10The Open Natural Products Journal, 2010, 3, 10-19

Open Access

An Initial Evaluation of the Safety, Efficacy and Purity of VigRX, an
Herbal Combination Formula, for the Enhancement of Male Sexual HealthY. Smitasiri1, P. D’Souza2, J. Neal-Kababick3 and A.G. Schauss*,41

Mae Fah Luang University, Muang District, Chiang Rai 57100, Thailand

2

Natural Remedies, Plot 5B, 19th K. M Stone, Hosur Road, Bangalore 560 100, India

3

Flora Research Laboratories, 1000 SE M Street, Grants Pass, Oregon 97526, USA

4

Natural and Medicinal Products Research, AIBMR Life Sciences, Inc., 4117 S. Meridian, Puyallup, Washington 98373,
USA
Abstract: An herbal combination formula, known as VigRX, has been studied for purity, safety and for efficacy in a
Sprague-Dawley rat model. Two separate assays determined that VigRX was free from pharmaceutical adulterants, including phosphodiesterase type 5 (PDE-5) inhibitors and related analogues. An in vitro assay determined that VigRX isable 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 statistically 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.
1. INTRODUCTION
Age-related decline in sexual function is a noted phenomenon worldwide. The prevalence in adult men has been
estimated at 20–30% [1,2]. Men aged 50–59 experienced
three times more problems related to erections and low sexual desire than their 18–29 year old counterparts [2]. The
pathophysiology of erectile dysfunction, a commonly experienced aspect of male sexual dysfunction, can include arteriogenic, neurogenic, endocrinologic, or psychogenic factors[3]. Multiple mechanisms may account for changes in thesesystems, including increased RhoA/Rho-kinase activity, impaired function of the nitric oxide (NO) and endothelial nitric-oxide synthase (eNOS) systems, and increased levels of
inflammatory and prothrombotic vascular endothelial compounds [4-6].
Men have long sought remedies for declining sexual
health. While prescription medications are often used, men
are increasingly turning to herbal alternatives. The dietary
supplement market includes a large number of products
claiming to benefit men seeking enhancement of their*Address correspondence to this author at the Natural and Medicinal Products Research, AIBMR Life Sciences, Inc., 4117 S. Meridian, Puyallup,
Washington 98373, USA; Tel: +1-253-286-2888; Fax: +1-253-286-2451;
E-mail: alex@aibmr.com
1874-8481/10

erectile function, sexual health and performance. While theconsumer may assume these “natural” products are safe, thiscategory of dietary supplements is marred with numerous
reports of adulteration with active pharmaceutical ingredients (APIs) [7]. Some APIs commonly discovered in thesemisleading supplements include phosphodiesterase (PDE-5)
inhibitors such as sildenafil (Viagra®), vardenafil (Levitra®),and tadalafil (Cialis®), or their analogues. Because these prescription drugs are used as effective treatments for erectile
dysfunction, their inclusion in herbal formulas may enhancethe efficacy of these formulas, misleading consumers to attribute positive results to the listed combination of vitamins
and herbs in the ingredients rather than the effect of thepharmaceutical adulterant. Furthermore, not disclosing thepresence of the API adulterants on the labeling of theseproducts puts consumers at considerable risk, including exposure to side effects such as flushing, headaches, palpitation, nasal congestion, and visual disturbances [8,9]. Of evengreater concern is the possibility of these pharmaceuticaladulterants interacting with other medications the consumer
could be taking [10]. For instance, the combination of nitroglycerine and PDE-5 inhibitors or their analogues can dangerously lower blood pressure. This is of particular relevancebecause the population that is prescribed nitrates also commonly suffers from erectile dysfunction. While doctors generally warn patients on nitrates against taking pharmaceutical2010 Bentham OpenAn Initial Evaluation of the Safety, Efficacy and Purity of VigRX

