitted by Freundlich isotherm model. Structurally similar compound of sildenafil and a reference compound protocatechuic acid were used for investing the selective recognition of MMIPs.

Simultaneous identification of hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, and thiodimethylsildenafil in dietary supplements using high-performance liquid chromatography-mass spectrometry.

We developed a method for the separation and identification of illegal adulterants (hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, and thiodimethylsildenafil) from dietary supplements using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a C18 column: the mobile phase consisted of 5 mM ammonium formate (pH 6.3)-acetonitrile (75 : 25, v/v) and acetonitrile, with gradient elution at a flow rate of 0.2 mL/min. The proposed method could also be used to separate vardenafil, homosildenafil, and dimethylsildenafil, all of which have the same molecular weight. Furthermore, the proposed method could simultaneously separate hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, thiodimethylsildenafil, vardenafil, and homosildenafil. Thus, this method may be useful to identify medicinal ingredients for erectile dysfunction and their analogs and to control the quality of dietary supplements.

Isolation and characterization of propoxyphenyl linked sildenafil and thiosildenafil analogues in health supplements.

Two new phosphodiesterase-5 inhibitors (PDE-5) which consist of one sildenafil analogue and one thiosildenafil analogue have been found in heath supplements. The structural properties of these analogues have been elucidated by NMR, high resolution MS, MS(2), UV and IR spectroscopy. The sildenafil analogue is very similar to aildenafil and the thiosildenafil analogue is similar to thioaildenafil, except the ethoxy group bonded to phenyl ring is replaced by a propoxy group. Hence, the sildenafil analogue is named as propoxyphenyl aildenafil or propoxyphenyl methisosildenafil and the thiosildenafil analogue as propoxyphenyl thioaildenafil or propoxyphenyl thiomethisosildenafil.