The Open Natural Products Journal, 2010, 3, 10-19 An Initial Evaluation of the Safety, Efficacy and Purity of VigRX, a Herbal Combination Formula, for the Enhancement of Male Sexual Health Y. Smitasirib, P. D'Souzac, J. Neal-Kababick d and A.G. Schauss,*a aNatural and Medicinal Products Research, AIBMR Life Sciences, Inc., 4117 S. Meridian, Puyal up, Washington 98373, USA bMae Fah Luang University, Muang District, Chiang Rai 57100, Thailand cNatural Remedies, Plot 5B, 1t9 h K. M Stone, Hosur Road, Bangalore 560 100, India dFlora Research Laboratories, 1000 SE M Street, Grants Pass, Oregon 97526, USA Abstract: A patented herbal combination formula, known as VigRX, has been studied for purity, and for safety and effi-cacy in a Sprague-Dawley rat model. Two separate assays determined that VigRX was free from pharmaceutical adulter-ants, including phosphodiesterase type 5 (PDE-5) inhibitors and related analogues. An in vitro assay determined that VigRX is able to inhibit the enzyme Rho-kinase, suggesting a potential mechanism of action for this product. A 2-week (14-day) study in rats demonstrated a marked enhancement in sexual behavior, including decreased intromission and ejaculation latencies, and increased intromission, ejaculation and mounting frequencies, upon oral administration of 30 mg/kg/day. A longer 12-week study using 15 mg/kg/day showed only a decrease in ejaculation latency with respect to sexual behavior. In both studies, the treatment led to increased intracavernosal pressure, increased sperm concentration, and increased width of erect penis (and an increase in erect penile length in the 14-day study only). There was a statisti-cal y significant increase in blood testosterone levels in rats at the end of the 12-week study, which did not occur in the 14-day study. A non-dose dependent decrease in kidney and liver weights was found in the 14-day study that was not seen in the 12-week study, and neither study found any notable histopathological changes in any tissues studied. In conclusion, these preliminary results demonstrate safety and efficacy of VigRx for use in supporting male erectile function, and justify further investigation in these areas. Keywords: Male erectile function, male sexual health, humans, active pharmaceutical ingredient, dietary supplement, herbal supplement, PDE-5 inhibitor, herbal medicine, Rho-kinase inhibition, penile intracavernosal pressure. erectile function, sexual health and performance. While the consumer may assume these "natural" products are safe, this Age-related decline in sexual function is a noted phe- category of dietary supplements is marred with numerous nomenon worldwide. The prevalence in adult men has been reports of adulteration with active pharmaceutical ingredi- estimated at 20–30% [1,2]. Men aged 50–59 experienced ents (APIs) [7]. Some APIs commonly discovered in these three times more problems related to erections and low sex- misleading supplements include phosphodiesterase (PDE-5) ual desire than their 18–29 year old counterparts [2]. The inhibitors such as sildenafil (Viagra®), vardenafil (Levitra®), pathophysiology of erectile dysfunction, a commonly expe- and tadalafil (Cialis®), or their analogues. Because these pre- rienced aspect of male sexual dysfunction, can include arte- scription drugs are used as effective treatments for erectile riogenic, neurogenic, endocrinologic, or psychogenic factors dysfunction, their inclusion in herbal formulas may enhance [3]. Multiple mechanisms may account for changes in these the efficacy of these formulas, misleading consumers to at- systems, including increased RhoA/Rho-kinase activity, im- tribute positive results to the listed combination of vitamins paired function of the nitric oxide (NO) and endothelial ni- and herbs in the ingredients rather than the effect of the tric-oxide synthase (eNOS) systems, and increased levels of pharmaceutical adulterant. Furthermore, not disclosing the inflammatory and prothrombotic vascular endothelial com- presence of the API adulterants on the labeling of these products puts consumers at considerable risk, including ex- Men have long sought remedies for declining sexual posure to side effects such as flushing, headaches, palpita- health. While prescription medications are often used, men tion, nasal congestion, and visual disturbances [8,9]. Of even are increasingly turning to herbal alternatives. The dietary greater concern is the possibility of these pharmaceutical supplement market includes a large number of products adulterants interacting with other medications the consumer claiming to benefit men seeking enhancement of their could be taking [10]. For instance, the combination of nitro- glycerine and PDE-5 inhibitors or their analogues can dan- gerously lower blood pressure. This is of particular relevance *Address correspondence to this author at the Natural and Medicinal Prod- because the population that is prescribed nitrates also com- ucts Research, AIBMR Life Sciences, Inc., 4117 S. Meridian, Puyal up, monly suffers from erectile dysfunction. While doctors gen- Washington 98373, USA; Tel: +1-253-286-2888; Fax: +1-253-286-2451; E-mail: [email protected] eral y warn patients on nitrates against taking pharmaceutical 2010 Bentham Open An Initial Evaluation of the Safety, Efficacy and Purity of VigRX The Open Natural Products Journal, 2010, Volume 3 11 PDE-5 inhibitors, these individuals may turn to dietary sup- Thus, agents that inhibit the Rho-kinase enzyme may plements as "natural" alternatives to treating their erectile have a direct effect on erectile function. This has been dem- dysfunction [7,11]. onstrated