PDE-5 inhibitors, these individuals may turn to dietary supplements as “natural” alternatives to treating their erectile
dysfunction [7,11].
Unfortunately, there is evidence that this illegal practiceof including undisclosed APIs in dietary supplements is
widespread [11-13]. This adulteration can occur at any stepof the manufacturing process, and may be unknown to thecompanies who are marketing and selling the product.
Therefore, conscientious dietary supplement manufacturers
should test every batch of finished products for APIs to assure no adulteration took place during any step of manufacturing. This serves to ensure the quality and identity of the
product being consumed as well as heighten the potential
level of safety of the formulation.
Long before the advent of synthetic pharmaceuticals forthe treatment of sexual dysfunction, herbal medications havebeen used as sexual health supportive agents in diverse cultures throughout the world. While the traditional use of suchplants is extensive, recent scientific investigations into individual herbs and combination formulas have yielded important positive data in terms of the ability of botanicals to support sexual wellness and the erectile process. There are many
medicinal plants that have been reported to possess aphrodisiac effects, such as Muira Puama (Ptychopetalum spp.) rootand Catuaba (Trichilia catigua) bark [13]. Recent researchhas demonstrated increased sexual activity in rats after oral
administration of an extract of Ginkgo biloba [14]. Other
medicinal plants, such as Panax ginseng root, are thought toenhance circulation through nitric oxide mediated vasodilation [15]. Hawthorn (Crataegus spp.) berry is also purported
to possess vasodilatory effects [16,17]. Ginkgo biloba leaf
and Panax ginseng root are known to have antioxidant properties [15]. These and other physiological actions highlightthe potential of botanicals to support and/or enhance erectile
function.
Penile erection occurs in response to cavernous smoothmuscle relaxation, increased blood flow to the penis, andrestriction of venous outflow. These events are regulated bya spinal reflex relying on visual, imaginative, and olfactorystimuli generated within the central nervous system (CNS)
and on tactile stimuli to the penis. Pharmaceutical or botanical drugs can have a stimulatory or inhibitory effect either on
the nerves regulating this reflex or directly on the cavernoussmooth muscle within the penis. A balance between contractile and relaxant factors governs penile flaccidity/rigidity.
Of the numerous biochemical reactions involved in regulating penile smooth muscle tone, the phosphorylation stateof myosin fibrils is of central importance. When phosphorylated, myosin induces the contraction of smooth muscle,
leading to vasoconstriction and penile detumescence. The
opposite effect is seen when myosin is dephosphorylated.
The phosphorylation state of myosin is controlled by the
enzyme myosin phosphatase, which is active in its dephosphorylated form. Rho-kinase is an enzyme that phosphorylates myosin phosphatase, thereby inactivating it. Inhibitionof Rho-kinase thus keeps myosin phosphatase in an active
state, leading to the dephosphorylation of myosin and consequently the relaxation of smooth muscle. Smooth muscle
relaxation in turn results in increased blood flow and turgidity of penile tissue [18].

The Open Natural Products Journal, 2010, Volume 3

11Thus, agents that inhibit the Rho-kinase enzyme may
have a direct effect on erectile function. This has been demonstrated in vivo following the administration of the selective
Rho-kinase inhibitor Y-27632 to rats [19]. Pharmaceuticals
and botanicals alike are known to possess Rho-kinase inhibitory activity as a means of facilitating the erectile process.
Assays such as the one measuring Rho-kinase inhibition
serve to elucidate potential mechanisms of action whereby
promising compounds could have beneficial effects on aparticular physiological function.
Once an in vitro evaluation has yielded insights into a
possible or likely mechanism of action, a logical next step inthe progression of research is to evaluate the efficacy of a
product in a valid animal model. The rat has been established
as a suitable animal model for the study of penile erection[20], and has been used to study the effects of a number of
medicinal plants on sexual behavior [21-25]. Research in rats
can yield important preliminary information regarding theeffects of a formula on various parameters associated withmale sexual function.
While many marketers of herbal sexual health aids citehistorical precedent as their evidence for efficacy, the needfor scientific investigation into the validity of these claims isnecessary. In vitro assays, animal studies and ultimately human clinical trials can help elucidate possible mechanisms ofaction, establish safety, and investigate the efficacy of dietary supplement products.
This report summarizes initial research directions investigating the quality, safety, efficacy and potential mechanisms of action of VigRX, an herbal formulation containinga variety of botanical ingredients with purported aphrodisiac
properties based on traditional use.
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
drive in the male rat. In addition, investigations were conducted to assess the purity of VigRX by analyzing the batch
used in the aforementioned studies for the presence or absence of pharmaceutical adulterants.
2. MATERIALS AND METHODS
2.1. Test Article
All five of the studies described used VigRX (Leading
Edge Herbals, Greeley, Colorado, USA, Lot #4242) as thetest article. VigRX is made up of a proprietary blend of
Panax ginseng root, Saw palmetto berry powder (Serenoa
repens), Gingko biloba leaf powder, Hawthorn berry(Crataegus laevigata), Muira Puama bark extract 4:1(Ptychopetalum olacoides), Catuaba bark extract 4:1 powder(Erythroxylum catuaba), Cuscuta seed extract 4:1 (Cuscutachinensis), and Epimedium sagittatum extract 20:1.
2.2. Assays for Pharmaceutical Adulteration
2.2.1. Assay for PDE-5 Inhibitor Analogues
VigRX was tested for the presence of PDE-5 inhibitors
and their analogues by Flora Research Laboratories (Grants
Pass, Oregon, USA). Samples were prepared by mixing thecontents of 20 VigRX capsules. The material was thensieved 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(Chromasolve®, Sigma-Aldrich®) and water (Type 1, generated in house using Easy Pure®, Barnstead, Dubuque,Iowa, USA) was added to the flask and swirled to wet thecontents. The flask was then shaken for 15 minutes on a Burrell Wrist Action® shaker followed by sonication at room
temperature for 15 minutes. After allowing the contents tocool to room temperature, the flask was diluted to volume,
mixed and approximately 10 mL was transferred to a 15 mL
BD Falcon polypropylene centrifuge tube (PPCT). This aliquot was centrifuged at 3500 rpm for 15 minutes (BeckmanGS-6). The supernatant was syringe filtered via a 0.45 micron 25 mm diameter polytetrafluoroethylene syringe filter,discarding the first few mL and collecting the remaining
sample in an amber screw cap vial for analysis. A second
aliquot of the test sample was placed into a second 15 mL
PPCT as above and spiked with 100 ppm sildenafil for quality control purposes.

