Chronic Administration of Propolis Improves Plasma Levels of Nitric

1797 Khartoum Medical Journal (2021) Vol. 14, No. 01, pp. 1797 - 1803

Chronic administration of propolis improves plasma levels of nitric oxide, calcium and cyclic guanosine monophosphate in selective serotonin reuptake inhibitors-induced erectile dysfunction in male Wistar rats Ayinde TO,1 Beheiry HM,2 Ojulari LS,1 Hussien MO,3* Afodun AM,4 Oluwasola A,5 Maryoud AH,2 Abdulwahab HM,6 Kardash MM.7 1Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Nigeria. 2Department of Physiology, Faculty of Medicine, International University of Africa, Khartoum, Sudan. 3Department of Microbiology, Central Laboratory, Ministry of Higher Education and Scientific Research, Khartoum, Sudan. 4Department of Anatomy and Cell Biology, Faculty of Health Sciences, Busitema University, Uganda. 5Department of Physiology, Faculty of Health Sciences, Al-Hikmah University, Ilorin, Nigeria. 6Department of Pharmacology, Faculty of Pharmacy, University of Ribat, Khartoum, Sudan. 7Department of Physiology, Faculty of Medicine, Ahlia University, Omdurman, Sudan.

ABSTRACT Background Sexual dysfunction is a common adverse effect for 50-80% of patients taking selective serotonin reuptake inhibitors. This could be very annoying and makes the patient scornful of his sexual aggrandizement despite thoughtlessly patronizing several sexual enhancing drugs. It also progressively leads to loss of confidence among couples with the psyche of searching for alternative sexual pleasure. The present study investigated the effect of chronic administration of propolis on plasma levels of nitric oxide, calcium and cyclic guanosine monophosphate which interplay in the physiological mechanism of erectile function. Methods Male Wistar rats (140-190g) that were sexually active and sexually unexposed females (120- 130g) were employed in this study. Induction of erectile dysfunction was done in the male rats in groups II- VII by paroxetine hydrochloride 10mg/kg body weight orally for 2 weeks and confirmed by copulatory test with females. They were then randomly divided into seven groups of six rats (n=6) each and administered sildenafil or graded concentrations of propolis or 0.9% sodium chloride (group I) over a duration of 60 days. Following this, the animals were euthanized and blood samples collected for nitric oxide, calcium and cyclic guanosine monophosphate analysis. Results There was significant (p<0.05) increase in the plasma levels of nitric oxide of sildenafil treated group (III), high dose propolis treated group (VI), propolis-sildenafil combination treated rats (group VII); and significant (p<0.05) reduction in paroxetine-induced untreated rats (group II) and low dose propolis treated rats (group IV) compared to control (group I). However, there was no significant (p>0.05) difference between moderate dose treated rats (group V) compared to control group. There was significant (p<0.05) increase in plasma levels of calcium in group III, VI and VII while there was significant (p<0.05) reduction in calcium level of group II. However, there was no significant (p>0.05) difference in group IV and V. There was significant (p<0.05) reduction of cyclic guanosine monophosphate in group II, IV, and V, while significant (p<0.05) increase was seen in cyclic guanosine monophosphate of groups III, VI and VII compared to control. Conclusion Propolis has a key role to play in sexual functions through enhancement of erectogenic ability if given at high dose for sexual dysfunction by modulation of nitric oxide, calcium and cyclic guanosine monophosphate pathways in the physiological mechanism of penile erection. *Correspondence to [email protected]. Ayinde TO, Beheiry HM, Ojulari LS, Hussien MO, Afodun AM, Oluwasola A,Maryoud AH, Abdulwahab HM, Kardash MM 1798