in vivo fol owing the administration of the selective Unfortunately, there is evidence that this il egal practice Rho-kinase inhibitor Y-27632 to rats [19]. Pharmaceuticals of including undisclosed APIs in dietary supplements is and botanicals alike are known to possess Rho-kinase inhibi- widespread [11-13]. This adulteration can occur at any step tory activity as a means of facilitating the erectile process. of the manufacturing process, and may be unknown to the Assays such as the one measuring Rho-kinase inhibition companies who are marketing and sel ing the product. serve to elucidate potential mechanisms of action whereby Therefore, conscientious dietary supplement manufacturers promising compounds could have beneficial effects on a should test every batch of finished products for APIs to as- particular physiological function. sure no adulteration took place during any step of manufac- Once an in vitro evaluation has yielded insights into a turing. This serves to ensure the quality and identity of the possible or likely mechanism of action, a logical next step in product being consumed as wel as heighten the potential the progression of research is to evaluate the efficacy of a level of safety of the formulation. product in a valid animal model. The rat has been established as a suitable animal model for the study of penile erection Long before the advent of synthetic pharmaceuticals for [20], and has been used to study the effects of a number of the treatment of sexual dysfunction, herbal medications have medicinal plants on sexual behavior [21-25]. Research in rats been used as sexual health supportive agents in diverse cul- can yield important preliminary information regarding the tures throughout the world. While the traditional use of such effects of a formula on various parameters associated with plants is extensive, recent scientific investigations into indi- male sexual function. vidual herbs and combination formulas have yielded impor-tant positive data in terms of the ability of botanicals to sup- While many marketers of herbal sexual health aids cite port sexual wel ness and the erectile process. There are many historical precedent as their evidence for efficacy, the need medicinal plants that have been reported to possess aphrodis-for scientific investigation into the validity of these claims is iac effects, such as Muira Puama (Ptychopetalum spp.) root necessary. In vitro assays, animal studies and ultimately hu-and Catuaba (Trichilia catigua) bark [13]. Recent research man clinical trials can help elucidate possible mechanisms of has demonstrated increased sexual activity in rats after oral action, establish safety, and investigate the efficacy of die-administration of an extract of Ginkgo biloba [14]. Other tary supplement products. medicinal plants, such as Panax ginseng root, are thought to This report summarizes initial research directions inves- enhance circulation through nitric oxide mediated vasodila- tigating the quality, safety, efficacy and potential mecha- tion [15]. Hawthorn (Crataegus spp.) berry is also purported nisms of action of VigRX, an herbal formulation containing to possess vasodilatory effects [16,17]. Ginkgo biloba leaf a variety of botanical ingredients with purported aphrodisiac and Panax ginseng root are known to have antioxidant prop- properties based on traditional use. erties [15]. These and other physiological actions highlight the potential of botanicals to support and/or enhance erectile Research summarized here includes the effects of VigRX on inhibition of Rho-kinase in vitro, and the evaluation of its effect on erectile function, tumescent penis size, and sex Penile erection occurs in response to cavernous smooth drive in the male rat. In addition, investigations were con- muscle relaxation, increased blood flow to the penis, and ducted to assess the purity of VigRX by analyzing the batch restriction of venous outflow. These events are regulated by used in the aforementioned studies for the presence or ab- a spinal reflex relying on visual, imaginative, and olfactory sence of pharmaceutical adulterants. stimuli generated within the central nervous system (CNS) and on tactile stimuli to the penis. Pharmaceutical or botani- 2. MATERIALS AND METHODS cal drugs can have a stimulatory or inhibitory effect either on 2.1. Test Article the nerves regulating this reflex or directly on the cavernous smooth muscle within the penis. A balance between contrac- Al five of the studies described used VigRX (Leading tile and relaxant factors governs penile flaccidity/rigidity. Edge Herbals, Greeley, Colorado, USA, Lot #4242) as the test article. VigRX is made up of a proprietary blend of Of the numerous biochemical reactions involved in regu- Panax ginseng root, Saw palmetto berry powder (Serenoa lating penile smooth muscle tone, the phosphorylation state repens), Gingko biloba leaf powder, Hawthorn berry of myosin fibrils is of central importance. When phosphory- (Crataegus laevigata), Muira Puama bark extract 4:1 lated, myosin induces the contraction of smooth muscle, (Ptychopetalum olacoides), Catuaba bark extract 4:1 powder leading to vasoconstriction and penile detumescence. The (Erythroxylum catuaba), Cuscuta seed extract 4:1 (Cuscuta opposite effect is seen when myosin is dephosphorylated. chinensis), and Epimedium sagittatum extract 20:1. The phosphorylation state of myosin is control ed by the enzyme myosin phosphatase, which is active in its dephos- 2.2. Assays for Pharmaceutical Adulteration phorylated form. Rho-kinase is an enzyme that phosphory- 2.2.1. Assay for PDE-5 Inhibitor Analogues lates myosin phosphatase, thereby inactivating it. Inhibition of Rho-kinase thus keeps myosin phosphatase in an active VigRX was tested for the presence of PDE-5 inhibitors state, leading to the dephosphorylation of myosin and conse- and their analogues by Flora Research Laboratories (Grants quently the relaxation of smooth muscle. Smooth muscle Pass, Oregon, USA). Samples were prepared by mixing the relaxation in turn results in increased blood flow and turgid- contents of 20 VigRX capsules. The material was then ity of penile tissue [18]. sieved via a 40 mesh sieve and blended on lab paper. 