Following data acquisition, the data were evaluated in thesoftware with MS/MS ion trigger masses sorted by weight. If
any masses were identified that matched up to the PDE-5
inhibitors or known analogues (based on the FDA FCC
Compound List), the ion was selected and the product spectrum ions were evaluated against the list. If all ions were
present in the relative ratios, the compound was consideredpreliminary positive. A second HPLC-MS/MS run was set
up for that exact compound and used for confirmation spectra in order to obtain an enhanced spectra for the exact compound. If this too was positive, then the sample was determined to be adulterated with that compound.

For HPLC analysis, an aliquot was transferred to an amber HPLC autosampler vial with snap cap (National Scientific). For HPLC-PDA-MSn analysis, 25 L was transferredto the same type of vial and diluted to 1 mL using the 50%
acetonitrile extraction solvent.

The standard solution was prepared by weighing 8 mg ofeach standard API and dissolving it in 4 mL of methanol(ChromAR®, LC grade, Mallinckrodt Chemicals) to makestock solution with the concentration of 2.0 mg/mL. The
stock solution was further diluted with 100X methanol toreach a concentration of 0.02 mg/ml. A quality control standard was also prepared by weighing 8 mg of each standard
pharmaceutical ingredient and dissolving it in 4 mL of
methanol to reach the concentration of 2.0 mg/mL. Quality
control testing using a standard API solution was performed
following every tenth sample tested, in order to measure thechange in the retention time of the HPLC apparatus (Hitachi
D-700 interface, L-7100 pump, L-7200 autosampler, L-7455
diode array detector).

Two separate screening methods were utilized to examine the product for PDE-5 inhibitors. Initially, analysis was
carried out using an HP100 HPLC system with a diode array
detector (Agilent). The separation was performed using a
Zorbax Eclipse XDB-Phenyl 4.5x150 5m at 40ºC. The mobile phase was a gradient consisting of 10 mM ammonium
acetate (Fluka) in water (A) and acetonitrile (B). The gradient went from 20% B to 80% B in 20 minutes. Detection was
carried out at 230 nm and 290 nm and peak spectra werecollected as well. This method elutes the bulk of the botanical phytochemicals early near the void volume allowing thePDE-5 inhibitor drugs to elute later in the middle or end of
the run. Spectra were acquired for the prescription PDE-5
inhibitors and sildenafil was used as a qualitative spike todetermine run performance and chromatogram alignment.
Spectra were compared to the parent approved prescription
PDE-5 inhibitors and to published spectra for known analogues. Two criteria were used to establish adulteration.
First, in almost all adulterated products tested, there was amajor peak eluting in the PDE-5 inhibitor window indicatinga pharmaceutical dosage level. The second criterion was thepresence of a spectra matching or related to the known PDE5 inhibitors and their analogues.
A second analysis was carried out using a Varian 500
Ion-Trap LC-MS system equipped with TurboDDS (Varian).
The system was also equipped with a Varian 335 Prostar
PDA Detector to collect PDA spectra during the runs. The
column used was a Varian Pursuit XRs 3u C-18 150x2 at
35ºC. The mobile phase was a gradient consisting of Type 1
water containing 0.1% formic acid (EMD Chemicals) (A)and acetonitrile containing 0.1% formic acid (B). The gradient was 20% B to 80% B over 20 minutes. The separation
allowed for phytochemical constituents to elute across therun time and thus required careful analysis of the PDA spectra and MS data. The TurboDDS mode was set to acquire
MS/MS spectra on the 3 largest ions detected in a given microscan. The largest product ion in any given MS/MS spectra
was further fragmented to provide MS3 spectra. The instrument was operated in ESI(+) mode.

2.2.2. Assay for Active Pharmaceutical Ingredients
A novel assay for inspecting 180 APIs, including steroids, CNS stimulants, hormones, and narcotics, was performed by General Standard Laboratory (Taipei, Taiwan)
using HPLC.