INTRODUCTION low libido, erectile dysfunction (ED), premature Propolis (PL) is a collection of mixtures from ejaculation, delayed orgasm with other associated 15 different parts of plants harvested by honey bees multifactorial underlying causes. and is currently of much research interests, from Part of the present interest resides in ED, which has both traditional and orthodox perspectives.1,2 It is been documented by some authorities as the most a waxy product which bees utilize for construction common sexual problem in men. It is currently and repairation of their hives,3 and also employed as defined as “the inability to achieve and/or maintain chemical tools by bees against invading organisms. penile erection sufficient to attain satisfactory It has also been explored by humans as antidote sexual intercourse”. ED is a complex multifactorial for several ailments.4 It is a complex biologically disease, and one of the commonest health problems active substance containing more than 80-100 in men, and even more common with increasing identifiable chemical compounds depending on age.16 It has a high worldwide prevalence, with a source of its plant.5 Raw PL is composed of 50% projection of increasing over the next 25 years. resin, flavonoids, and related phenolic acids, In a community-based study ED affected 50-69% 30% wax, 10% essential oils, 5% pollen, and 5% and found to be more in 30% of men between 40 various organic compounds.6 However, a study and 70 years of age.17 It is a globally increasing also discovered that the major phytochemical problem, and because it is not a life threatening components of propolis are caffeic acid phenethyl disease on one hand, and due to its associated ester (CAPE), phenolics, hydrocarbons, aromatic stigma on the other hand, the sufferers seldom seek and aliphatic acids, flavonoids (Pinocembrin, for medical intervention which further deteriorates Galangin and Chrysin).7 Propolis exhibits anti- the situation.18 Thus, ED is known to be hiddenly inflammatory and antioxidant properties8, because associated with high level of psychological trauma it inhibits nuclear factor kappa light chain enhancer in affected men, and this precipitates a low level of activated B cells (NF- B) and subsequently K of self esteem and ecstasy, and poor relationship production of interleukins.9,10,11 among the sufferers, especially with their spouses.15 In another study, the Brazilian green propolis Most of the orthodox oral preparations are of cyclic has been explored to have caused lengthening in nucleotide phosphodiesterases (PDE) which are epididymal epithelium that is free of oxidative enzymes that catalyze the hydrolysis of the cyclic stress as well as sperm production in rats in a nucleotides, cyclic adenosine monophosphate morphometric-stereological study.12,13 PL has (cAMP) and 3’, 5’-cyclic guanosine monophosphate also been observed to increase semen quality and (cGMP); and they appear in different molecular seminal plasma enzymes. In a similar in vitro study, forms such as phosphodiesterase. An important it was discovered that PL can improve the total subtype distributed throughout the body is PDE5. mitochondrial respiratory efficiency in the human Sildenafil (Viagra), vardenafil (Levitra) and spermatozoa, suggesting its potential to improve tadalafil (Cialis) are common PDE5 inhibitors sperm motility,14 which is an important parameter prescription drugs which are taken orally.19 They for the fertilizing power of spermatozoa. Apart work by blocking the action of PDE5, which causes from the sperm produced by the male reproductive degradation of cGMP. Alprostadil (Vitaros) is a system, having a sustained penile erection is a big similar topical cream approved in Canada for erectile psychosexual phenomenon that plays a key role dysfunction.20 Many traditional concoctions from in sociocultural definition of a complete man. The parts of plants and animal secretions have been major shortfall of this system is a constellation used before the advent of PDE5 inhibitors, and of manifestations collectively termed sexual still much prevalent in many African communities dysfunction experienced by men at a point in as antidote for various conditions of sexual life, though of varying degrees which include: Chronic administration of propolis improves plasma levels of nitric oxide, calcium and cyclic guanosine monophosphate in selective 1799 serotonin reuptake inhibitors-induced erectile dysfunction in male Wistar rats dysfunction. For example, some people believe as Sources of kits and drugs folktales that the toxin in the venom of Brazilian Nitric oxide, Calcium and cGMP were products of 21 spider has erectogenic activity. Fortress Diagnostics Limited, Antrim Technology Park, Antrim, BT41 IQ3, United Kingdom and The burden of high cost and desensitization to purchased at Hisam Medical Diagnostic Equipment, availability of current therapies among sufferers in Khartoum, Sudan. our environment, especially in rural communities, necessitates the need for indigenous, inexpensive, Propolis extract was a product of Y.S. Organic Bee natural sources that is almost free of side effects as Farms 2774 N 4351 Rd. Sheridan, IL 60551 USA. intervention. This is a big gap in the knowledge of It is commercially prepared as 1000mg per capsule the present available clinical intervention for the and reconstituted by dissolving in 10ml of normal victims of ED and by extension sexual dysfunction saline and delivered as 100mg/ml. as a stigmatizing menace. The current study was carried out to explore and evaluate the medicinal Paroxetine hydrochloride was a product of activities of propolis using rat model with the Pharaonia Pharmaceuticals, New Borg El- Arab view to justify part of its ethno-medicinal claim City, 3rd Industrial Zone, Block 16, Alexandra. It to be erectogenic. Therefore, we hypothesized was purchased at Hashmik Pharmacy, Maknimir that propolis is beneficial in improving erectile Street, Khartoum Sudan. Ten milligram of the drug dysfunction in male Wistar rats. was dissolved in 10ml normal saline and delivered as 1mg/ml. MATERIALS AND METHODS Experimental animals Sildenafil citrate (Viagra) was a product manufactured by Cadila Health Care Limited, Sexually potent male rats weighing between 140– Sharkhe-Bavla N.H. No. 8A, Moraiya Tal: Sanand 190g, and equal number of sexually unexposed Dist: Ahmedabad 382210, India. It was purchased at female (120-130g) purchased from National Center the pharmacy outlet of the International University for Research, Khartoum, Sudan were used for this of Africa, Khartoum, Sudan. Twenty-five milligram study. They were housed separately, and maintained of the tablet was reconstituted in 10ml of normal in the animal house of the Faculty of Veterinary saline and delivered as 2.5mg/ml. Medicine, University of Khartoum, Shambat, Sudan. The animals were kept in standard plastic cages Animal grouping containing wood chips (sawdust) bedding, with Forty-two (42) male rats were finally recruited; good ventilation, free access to standard rat pellet and randomly distributed into seven (7) groups feeds (National Centre for Research) and water ad containing six rats each (n=6). Following induction libitum. The animals were subjected to artificial day of erectile dysfunction of groups II-VII with 10mg/ and night cycle of 12 hours by 12 hours, light off kg of paroxetine (PT) for two weeks.15 They at 08:00pm, under an optimum temperature of 25- were either left with the vehicle alone, or orally 30oC. The animals were acclimatized for two weeks administered sildenafil (SF), or orally administered prior to commencement of the experiment. The different graded concentrations of propolis, or experimental protocols which involved invasive orally co-administered sildenafil and propolis for a and non-invasive procedures were approved by duration of 60 days is as itemized below: the Animal Research Ethical Committee of the International University of Africa, Khartoum, Group I: Normal saline (0.9% NaCl) Normal Sudan, and were conducted in accordance with control internationally accepted principle for laboratory Group II: PT (10mg/kg) animal use and care. Group III: PT (10mg/kg) + SF (25mg/kg) Ayinde TO, Beheiry HM, Ojulari LS, Hussien MO, Afodun AM, Oluwasola A,Maryoud AH, Abdulwahab HM, Kardash MM 1800