0.5 grams of the sample was then weighed into a 100 mL volu- 12 The Open Natural Products Journal, 2010, Volume 3 Smitasiri et al. metric flask and 80 mL of a 1:1 mixture of acetonitrile Fol owing data acquisition, the data were evaluated in the (Chromasolve®, Sigma-Aldrich®) and water (Type 1, gener- software with MS/MS ion trigger masses sorted by weight. If ated in house using Easy Pure®, Barnstead, Dubuque, any masses were identified that matched up to the PDE-5 Iowa, USA ) was added to the flask and swirled to wet the inhibitors or known analogues (based on the FDA FCC contents. The flask was then shaken for 15 minutes on a Bur- Compound List), the ion was selected and the product spec- rel Wrist Action ® shaker fol owed by sonication at room trum ions were evaluated against the list. If al ions were temperature for 15 minutes. After al owing the contents to present in the relative ratios, the compound was considered cool to room temperature, the flask was diluted to volume, preliminary positive. A second HPLC-MS/MS run was set mixed and approximately 10 mL was transferred to a 15 mL up for that exact compound and used for confirmation spec- BD Falcon polypropylene centrifuge tube (PPCT). This ali- tra in order to obtain an enhanced spectra for the exact com- quot was centrifuged at 3500 rpm for 15 minutes (Beckman pound. If this too was positive, then the sample was deter- GS-6). The supernatant was syringe filtered via a 0.45 mi- mined to be adulterated with that compound. cron 25 mm diameter polytetrafluoroethylene syringe filter, discarding the first few mL and col ecting the remaining 2.2.2. Assay for Active Pharmaceutical Ingredients sample in an amber screw cap vial for analysis. A second A novel assay for inspecting 180 APIs, including ster- aliquot of the test sample was placed into a second 15 mL oids, CNS stimulants, hormones, and narcotics, was per- PPCT as above and spiked with 100 ppm sildenafil for qual- formed by General Standard Laboratory (Taipei, Taiwan) ity control purposes. For HPLC analysis, an aliquot was transferred to an am- The standard solution was prepared by weighing 8 mg of ber HPLC autosampler vial with snap cap (National Scien- each standard API and dissolving it in 4 mL of methanol tific). For HPLC-PDA-MSn analysis, 25 !L was transferred (ChromAR®, LC grade, Mal inckrodt Chemicals) to make to the same type of vial and diluted to 1 mL using the 50% stock solution with the concentration of 2.0 mg/mL. The acetonitrile extraction solvent. stock solution was further diluted with 100X methanol to Two separate screening methods were utilized to exam- reach a concentration of 0.02 mg/ml. A quality control stan- ine the product for PDE-5 inhibitors. Initial y, analysis was dard was also prepared by weighing 8 mg of each standard carried out using an HP100 HPLC system with a diode array pharmaceutical ingredient and dissolving it in 4 mL of detector (Agilent). The separation was performed using a methanol to reach the concentration of 2.0 mg/mL. Quality Zorbax Eclipse XDB-Phenyl 4.5x150 5!m at 40ºC. The mo- control testing using a standard API solution was performed bile phase was a gradient consisting of 10 mM ammonium fol owing every tenth sample tested, in order to measure the acetate (Fluka) in water (A) and acetonitrile (B). The gradi- change in the retention time of the HPLC apparatus (Hitachi ent went from 20% B to 80% B in 20 minutes. Detection was D-700 interface, L-7100 pump, L-7200 autosampler, L-7455 carried out at 230 nm and 290 nm and peak spectra were diode array detector). col ected as wel . This method elutes the bulk of the botani- To prepare the test material, the content of ten VigRX cal phytochemicals early near the void volume al owing the capsules was emptied and wel mixed. Subsequently, 0.5 g of PDE-5 inhibitor drugs to elute later in the middle or end of the sample was measured, added to 5.0 mL of methanol and the run. Spectra were acquired for the prescription PDE-5 vortexed. The sample solution was then sonicated for 15 inhibitors and sildenafil was used as a qualitative spike to determine run performance and chromatogram alignment. minutes and centrifuged for 10 minutes at 3000 rpm. Liquid Spectra were compared to the parent approved prescription from the upper layer of the test tube was col ected and di-PDE-5 inhibitors and to published spectra for known ana- luted with 5X methanol. logues. Two criteria were used to establish adulteration. The analysis column used was Supelco Ascentis C18 First, in almost all adulterated products tested, there was a (5!m 25cm"4.6mm) (Sigma-Aldrich ®). The injection vol- major peak eluting in the PDE-5 inhibitor window indicating ume was 20 !L. Data acquisition was preformed by Hitachi a pharmaceutical dosage level. The second criterion was the D-7000 HPLC System Management (Suzuka, Japan). presence of a spectra matching or related to the known PDE- Identification of pharmaceutical adulterants present in the 5 inhibitors and their analogues. sample was made by comparing the UV spectrum of each A second analysis was carried out using a Varian 500 sample peak above 0.05 absorbance units at 200 nm with the Ion-Trap LC-MS system equipped with TurboDDS (Varian). spectral library of the standard APIs. A second validation The system was also equipped with a Varian 335 Prostar was performed when the retention time of sample peaks dif- PDA Detector to col ect PDA spectra during the runs. The fered by ±10% and the similarity of the sample spectrum column used was a Varian Pursuit XRs 3u C-18 150x2 at with the standard library reached above 92%. 