To prepare the test material, the content of ten VigRX
capsules was emptied and well mixed. Subsequently, 0.5 g of
the sample was measured, added to 5.0 mL of methanol andvortexed. The sample solution was then sonicated for 15
minutes and centrifuged for 10 minutes at 3000 rpm. Liquidfrom the upper layer of the test tube was collected and diluted with 5X methanol.
The analysis column used was Supelco Ascentis C18(5μm 25cm4.6mm) (Sigma-Aldrich®). The injection volume was 20 L. Data acquisition was preformed by Hitachi
D-7000 HPLC System Management (Suzuka, Japan).
Identification of pharmaceutical adulterants present in thesample was made by comparing the UV spectrum of eachsample peak above 0.05 absorbance units at 200 nm with thespectral library of the standard APIs. A second validation
was performed when the retention time of sample peaks differed by ±10% and the similarity of the sample spectrumwith the standard library reached above 92%.
The criteria for positive identification of pharmaceuticaladulteration was: a) the result showed only peaks of the suspicious adulterants; b) the concentration of a suspicious peak
was increased; and, c) the similarity of the UV spectrum
between a suspicious peak and standard library exceeded95%.
2.3. Rho-Kinase II Enzyme Inhibition Assay
The Rho-kinase enzyme inhibition assay was conductedby Natural Remedies (Bangalore, India). This assay is based

An Initial Evaluation of the Safety, Efficacy and Purity of VigRXThe Open Natural Products Journal, 2010, Volume 3

13

on ELISA and operates according to the following principles. Plates are pre-coated with a substrate corresponding to
the recombinant C terminus of the myosin–binding subunit(MBS) of myosin phosphatase. The threonine residue of the
MBS is phosphorylated by members of the myotonic dystrophy protein kinase family, including Rho-kinase. The detector antibody AF20 specifically detects only the phosphorylated form of threonine-696 on MBS. The amount of phosphorylated substrate is measured by binding it with an antiphospho-MBS threonine-696 specific antibody conjugatedwith horseradish peroxidase, which then catalyzed the conversion of the chromogenic substrate tetramethylbenzidinefrom a colorless solution to a blue solution (or yellow after
addition of stopping solution). The color is quantified byspectrophotometry and reflects the relative amount of Rhokinase activity.

with proper ventilation and a daily light-dark cycle of 12hours. All of the rats were fed rodent feed Number 082(Pokkapan Animal Feed Co. Ltd.). Water was provided to
the rats ad libitum during the entire period of the study. The
university’s board authorized to regulate animal experiments
approved all experiments conducted.

A Rho-kinase assay kit CY-1160 (Cyclex Co. Ltd., Nagano, Japan) was used. The components of the kit included
microplate-wells coated with recombinant MBS C terminus,
10X wash buffer, kinase buffer, 20X ATP, HRP conjugateddetection antibody, substrate solution, stop solution, Rhokinase II (CY-E 1160-1, Cyclex Co.), and Rho-kinase specific inhibitor Y-27632 (Calbiochem, cat. no. 688001).

The rats were divided into 3 groups (10 rats/group):Group 1 received 1 mL/day of distilled water administeredorally for 14 consecutive days. Group 2 and 3 were similar tothe control group, but received VigRX at either 15 or 30
mg/kg/day respectively for 14 consecutive days. Each rat
was weighed every three days until the test was completed
on Day 14. On Day 14, between 7–9 p.m., the sexual behavior of each adult male rat exposed to a female rat in-estrus
was observed. (Female rats were induced into estrus with
subcutaneous doses of estradiol benzoate and progesterone
as described by Islam et al. [26]). Each adult male rat wasplaced into an individual glass cupboard and a dim light was
turned on for 5 minutes prior to the start of observation. Subsequently, the female rats in estrus were caged with male rats
at a ratio of 1:1. Sexual behavior of the male rat was observed and recorded with a video recorder for 30 minutes.
The latency and frequency of mounting, intromission andejaculation were then assessed. Statistical analysis was performed using ANOVA and LSD.

Two grams of VigRX were sonicated for 8 minutes in 20
mL of methanol. Ultra-pure water was added to attain a total
volume of 100 mL of solution. The solution was filtered andthe resulting filtrate was used for the assay. It was found that
the VigRX capsule content was 19.3% soluble (385.94 mg
was soluble out of 2000 mg). For calculation purposes, thestock solution was considered to be 20 mg/mL.
The assay was carried out as follows: a total reaction
mixture of 100 μL, containing kinase reaction buffer, varying concentrations of VigRX (125, 250, 500, 1000, and 2000
g/mL) or Y-27632 (the positive control), 10 munits of Rhokinase II enzyme, and 100 μM of ATP, was combined andincubated at 30°C for 30 minutes in a substrate coated plate.
Following incubation, the contents of the wells were discarded and washed 200 L X 5 times with 1X wash buffer.
100 μL of HRP conjugated detection antibody was added
and incubated at 25°C for 60 minutes. The contents werediscarded and the wells were washed 200L X 5 times with
1X wash buffer. 100 μL of substrate solution was added andincubated at 25°C for 15 minutes. Finally 100 L of stop
solution was added and the absorbance was measured at 450
nm in a spectrophotometer (Versamax microplate reader,
Molecular Devices). The % inhibition was calculated as follows: % inhibition = {[Absorbance of control –Absorbanceof test] / Absorbance of control} X 100. The IC50 was calculated using log-probit analysis.
2.4. Aphrodisiac Activity Studies in Male SpragueDawley Rats
2.4.1. Test Animals
Sprague-Dawley adult male (250–280 g) and female rats
(200–240 g) were used in this study. All of the rats weresourced from the National Laboratory Animal Center at Mahidol University, Nakhon Pathom Province, Thailand. They
were transferred to Mae Fah Luang University, Chiang Rai,
Thailand, by air and reared in the Laboratory Animal Housein a temperature-controlled room (approximately 24 ±1°C)