Group IV: PT (10mg/kg) + PL (50mg/kg) Drive, Suite 216, Houston, Texas 77079. Briefly, Group V: PT (10mg/kg) + PL (100mg/kg) calcium standard was prepared firstly, 90 ul of the chromogenic reagent was added to each well Group VI: PT (10mg/kg) + PL (200mg/kg) containing standards, samples, or controls and Group VII: PT (10mg/kg) + PL (200mg/kg) + SF then mixed gently. 60 ul of calcium assay buffer (25mg/kg) was then added to each well and mixed gently. The reaction was then incubated for 5–10 min at Following this, the animals were euthanized under room temperature in dark. The absorbance was then ketamine anesthesia 50mg/kg, and blood samples measured at 575 nm. collected for determination of NO, calcium and Determination of plasma levels of cyclic cGMP. guanosine monophosphate Blood sample collection Plasma cGMP levels were measured using an Following laparotomy and adequate exposure of immunoradiometric assay kit according to the the heart while still beating, the right ventricle manufacturer’s protocol supplied by IBL GmbH was located and carefully punctured with a 21G (Cat. #29071/75, Flughafenstrasse, Hamburg, needle attached to a 5ml syringe. Blood sample was Germany). Briefly, 50 µL of plate primer was added collected and put inside EDTA sample bottle. It was into all wells used. 75 µL of sample diluent was then centrifuged at 3000 revolutions for 10 minutes pipetted into the non-specific binding (NSB) wells. for separation of plasma ahead of nitric oxide, 50 µL of sample diluent was also pipetted into the calcium and cGMP assay. maximum binding (Zero standard) wells. 50 µL of Determination of plasma levels of total samples or standards were similarly pipetted into nitric oxide derivatives (Nitrite+Nitrate) wells in the plate. Sample diluent was then turned Plasma nitric oxide was measured using ELISA from orange to bright pink upon sample or standard kit (KGE001), a product of R and D system addition to the plate primer in the wells. 25 µL of Biocompare, 614 Mckinley Place N.E, Minneapolis, the cGMP conjugate was then added to each well. Minnesota 55413, United State. Briefly, standards 25 µL of the cGMP antibody was then added to each were prepared firstly, 40ul of sample dilution well, except the NSB wells. The plate was covered was pipetted to testing sample well, then 10ul of with the plate sealer and shaken at room temperature the sample was added and mixed gently bringing for 2 hours. The plate was then washed 4 times sample final dilution to 5-fold. The plate was with 300 µL of wash buffer. 100 µL of the TMB covered with adhesive strip, and incubated for 30 Substrate was added to each well and incubated at minutes (min) at 37oC. The plate was then washed room temperature for 30 minutes without shaking. 5 times using wash buffer diluted 30-fold with 50 µL of the Stop Solution was then added to each distilled water. This was followed by pipetting of well and optical density (O.D) generated was 50ul chromogen solution A and 50ul of chromogen measured using ELISA reader at 450 nm. The plate solution B to each well, and incubated for 15 min reader’s built-in 4PLC software capabilities were at 37oC at dark. Stop solution 50ul was then added used to calculate cGMP concentration for each to each well to stop the reaction. Finally, blank well unknown sample. was taken as zero, and absorbance was measured Statistical analysis at 450 nm after pipetting stop solution within 15 Analysis of data was done using SPSS version min. 20. Results were presented as mean ± standard Determination of plasma levels of error of mean (SEM). Differences between groups calcium were analyzed by one-way analysis of variance Calcium level was determined quantitatively using (ANOVA) followed by LSD post-hoc test. In all calcium assay kit (colorimetric) (E-BC-K103-M), statistical analyses, differences were considered a product of Elabscience, 14780 Memorial significant when p was less than 0.05 (p<0.05). Chronic administration of propolis improves plasma levels of nitric oxide, calcium and cyclic guanosine monophosphate in selective 1801 serotonin reuptake inhibitors-induced erectile dysfunction in male Wistar rats