35ºC. The mobile phase was a gradient consisting of Type 1 water containing 0.1% formic acid (EMD Chemicals) (A) The criteria for positive identification of pharmaceutical and acetonitrile containing 0.1% formic acid (B). The gradi- adulteration was: a) the result showed only peaks of the sus- ent was 20% B to 80% B over 20 minutes. The separation picious adulterants; b) the concentration of a suspicious peak al owed for phytochemical constituents to elute across the was increased; and, c) the similarity of the UV spectrum run time and thus required careful analysis of the PDA spec- between a suspicious peak and standard library exceeded tra and MS data. The TurboDDS mode was set to acquire MS/MS spectra on the 3 largest ions detected in a given mi- 2.3. Rho-Kinase II Enzyme Inhibition Assay croscan. The largest product ion in any given MS/MS spectra was further fragmented to provide MS3 spectra. The instru- The Rho-kinase enzyme inhibition assay was conducted ment was operated in ESI(+) mode. by Natural Remedies (Bangalore, India). This assay is based An Initial Evaluation of the Safety, Efficacy and Purity of VigRX The Open Natural Products Journal, 2010, Volume 3 1 3 on ELISA and operates according to the fol owing princi- with proper ventilation and a daily light-dark cycle of 12 ples. Plates are pre-coated with a substrate corresponding to hours. Al of the rats were fed rodent feed Number 082 the recombinant C terminus of the myosin–binding subunit (Pokkapan Animal Feed Co. Ltd.). Water was provided to (MBS) of myosin phosphatase. The threonine residue of the the rats ad libitum during the entire period of the study. The MBS is phosphorylated by members of the myotonic dystro- university's board authorized to regulate animal experiments phy protein kinase family, including Rho-kinase. The detec- approved al experiments conducted. tor antibody AF20 specifical y detects only the phosphory- 2.4.2. Preparation of Test Material lated form of threonine-696 on MBS. The amount of phos-phorylated substrate is measured by binding it with an anti- The contents of the VigRX capsules (Lot #4242) were phospho-MBS threonine-696 specific antibody conjugated mixed with distil ed water and prepared into two dosages to with horseradish peroxidase, which then catalyzed the con- be administered at 15 and 30 mg/kg/day. These dosages were version of the chromogenic substrate tetramethylbenzidine chosen to al ometrical y reflect the current recommended from a colorless solution to a blue solution (or yel ow after doses of VigRX in adult human males (2 or 4 capsules per addition of stopping solution). The color is quantified by day), using an average body weight of 70 kg. spectrophotometry and reflects the relative amount of Rho- 2.4.3. 14-Day Study in Sprague-Dawley Rats kinase activity. A Rho-kinase assay kit CY-1160 (Cyclex Co. Ltd., Na- The rats were divided into 3 groups (10 rats/group): gano, Japan) was used. The components of the kit included Group 1 received 1 mL/day of distil ed water administered microplate-wel s coated with recombinant MBS C terminus, oral y for 14 consecutive days. Group 2 and 3 were similar to 10X wash buffer, kinase buffer, 20X ATP, HRP conjugated the control group, but received VigRX at either 15 or 30 detection antibody, substrate solution, stop solution, Rho- mg/kg/day respectively for 14 consecutive days. Each rat kinase II (CY-E 1160-1, Cyclex Co.), and Rho-kinase spe- was weighed every three days until the test was completed cific inhibitor Y-27632 (Calbiochem, cat. no. 688001). on Day 14. On Day 14, between 7–9 p.m., the sexual behav- ior of each adult male rat exposed to a female rat in-estrus Two grams of VigRX were sonicated for 8 minutes in 20 was observed. (Female rats were induced into estrus with mL of methanol. Ultra-pure water was added to attain a total subcutaneous doses of estradiol benzoate and progesterone volume of 100 mL of solution. The solution was filtered and as described by Islam et al. [26]). Each adult male rat was the resulting filtrate was used for the assay. It was found that placed into an individual glass cupboard and a dim light was the VigRX capsule content was 19.3% soluble (385.94 mg turned on for 5 minutes prior to the start of observation. Sub- was soluble out of 2000 mg). For calculation purposes, the sequently, the female rats in estrus were caged with male rats stock solution was considered to be 20 mg/mL. at a ratio of 1:1. Sexual behavior of the male rat was ob- The assay was carried out as fol ows: a total reaction served and recorded with a video recorder for 30 minutes. mixture of 100 ! L, containing kinase reaction buffer, vary- The latency and frequency of mounting, intromission and ing concentrations of VigRX (125, 250, 500, 1000, and 2000 ejaculation were then assessed. Statistical analysis was per-!g/mL) or Y-27632 (the positive control), 10 munits of Rho- formed using ANOVA and LSD. kinase II enzyme, and 100 ! M of ATP, was combined and On Day 15, after weighing, the erect penis was measured incubated at 30°C for 30 minutes in a substrate coated plate. per the fol owing protocol. Each rat was put in supine posi-Fol owing incubation, the contents of the wel s were dis- tion, the anterior portion of its body was inserted into a plas- carded and washed 200 !L X 5 times with 1X wash buffer. tic cylinder, and the trunk, limbs and tail were restrained. 