2.4.2. Preparation of Test Material
The contents of the VigRX capsules (Lot #4242) weremixed with distilled water and prepared into two dosages tobe administered at 15 and 30 mg/kg/day. These dosages werechosen to allometrically reflect the current recommended
doses of VigRX in adult human males (2 or 4 capsules perday), using an average body weight of 70 kg.
2.4.3. 14-Day Study in Sprague-Dawley Rats

On Day 15, after weighing, the erect penis was measured
per the following protocol. Each rat was put in supine position, the anterior portion of its body was inserted into a plastic cylinder, and the trunk, limbs and tail were restrained.
The penile sheath was retracted to expose the glans penis,
and this position was maintained using forceps. Light tactile
stimulation was applied to the base of the glans penis for 1–2
minutes until penis became fully erect. The width of the baseof the glans penis and the length between the tip of glans
penis and base of penis were then measured. Subsequently,
the rat was anesthetized with an intraperitoneal injection of
Nembutal (pentobarbital), at which point the intracavernouspressure (ICP) was recorded using the method described by
Tocharus et al. [27]. Blood was then collected via cardiac
puncture and testosterone levels were measured using electrochemical luminescence. The animals were euthanized andthe 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
weighing the epididymis, the cauda epididymis was removed
and the sperm concentration was counted using a Neubauer
hemacytometer. A histopathological examination was conducted on all organs that demonstrated a weight change.
2.4.4. 12-Week Study in Sprague-Dawley Rat
The rats were divided into two groups consisting of
twelve rats each. Group 1 served as the control and received

14 The Open Natural Products Journal, 2010, Volume 3

1 mL of distilled water administered orally each day for 84
consecutive days (12 weeks). Group 2 received VigRX administered orally at the dosage of 15 mg/kg/day for 84 consecutive days (12 weeks). Each rat was weighed every sevendays 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 using ANOVA and LSD.
On Day 85, after weighing, erect penile size was measured as in the 14-day study. The rats were then anaesthetizedand the intracavernous pressure (ICP) was recorded. Blood
was collected via cardiac puncture to measure testosterone
levels (using electrochemical luminescence), and clinicalblood chemistry parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase, creatinine, blood urea nitrogen (BUN), cholesterol, triglycerides, total protein, albumin, and glucose wereassessed. The animals were subsequently euthanized and thepenis, testes, epididymis, prostate gland, seminal vesicle,
pituitary gland, adrenal gland, liver, kidney and spleen were
removed and weighed. After weighing, the epididymis andthe cauda epididymis were removed and the sperm concentration was counted using a Neubauer hemacytometer in thesame manner as the 14-day study. The liver, kidney and testes were sectioned for histopathological studies. The liver
was specifically examined for fatty degeneration, hepatocyte
megalocytosis, lymphoid aggregated periportal area, bile
duct proliferation and peliosis hepatitis. The kidneys were
specifically examined for multifocal tubular cysts, tubular
casts and tubulonephrosis. The testes were specifically examined for signs of interstitial edema, seminiferous tubule
degeneration and congestion. All collected data was statistically analyzed using ANOVA and LSD.
3. RESULTS
3.1. Assays for Product Adulteration with PDE-5 Inhibitor Analogues and APIs
VigRX was found to contain no detectable levels ofknown PDE-5 inhibitors including sildenafil, tadalafil,
vardenafil or related analogues known at the time of thestudy (Table 1).
A separate assay determined that VigRX was free fromadulteration with any of the 180 tested APIs representing thefollowing 19 drug categories: steroids, diuretics, gastrointestinal drugs, CNS stimulants, local anesthetics, antilipemic
drugs, antibiotics, CNS depressants, muscle relaxants,
antigout drugs, hormone drugs, antidiabetics, analgesics/NSAIDs, bronchodilators, abused drugs, narcotics, antihistamines, cardiovascular drugs, and antitussives/expectorants.
3.2. Rho-Kinase II Enzyme Inhibition Assay
VigRX exhibited concentration-dependant inhibitory
effects in the Rho-kinase assay with a fifty percent inhibitory
concentration (IC50) of 1673.18 g/mL (95% confidence
interval of 1216.28–2710.01 g/mL) (Table 2).

Smitasiri et al.

Table 1.