RESULTS Table. Effect of propolis on selective serotonin reuptake inhibitors-induced erectile dysfunction on plasma levels of nitric oxide, calcium and cyclic guanosine monophosphate (cGMP)

I II III IV V VI VII Nitric oxide 14.11±0.18 8.94±0.33* 17.33±1.40* 10.04±0.45* 13.92±0.40 18.69±0.34* 18.98±0.84* (µmol/L) Calcium 10.6923..0± 4.720.26±* 12.450.12±* 10.761.03± 10.831.06± 12.180.47±* 12.91±0.31* (mmol/L) cGMP pmol/ml 2.470.07± 0.970.12±* 2.980.23± 1.590.31±* 1.970.28±* 2.990.09±* 3.01±0.06* Values are Mean ± SEM (n=6). *p<0.05= significant compared to control. There was significant (p<0.05) increase in the mean DISCUSSION values of sildenafil treated rats (group III), high Oral administration of high dose propolis, propolis- dose propolis treated rats (group VI), propolis- sildenafil combination or sildenafil citrate alone in sildenafil combination treated rats (group VII); induced rats was found to remarkably elevate the and significant (p<0.05) reduction in paroxetine- plasma level of nitric oxide. Elevation of nitric induced untreated rats (group II) and low dose oxide by propolis at only high dose was possibly propolis treated rats (group IV) when compared due to expression of endothelial nitric oxide to the control (group I). However, there was no synthase (eNOS), though not quantified in this significant (p>0.05) difference between moderate study or perhaps due to inhibition of PDE so as to dose treated rats (group V) and control (group I). make more cGMP available to prevent influx of It was also noticed that the significant increase in calcium into the vascular smooth muscle cells of propolis-sildenafil combination treated rats (group penile. This was evident from the increase in the VII) was remarkably higher than either sildenafil treated rats (group III) or high dose treated rats plasma levels of cGMP and calcium assessment (group VI) alone. seen in this work. The increased plasma calcium was a reflection of reduced cytoplasmic calcium The plasma levels of calcium were significantly essentially needed for endothelial smooth muscle (p<0.05) increased in sildenafil treated (group III), relaxation to precipitate penile erection. The work high dose treated (group VI), and propolis-sildenafil also confirmed previous studies22 which reported combination treated rats (group VII), while there that sildenafil can prevent degradation of cGMP by was significant reduction (p<0.05) in calcium inhibiting PDE to cause penile erection. The result level of paroxetine-induced untreated rats (group also showed that the resultant erectogenic effect II). However, there was no statistical significant of propolis as a consequence of alteration in the (p>0.05) difference in low dose propolis treated rats concerned biomolecules was dose dependent, and (groups IV) and moderate dose propolis treated rats has synergistic activity with sildenafil citrate if given (group V). at appropriate dose. It was also evident that propolis was able to increase plasma calcium remarkably The plasma values of cGMP were significantly only at high dose as seen in group VI which also (p<0.05) reduced in paroxetine-induced untreated accounted for its synergy with sildenafil in group rats (group II), low dose propolis treated rats VII. The activity of PL at high dose in elevating (group IV), and moderate dose propolis treated rats NO level in this study is similar to the work23 where (group V). However, there was significant increase testosterone was found to up-regulate NO signaling (p<0.05) in cGMP level of sildenafil treated rats through increased nitric oxide synthase-2 (NOS2) (group III), high dose propolis treated rats (group expression. VI), and propolis-sildenafil combination treated rats (group VII) compared to the control (Table) Ayinde TO, Beheiry HM, Ojulari LS, Hussien MO, Afodun AM, Oluwasola A,Maryoud AH, Abdulwahab HM, Kardash MM 1802