100 ! L of HRP conjugated detection antibody was added The penile sheath was retracted to expose the glans penis, and incubated at 25°C for 60 minutes. The contents were and this position was maintained using forceps. Light tactile discarded and the wel s were washed 200!L X 5 times with stimulation was applied to the base of the glans penis for 1–2 1X wash buffer. 100 ! L of substrate solution was added and minutes until penis became ful y erect. The width of the base incubated at 25°C for 15 minutes. Final y 100 !L of stop of the glans penis and the length between the tip of glans solution was added and the absorbance was measured at 450 penis and base of penis were then measured. Subsequently, nm in a spectrophotometer (Versamax microplate reader, the rat was anesthetized with an intraperitoneal injection of Molecular Devices). The % inhibition was calculated as fol- Nembutal (pentobarbital), at which point the intracavernous lows: % inhibition = {[Absorbance of control –Absorbance pressure (ICP) was recorded using the method described by of test] / Absorbance of control} X 100. The IC Tocharus et al. [27]. Blood was then col ected via cardiac lated using log-probit analysis. puncture and testosterone levels were measured using elec-trochemical luminescence. The animals were euthanized and 2.4. Aphrodisiac Activity Studies in Male Sprague- the penis, testes, epididymis, prostate gland, seminal vesicle, pituitary gland, adrenal gland, liver, kidney and spleen were removed and weighed (Mettler Toledo AB 204-S). After 2.4.1. Test Animals weighing the epididymis, the cauda epididymis was removed Sprague-Dawley adult male (250–280 g) and female rats and the sperm concentration was counted using a Neubauer (200–240 g) were used in this study. Al of the rats were hemacytometer. A histopathological examination was con- sourced from the National Laboratory Animal Center at Ma- ducted on all organs that demonstrated a weight change. hidol University, Nakhon Pathom Province, Thailand. They 2.4.4. 12-Week Study in Sprague-Dawley Rat were transferred to Mae Fah Luang University, Chiang Rai, Thailand, by air and reared in the Laboratory Animal House The rats were divided into two groups consisting of in a temperature-control ed room (approximately 24 ±1°C) twelve rats each. Group 1 served as the control and received 14 The Open Natural Products Journal, 2010, Volume 3 Smitasiri et al. 1 mL of distil ed water administered oral y each day for 84 Table 1. List of PDE-5 Inhibitor Drugs and Known Ana- consecutive days (12 weeks). Group 2 received VigRX ad- logues Screened for (Detection Limit= 100 ppm) ministered oral y at the dosage of 15 mg/kg/day for 84 con-secutive days (12 weeks). Each rat was weighed every seven PDE-5 Inhibitor days until the test was completed. On Day 28 (4 weeks), 56 (8 weeks) and 84 (12 weeks), between 7–9 p.m., the sexual behavior of each adult male rat exposed to a female rat in estrus was observed in the same manner as in the 14-day study reported above. Statistical analysis was performed us- ing ANOVA and LSD. Dimethyl sildenafil (aildenafil) On Day 85, after weighing, erect penile size was meas- Dimethyl sildenafil thione (sulfoaildenafil) ured as in the 14-day study. The rats were then anaesthetized and the intracavernous pressure (ICP) was recorded. Blood was col ected via cardiac puncture to measure testosterone Hydroxyacetildenafil levels (using electrochemical luminescence), and clinical blood chemistry parameters including aspartate aminotrans- Hydroxyhomosildenafil ferase (AST), alanine aminotransferase (ALT), alkaline Noracetildenafil phosphatase, creatinine, blood urea nitrogen (BUN), choles-terol, triglycerides, total protein, albumin, and glucose were Piperadino acetildenafil assessed. The animals were subsequently euthanized and the Piperadino vardenafil penis, testes, epididymis, prostate gland, seminal vesicle, pituitary gland, adrenal gland, liver, kidney and spleen were removed and weighed. After weighing, the epididymis and Sildenafil thione (sulfosildenafil) the cauda epididymis were removed and the sperm concen-tration was counted using a Neubauer hemacytometer in the Sildenafil thione (sulfohomosildenafil) same manner as the 14-day study. The liver, kidney and tes-tes were sectioned for histopathological studies. The liver was specifical y examined for fatty degeneration, hepatocyte megalocytosis, lymphoid aggregated periportal area, bile ND=not detected. duct proliferation and peliosis hepatitis. The kidneys were specifical y examined for multifocal tubular cysts, tubular 3.3. Aphrodisiac Activity Study in Male Sprague-Dawley casts and tubulonephrosis. The testes were specifical y ex- amined for signs of interstitial edema, seminiferous tubule degeneration and congestion. Al col ected data was statisti- 3.3.1. 