List of PDE-5 Inhibitor Drugs and Known Analogues Screened for (Detection Limit= 100 ppm)

PDE-5 Inhibitor

ResultAcetildenafil

ND

Aminotadalafil

ND

Benzamidenafil

ND

Dimethyl sildenafil (aildenafil)

ND

Dimethyl sildenafil thione (sulfoaildenafil)

ND

Homosildenafil

ND

Hydroxyacetildenafil

ND

Hydroxyhomosildenafil

ND

Noracetildenafil

ND

Piperadino acetildenafil

ND

Piperadino vardenafil

ND

Sildenafil

ND

Sildenafil thione (sulfosildenafil)

ND

Sildenafil thione (sulfohomosildenafil)

ND

Tadalafil

ND

Vardenafil

ND

ND=not detected.

3.3. Aphrodisiac Activity Study in Male Sprague-Dawley
Rats
3.3.1. 14-Day Study
Male rats fed VigRX at a dose of 30 mg/kg/day for 14
consecutive days experienced a significant decrease in intromission latency and ejaculation latency and an increase in
intromission frequency, ejaculation frequency and mounting
frequency as compared to the control group (p <0.05) (Table3). Rats fed VigRX at 30 mg/kg/day also experienced a statistically significant increase in the length and width of theirerect penis when compared with the control group (p <0.05),
whereas the rats fed VigRX at 15 mg/kg/day experienced anincrease only in the length of the erect penis (p <0.05). Bothdosages of VigRX were shown to significantly increase theintracavernosal pressure (ICP) compared to control group (p<0.001). An increase in sperm concentration was noted onlyin the group treated with VigRX at 30 mg/kg/day (p <0.05).
However, no significant difference was seen in the testosterone level in either the VigRX treated groups or the control
group.
There was no significant difference between the bodyweight gained of the VigRX-treated groups and the control
group (Table 4). The organ weights of both treatment groups
did not significantly differ from the control group after 14days, with the exception of a decrease in liver weight experienced by both the 15 and 30 mg/kg/day treatment groups anda decrease in kidney weight experienced only by the 15
mg/kg/day group (p <0.01). Neither of these decreases was

An Initial Evaluation of the Safety, Efficacy and Purity of VigRX

Table 2.

15

Rho-Kinase II Inhibition Assay

Rho-kinase specificinhibitor (Y-27632)

VigRX

Table 3.

The Open Natural Products Journal, 2010, Volume 3

Concentration(g/mL)

Percent Inhibition (mean ±SD)
n=20.0032

4.06 ±0.52

0.032

33.40 ±1.64

0.20

0.32

57.22 ±0(0.13–0.319)

3.2

79.19±3.8

12511.5 ±0.9

25018.38 ±1.73

500

29.98 ±0.82

100035.36 ±2.86

2000

56.39 ±0.83

IC50 as g/mL(95% confidence interval)

1673.18(1216.28–2710.01)

Sexual Behavior in Rat Studies

Sexual Behavior Paramater

2-week (14-day) study
n=10 per group12-week study
n=12 per group

Control

VigRX
15 mg/kg/day

VigRX
30 mg/kg/day

Control

VigRX
15 mg/kg/day

Latency

Mounta67.8 ±36.6

48 ±25.8

39.6 ±22.2

126.25 ±40.93

122.00 ±24.40(seconds)

Intromissionb

345.6 ±159.6

229.2 ±94.2

109.2 ±32.4**

228.67 ±55.58

231.75 ±87.13

Ejaculationc

954 ±321

736.8 ±169.2

539.4 ±142.2*

1730.00 ±49.87

1474.00 ±149.17*

34.80 ±8.4743.00 ±10.63

50.57 ±10.28*

47.00 ±5.2337.60 ±9.58dFrequency

Mount(number oftimes)

Intromissione

19.00 ±8.1228.20 ±6.69

35.71 ±10.13**

27.00 ±9.56

23.00 ±5.61

f1.25 ±0.50

2.75 ±1.50

3.14 ±1.21*

1.00 ±0.00

1.50 ±1.00

Width5.60 ±0.445.80 ±0.45

6.03 ±0.21*

5.81 ±0.40

6.38 ±0.43**

Length10.31 ±0.80

11.38 ±1.16*

12.57 ±1.12**

11.36 ±1.1112.00 ±0.84

Intracavernous pressure (mmHg)

49.70 ±2.4970.68 ±3.66***

83.56 ±5.33***

58.08 ±4.60

85.75 ±6.14*

Sperm Density (x107/mL)

21.00 ±2.95

22.67 ±1.64

24.05 ±3.76*

20.41 ±3.30

24.57 ±3.64**

Ejaculation
Erect Penile
Size (mm.)