In contrary, propolis administration at low and 2. Paulis G, D’Ascenzo R, Nupieri P, et al. moderate doses in paroxetine-induced rats has little Effectiveness of antioxidants (propolis, or no effect in the alteration of biomolecules of blueberry, vitamin E) associated with verapamil erectile functions, though not as poorly reflected in the medical management of Peyronie’s in the paroxetine-induced untreated rats (group disease: a study of 151 cases. International II). This could have been possibly due to the Journal of Andrology 2012; 35:521–527. overwhelming side effect of residual paroxetine in 3. Bankova V, Popova M, Bogdanov S, Sabatini the animals. It is a clear submission that paroxetine AG. Chemical composition of European which antagonizes serotonergic and cholinergic propolis: expected and unexpected results. receptors to inhibit nitric oxide synthase failed Zeitschrift für Naturforschung C. Journal of to initiate or sustain this blockade as a result of Biosciences 2002; 57(5-6):530–533. availability of propolis at high doses. This is in tandem with a previous study that found that high 4. Wollenweber E, Hausen BM, Greenaway W. dose propolis has erectogenic effect via hormonal Phenolic constituents and sensitizing properties modulations.15 In a closely related human study of propolis, poplar balsam and balsam of Peru. where patients were treated at different times and Bull Groupe Polyphenols 1990; 15:112–20. with different combinations of antioxidants for 5. Marcucci MC, Ferreres F, García-Viguera correction of Peyronie’s disease. Propolis and C, Bankova VS, De Castro SL, Dantas AP. vitamin E combination containing group acquired Phenolic compounds from Brazilian propolis the highest rate where penile curvature typical of with pharmacological activities. Journal of the disease disappeared.24 In another similar study, Ethnopharmacology 2001; 74:105-12. royal jelly also a product of bees-like propolis with antioxidant property recovered erectile function by 6. Pietta PG, Gardana C, Pietta AM. Analytical improving endothelial function of pudendal artery.25 methods for quality control of propolis. The efficacy of PL as erectogenic was perhaps due Fitoterapia 2002; 73:S7-20. to part of its phytochemical constituent, caffeic 7. Schnitzler P, Neuner A, Nolkemper S, et al. acid phenethyl ester (CAPE), and this empowers Antiviral activity and mode of action of propolis PL its antioxidant capacity. Invariably, this could extracts and selected compounds. Phytotherapy have accounted for the positive alterations seen in Research 2010; 24(1):S20-8. biomolecules involved in the physiological pathway of penile erection. 8. Russo A, Longo R, Vanella A. Antioxidant CONCLUSIONS activity of propolis: role of caffeic acid It could be concluded that propolis has a key role to phenethyl esther and galangin. Fitoterapia play in reproductive physiology. It has tendency to 2002; 73(1):S21–S29. sustain biomolecules of erectile function which are 9. Natarajan K, Singh S, Burke TR Jr, Grunberger critical for maintenance of adequate penile erection D, Aggarwal BB. Caffeic acid phenethyl and sexual vigour of an individual. Similarly, the ester is a potent and specific inhibitor of increased plasma level of nitric oxide by propolis activation of nuclear transcription factor NF- observed in this work correlates well with its kappa B. Proceedings of National Academy of role in reproductive physiology in enhancing Sciences of the United States of America 1996; erectogenicity of penile. 93(17):9090-5.

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