14-Day Study cal y analyzed using ANOVA and LSD. Male rats fed VigRX at a dose of 30 mg/kg/day for 14 consecutive days experienced a significant decrease in in- tromission latency and ejaculation latency and an increase in 3.1. Assays for Product Adulteration with PDE-5 Inhibi- intromission frequency, ejaculation frequency and mounting tor Analogues and APIs frequency as compared to the control group (p <0.05) (Table 3). Rats fed VigRX at 30 mg/kg/day also experienced a sta- VigRX was found to contain no detectable levels of tistical y significant increase in the length and width of their known PDE-5 inhibitors including sildenafil, tadalafil, erect penis when compared with the control group (p <0.05), vardenafil or related analogues known at the time of the whereas the rats fed VigRX at 15 mg/kg/day experienced an study (Table 1). increase only in the length of the erect penis (p <0.05). Both A separate assay determined that VigRX was free from dosages of VigRX were shown to significantly increase the adulteration with any of the 180 tested APIs representing the intracavernosal pressure (ICP) compared to control group (p fol owing 19 drug categories: steroids, diuretics, gastrointes- <0.001). An increase in sperm concentration was noted only tinal drugs, CNS stimulants, local anesthetics, antilipemic in the group treated with VigRX at 30 mg/kg/day (p <0.05). drugs, antibiotics, CNS depressants, muscle relaxants, However, no significant difference was seen in the testoster- antigout drugs, hormone drugs, antidiabetics, analge- one level in either the VigRX treated groups or the control sics/NSAIDs, bronchodilators, abused drugs, narcotics, anti- group. histamines, cardiovascular drugs, and antitussives/expec- There was no significant difference between the body weight gained of the VigRX-treated groups and the control 3.2. Rho-Kinase II Enzyme Inhibition Assay group (Table 4). The organ weights of both treatment groups did not significantly differ from the control group after 14 VigRX exhibited concentration-dependant inhibitory days, with the exception of a decrease in liver weight experi- effects in the Rho-kinase assay with a fifty percent inhibitory enced by both the 15 and 30 mg/kg/day treatment groups and concentration (IC50) of 1673.18 !g/mL (95% confidence a decrease in kidney weight experienced only by the 15 interval of 1216.28–2710.01 !g/mL) (Table 2). mg/kg/day group (p <0.01). Neither of these decreases was An Initial Evaluation of the Safety, Efficacy and Purity of VigRX The Open Natural Products Journal, 2010, Volume 3 1 5 Table 2. Rho-Kinase II Inhibition Assay Percent Inhibition (mean ±SD) (95% confidence interval) Rho-kinase specific inhibitor (Y-27632) (1216.28–2710.01) Table 3. Sexual Behavior in Rat Studies Sexual Behavior Paramater 2-week (14-day) study 1474.00 ±149.17* Intracavernous pressure (mmHg) Sperm Density (x107/mL) *p <0.05 **p <0.01 ***p <0.001. aTime interval between introduction of the female and first mount by the male. bTime interval between introduction of the female and first intromission by the male. cTime interval between introduction of the female and first ejaculation by the male. dNumber of mounts with intromission from the time of introduction of the female until ejaculation. eNumber of intromissions from the time of introduction of the female until ejaculation. fNumber of ejaculations (characterized by longer, deeper pelvic thrusting and slow dismount fol owed by a period of inac tivity).
observed to be dose-dependent, and histopathological ex- The VigRX-treated animals in this study experienced amination of the liver and kidney revealed no differences significant increases in sperm concentration, width of the between treatment and control groups. erect penis, and intracavernosal pressure compared to control 3.3.2. 12-Week Study animals. Mean sperm concentration values after 12 weeks were 24.57 ±3.64 x 107/mL in the VigRX treated animals The ejaculation latency of the rats treated with a VigRX while the values were 20.41 ±3.30 x 107/mL in the control dose of 15 mg/kg/day was significantly less than the control group (p <0.01). The mean values for erect penile width group after 12 weeks of treatment (p <0.05) (Table 3). No were 6.38 ±0.43 mm in the VigRX treated group and 5.81 other significant differences in sexual behavior were noted in ±0.40 mm in the control group (p <0.01). The mean intra-male rats treated with VigRX for 4, 8 or 12 weeks when cavernosal pressure measurement in the VigRX treated compared to controls. 16 The Open Natural Products Journal, 2010, Volume 3 Smitasiri et al. Table 4. Body and Organ Weights in Rat Studies 2-week (14-day) study Epididymis (mg%) Seminal vesicle (mg%) Prostate gland (mg%) 4010.33 ±498.80 3512.82 ±197.19** 3552.43 ±220.40** 3271.31 ±167.72 3268.14 ±180.61 664.68 ±24.80** Adrenal gland (mg%) Pituitary gland (mg%) group was 85.75 ±6.14 mm Hg, while in the control group the best way to determine that no adulteration occurred dur- the mean value was 58.08 ±4.60 mm Hg (p <0.05). ing any step of the manufacturing process. Not only does this Treatment with VigRX for 12 weeks significantly in- protect consumers from unexpected side-effects and drug- creased levels of testosterone compared to the control (p interactions, it also assures that any treatment effects ob- <0.05) (Table 5). Mean serum testosterone levels in the served clinical y are due to the product's stated formulation VigRX treated rats were 6.20 ±2.57 ng/mL after 12 weeks of and not to unnamed and unlisted pharmaceutical agents. treatment while mean levels in the control group were 3.66 While there are a number of dietary supplements on the market making claims about erectile function and sexual There were no significant differences in weight gained in health, most have yet to be substantiated by scientific re- the male rats treated with VigRX daily for 12 weeks com- search performed on the end product. pared to the control group (Table 4). VigRX had no effect on The Rho-kinase assay can help reveal one of the potential the weights of any organs measured, including the penis, mechanisms of action behind the efficacy of an erectile testes, epididymis, seminal