Values are mean ±SD.
*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.
e
Number of intromissions from the time of introduction of the female until ejaculation.
fNumber of ejaculations (characterized by longer, deeper pelvic thrusting and slow dismount followed by a period of inactivity).

observed to be dose-dependent, and histopathological examination of the liver and kidney revealed no differences
between treatment and control groups.
3.3.2. 12-Week Study
The ejaculation latency of the rats treated with a VigRX
dose of 15 mg/kg/day was significantly less than the control
group after 12 weeks of treatment (p <0.05) (Table 3). No
other significant differences in sexual behavior were noted inmale rats treated with VigRX for 4, 8 or 12 weeks whencompared to controls.

The VigRX-treated animals in this study experienced
significant increases in sperm concentration, width of theerect penis, and intracavernosal pressure compared to control
animals. Mean sperm concentration values after 12 weeks
were 24.57 ±3.64 x 107/mL in the VigRX treated animals
while the values were 20.41 ±3.30 x 107/mL in the control
group (p <0.01). The mean values for erect penile width
were 6.38 ±0.43 mm in the VigRX treated group and 5.81
±0.40 mm in the control group (p <0.01). The mean intracavernosal pressure measurement in the VigRX treated16 The Open Natural Products Journal, 2010, Volume 3

Table 4.

Smitasiri et al.

Body and Organ Weights in Rat Studies
2-week (14-day) study
n=10 per group12-week study
n=12 per group

Control

VigRX
15 mg/kg/day

VigRX
30 mg/kg/day

Control

VigRX
15 mg/kg/day

Body weight (g)398.20 ±19.59

393.40 ±16.90

392.30 ±11.53

488.00 ±43.63

467.67 ±34.32

Penis (mg%)

71.00 ±11.81

75.82 ±9.8671.22 ±5.79

66.66 ±9.65

71.81 ±7.86

Testes (mg%)

928.29 ±60.25

934.52 ±51.29

929.01 ±18.13

814.61 ±89.97

811.84 ±107.07

Epididymis (mg%)

256.99 ±14.53

265.30 ±16.84

250.50 ±10.91

281.01 ±23.14

281.01 ±23.14

Seminal vesicle (mg%)

313.30 ±47.27

340.56 ±45.77

332.02 ±40.94

253.27 ±73.08

271.08 ±74.73

Prostate gland (mg%)

96.13 ±18.68

106.30 ±14.66

110.37 ±25.25

71.72 ±16.07

84.62 ±17.80

Liver (mg%)

4010.33 ±498.80

3512.82 ±197.19**

3552.43 ±220.40**

3271.31 ±167.72

3268.14 ±180.61

Kidney (mg%)

710.38 ±38.34

664.68 ±24.80**

685.69 ±27.64

585.11 ±34.35

607.48 ±36.96

Adrenal gland (mg%)

16.08 ±1.7715.53 ±1.47

14.98 ±1.4212.21 ±1.0312.35 ±1.60

Spleen (mg%)

232.80 ±22.06

233.01 ±11.42

247.74 ±25.64

198.17 ±15.22

196.05 ±17.24

Pituitary gland (mg%)

2.65 ±0.45

2.65 ±0.49

2.79 ±0.48

2.32 ±0.29

2.47 ±0.31

Values are mean ±SD.
**p <0.01.

group was 85.75 ±6.14 mm Hg, while in the control groupthe mean value was 58.08 ±4.60 mm Hg (p <0.05).
Treatment with VigRX for 12 weeks significantly increased levels of testosterone compared to the control (p<0.05) (Table 5). Mean serum testosterone levels in the
VigRX treated rats were 6.20 ±2.57 ng/mL after 12 weeks oftreatment while mean levels in the control group were 3.66
±1.10 ng/mL.
There were no significant differences in weight gained in
the male rats treated with VigRX daily for 12 weeks compared to the control group (Table 4). VigRX had no effect on
the weights of any organs measured, including the penis,
testes, epididymis, seminal vesicles, prostate gland, liver,kidney, adrenal gland, spleen and pituitary glands. There
were also no significant histopathological findings in either
the control or treatment groups.
VigRX had no effect on any other blood chemistries or
blood parameters measured, including those of glucose,
BUN, creatinine, cholesterol, triglycerides, AST, ALT, alkaline phosphatase, total protein, or albumin, with the exception of a statistically significant decrease in lymphocytes
found in the treatment group (Table 5). However, because noother clinically significant findings accompanied this result,including an absence of histopathological abnormalities, thedecrease in lymphocytes may be an incidental finding and
not related to the treatment article.
4. DISCUSSION
Two separate analyses from different labs determinedthat VigRX (Lot # 4242) was not adulterated with any of thetested pharmaceutical ingredients, including drugs commonly used for the treatment of erectile dysfunction or theiranalogues. Performing these analyses on the end product isthe best way to determine that no adulteration occurred during any step of the manufacturing process. Not only does thisprotect consumers from unexpected side-effects and druginteractions, it also assures that any treatment effects observed clinically are due to the product’s stated formulationand not to unnamed and unlisted pharmaceutical agents.
While there are a number of dietary supplements on themarket making claims about erectile function and sexual
health, most have yet to be substantiated by scientific research performed on the end product.
The Rho-kinase assay can help reveal one of the potential
mechanisms of action behind the efficacy of an erectile
health product. In this study, VigRX was shown to have an
IC50 of 1673.18 g/mL. While this result shows an inhibitory
action against the Rho-kinase enzyme in vitro, the relatively
high IC50 suggests that a large dose would be necessary toachieve similar results in a living system. This may imply
that, while inhibition of the Rho-kinase enzyme is a possible
partial explanation for the activity of VigRX, other mechanisms may also be at play. Future research into the effect ofVigRX on other aspects of erectile health, such as the nitricoxide pathway, may help further explain its efficacy as observed in the rat.
The 14-day study revealed that the rats fed VigRX at the
dosage of 30 mg/kg/day showed an increased sex drive as
reflected by the increased frequency of every measured parameter, including mounting, intromission, and ejaculation
frequencies, as well as short intromission and ejaculation
latencies during the observed 30-minutes of sexual behavior.
In addition to increased sex drive, VigRX treatment also
increased the erect penile size, the intracavernosal pressure
and sperm concentration, without affecting testosterone levels. One hypothesis for the increased sperm concentration isAn Initial Evaluation of the Safety, Efficacy and Purity of VigRX