vesicles, prostate gland, liver, health product. In this study, VigRX was shown to have an kidney, adrenal gland, spleen and pituitary glands. There IC50 of 1673.18 !g/mL. While this result shows an inhibitory were also no significant histopathological findings in either action against the Rho-kinase enzyme in vitro, the relatively the control or treatment groups. high IC50 suggests that a large dose would be necessary to VigRX had no effect on any other blood chemistries or achieve similar results in a living system. This may imply blood parameters measured, including those of glucose, that, while inhibition of the Rho-kinase enzyme is a possible BUN, creatinine, cholesterol, triglycerides, AST, ALT, alka- partial explanation for the activity of VigRX, other mecha- line phosphatase, total protein, or albumin, with the excep- nisms may also be at play. Future research into the effect of tion of a statistical y significant decrease in lymphocytes VigRX on other aspects of erectile health, such as the nitric found in the treatment group (Table 5). However, because no oxide pathway, may help further explain its efficacy as ob- other clinical y significant findings accompanied this result, served in the rat. including an absence of histopathological abnormalities, the The 14-day study revealed that the rats fed VigRX at the decrease in lymphocytes may be an incidental finding and dosage of 30 mg/kg/day showed an increased sex drive as not related to the treatment article. reflected by the increased frequency of every measured pa-rameter, including mounting, intromission, and ejaculation frequencies, as wel as short intromission and ejaculation Two separate analyses from different labs determined latencies during the observed 30-minutes of sexual behavior. that VigRX (Lot # 4242) was not adulterated with any of the In addition to increased sex drive, VigRX treatment also tested pharmaceutical ingredients, including drugs com- increased the erect penile size, the intracavernosal pressure monly used for the treatment of erectile dysfunction or their and sperm concentration, without affecting testosterone lev-analogues. Performing these analyses on the end product is els. One hypothesis for the increased sperm concentration is An Initial Evaluation of the Safety, Efficacy and Purity of VigRX The Open Natural Products Journal, 2010, Volume 3 1 7 Table 5. Blood Parameters in Rat Studies 2-week (14-day) study Blood Urea Nitrogen (BUN) (mg/dL) Creatine (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/dL) Aspartate aminotransferase (AST) (U/L) Alanine Aminotransferase (ALT) (U/L) Alkaline phosphatase (U/L) White blood cel (cel s/mm3) 2436.36 ±490.45 2522.22 ±618.02 Hemoglobin (g/dL) Red blood cel (mil /!L) Platelet (thsnd/!L) Polymorphonuclear granular leucocyte (%) Mean corpuscular volume (fL) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentra- Testosterone (ng/mL) NP = not performed. that VigRX treatment leads to an increased blood flow to the ing behavior, blood flow, and other demands of increased testes and consequently increases spermatogenesis. Herbs sex drive experienced by the rats treated with VigRX created found in VigRX that have demonstrated the ability to in- an increased energy need, thus increasing the rate of glyco- crease circulation include hawthorn berry and ginkgo leaf genolysis in the liver. Further investigation of this hypothesis [15,16]. However, no research has been published specifi- is needed. However, the decrease in liver and kidney sizes cal y on the effect of these herbs on testicular blood flow or were not found to be dose dependent. This, coupled with the spermatogenesis. relatively smal number of test animals, makes it difficult to VigRX had no effect on the weights of the majority of determine whether this finding was due to a treatment effect organs measured in the 14-day study, with the exception of a of VigRX or spontaneous findings in individual test animals. statistical y significant decrease in liver weight (both treat- Unlike the 14-day study, the 12-week study on VigRX ment groups) and kidney weight (15 mg/kg group only). The found no significant effect on the sexual behavior of male mechanisms by which VigRX could decrease the liver and rats. However, this study did corroborate the earlier findings kidney weight are stil unknown. One possible explanation of increased sperm concentration, width of erect penis (but for the decrease in liver weight is that the increased mount- not length), and intracavernosal pressure. Because the 12- 18 The Open Natural Products Journal, 2010, Volume 3 Smitasiri et al. week study only examined the effects of 15 mg/kg/day, = active pharmaceutical ingredients while the 14-day study found 30 mg/kg/day to be the more = alanine aminotransferase effective dose, perhaps it is the lower dose that could ac-count for the decreased treatment response in the 12-week AST = aspartate aminotransferase = adenosine triphosphate The 12-week study did not show the decreases in liver = blood urea nitrogen and kidney weights found in the 14-day study, and histopa-thological examination revealed no abnormalities in any or- = central nervous system gans. This may lend further credence to the hypothesis that eNOS = endothelial nitric-oxide synthase the initial findings in the shorter study were sporadic and not treatment related. = enzyme-linked-immunosorbent serologic Two significant hematological findings were noted in the 12-week study. The first was an increase in testosterone, = electrospray ionization which was not found in the 14-day study. While there were no concomitant increases in prostate or seminal vesicle weight, this finding warrants further investigation into the = high performance liquid chromatography long-term effects of VigRX on prostate health. Recent long- = high performance liquid chromatography- term studies in humans have questioned whether elevated photodiode array-mass spectrometry testosterone levels contribute to adverse prostate health or an increased risk of cancer, hence the clinical significance of = horseradish peroxidase elevated testosterone remains unknow[n 2 8,29]. A retrospec- = 50% inhibitory concentration tive analysis of numerous studies found no causal relation- = intracavernous pressure ship between testosterone replacement and prostate cancer or heart disease risk, nor did another study that reviewed decades = liquid chromatography-mass spectrometry of research find compel ing evidence that higher than normal LSD = least significant difference testosterone levels increased the risk of prostate cancer or cardiovascular disease [28,2 9].
= myosin–binding subunit The second statistical y significant hematological finding mg was a decrease in lymphocyte production. However, because mL no other clinical y significant findings accompanied this re-sult, including histopathological abnormalities, the decrease mm in lymphocytes may be an incidental finding, not related to the treatment article. No other blood parameter measured differed significantly from control. = mass spectrometry Overal this research provides scientific evidence in sup- port of the purity, safety, and potential efficacy of this herbal NO formulation in supporting male sexual health. Inhibition of NSAIDs = non-steroidal anti-inflammatory drugs the enzyme Rho-kinase, and increases in serum testosterone are two possible mechanisms of action for this formula. It is PDA = photodiode array likely that additional mechanisms play a role in the efficacy = phosphodiesterase type 5 as seen in the 14-day and 12-week rat studies, which require further investigation. Research exploring other physiological PPCT = polypropylene centrifuge tube effects, as wel as human clinical trials, are needed to better ppm = parts per mil ion understand the role VigRX may play in supporting and/or enhancing male sexual function. Additional in vivo toxicol- = revolutions per minute ogy studies would be beneficial to more ful y evaluate the safety of long-term consumption of this product. CONFLICT OF INTEREST STATEMENT Lewis, R.W.; Fugl-Meyer, K.S.; Bosch, R.; Fugl-Meyer, A.R.; AIBMR Life Sciences, Inc. was hired as an independent Laumann, E.O.; Lizza, E.; Martin-Morales, A. Epidemiology/risk consulting agency to design and implement studies on factors of sexual dysfunction. J Sex Med 2004, 1(1), 35–39. Laumann, E.O.; Paik, A.; Rosen R.C. Sexual dysfunction in the VigRX. None of the authors have any financial interest in United States: prevalence and predictors. JAMA 1999, 281(6), 537– Dean, R.C.; Lue, T.F. Physiology of penile erection and patho- physiology of erectile dysfunction. Urol Clin North Am 2005, 32(4), 379–95, v. Vlachopoulos, C.; Aznaouridis, K.; Ioakeimidis, N.; Rokkas, K.; Vasiliadou, C.; Alexopoulos, N.; Stefanadi, E.; Askitis, A.; Stefa-nadis, C. Unfavorable endothelial and inflammatory state in erectile = analysis of variance An Initial Evaluation of the Safety, Efficacy and Purity of VigRX The Open Natural Products Journal, 2010, Volume 3 1 9 dysfunction patients with or without coronary artery disease. Eur Vierling, W.; Brand, N.; Gaedcke, F.; Sensch, K.H.; Schneider, E.; Heart J 2006, 27(22), 2640–8. Scholz, M. Investigation of the pharmaceutical and pharmacologi- Jin, L.; Liu, T.; Lagoda, G.A.; Champion, H.C.; Bivalacqua, T.J.; cal equivalence of different Hawthorn extracts. Phytomedicine Burnett, A.L.; Elevated RhoA/Rho-kinase activity in the aged rat 2003, 10(1), 8–16. penis: mechanism for age-associated erectile dysfunction. Faseb J [18] Steers, W.D. Pharmacologic treatment of erectile dysfunction. Rev 2006, 20(3), 536–8. Urol 2002, 4 Suppl 3, S17–25. Haas, C.A.; Seftel, A.D.; Razmjouei, K.; Ganz, M.B.; Hampel, N.; Rajasekaran, M.; White, S.; Baquir, A.; Wilkes, N. Rho-kinase Ferguson, K. Erectile dysfunction in aging: upregulation of endo- inhibition improves erectile function in aging male Brown-Norway thelial nitric oxide synthase. Urology 1998, 51(3), 516–22. rats. J Androl 2005, 26(2), 182–8. United States Food and Drug Administration, FDA News. FDA Quinlan, D.M.; Nelson, R.J.; Partin, A.W.; Mostwin, J.L.; Walsh, Warns Consumers Not to Use Super Shangai, Strong Testis, Shan- P.C. The rat as a model for the study of penile erection. J Urol gai Ultra, Shangai Ultra X, Lady Shangai, and Shangai Regular 1989, 141(3), 656–61. Ang, H.H.; Ngai, T.H. Aphrodisiac evaluation in non-copulator male rats after chronic administration of Eurycoma longifolia Jack. cessed 13 November 2008). Fundam Clin Pharmacol 2001, 15(4), 265–8. 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McKenna, D.J.; Jones, K.; Hughes, K.; Humphrey, S. Botanical Medicines: The Desk Reference for Major Herbal Supplements, 2nd ed. The Haworth Herbal Press: New York, 20;0 Received: December 10, 2009 Revised: January 19, 2010 Accepted: March 04, 2010 Smitasiri et al.; Licensee Bentham Open. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


Download the original attachment Criminal Charges concerning Bioterrorism Acts and Mass Submitted: FBI OFFICER EMBASSY OF THE UNITED STATES Boltzmanngasse 16 A-1090 Vienna Date: June 10, 2009 I. Introduction: Summary of Claims II. Factual Background III. Evidence the "swine flu" vaccines are bioweapons IV. Scientific evidence the "swine flu" virus is an