Table 5.

The Open Natural Products Journal, 2010, Volume 3

17

Blood Parameters in Rat Studies
2-week (14-day) study
n=10 per group12-week study
n=12 per group

Control

VigRX
15 mg/kg/day

VigRX
30 mg/kg/day

Control

VigRX
15 mg/kg/day

Glucose (mg/dL)

NP

NP

NP

126.92 ±25.89

126.25 ±16.21

Blood Urea Nitrogen (BUN) (mg/dL)

NP

NP

NP

27.33 ±2.4227.58 ±2.50

Creatine (mg/dL)

NP

NP

NP

0.36 ±0.08

0.39 ±0.08

Cholesterol (mg/dL)

NP

NP

NP

77.67 ±7.63

71.58 ±7.69

Triglyceride (mg/dL)

NP

NP

NP

22.70 ±9.80

20.57 ±14.76

Aspartate aminotransferase (AST) (U/L)

NP

NP

NP

85.90 ±26.51

108.45 ±40.25

Alanine Aminotransferase (ALT) (U/L)

NP

NP

NP

59.90 ±19.16

54.22 ±13.15

Alkaline phosphatase (U/L)

NP

NP

NP

89.00 ±15.85

93.17 ±20.64

Total protein

NP

NP

NP

5.95 ±0.47

5.73 ±0.23

NP

NP

NP

3.75 ±0.13

3.72 ±0.12White blood cell (cells/mm )

NP

NP

NP

2436.36 ±490.45

2522.22 ±618.02

Hemoglobin (g/dL)

NP

NP

NP

13.80 ±1.51

14.42 ±0.42

Hematocrit (%)

NP

NP

NP

42.43 ±4.7845.11 ±2.27

Red blood cell (mill/L)

NP

NP

NP

7.92 ±0.858.33 ±0.52

Platelet (thsnd/L)

NP

NP

NP

652.17 ±78.17

646.75 ±54.74

Polymorphonuclear granular leucocyte (%)

NP

NP

NP

14.67 ±9.23

22.20 ±8.92

Lymphocytes (%)

NP

NP

NP

83.58 ±9.4474.40 ±9.87*

Monocytes (%)

NP

NP

NP

3.00 ±0.00

2.38 ±1.06

Eosinophils (%)

NP

NP

NP

1.33 ±0.50

2.50 ±1.22

Mean corpuscular volume (fL)

NP

NP

NP

53.50 ±0.5253.80 ±0.92

Mean corpuscular hemoglobin (pg)

NP

NP

NP

17.43 ±0.36

17.23 ±0.65

Mean corpuscular hemoglobin concentration (%)

NP

NP

NP

32.37 ±0.32

31.97 ±0.84

Testosterone (ng/mL)

5.79 ±2.154.69 ±2.37

5.51 ±2.18

3.66 ±1.10

6.20 ±2.57*

Albumin (g/dL)
3

Values are mean ±SD.
NP = not performed.
*p <0.05.

that VigRX treatment leads to an increased blood flow to thetestes and consequently increases spermatogenesis. Herbs
found in VigRX that have demonstrated the ability to increase circulation include hawthorn berry and ginkgo leaf
[15,16]. However, no research has been published specifically on the effect of these herbs on testicular blood flow or
spermatogenesis.
VigRX had no effect on the weights of the majority oforgans measured in the 14-day study, with the exception of astatistically significant decrease in liver weight (both treatment groups) and kidney weight (15 mg/kg group only). The
mechanisms by which VigRX could decrease the liver andkidney weight are still unknown. One possible explanation
for the decrease in liver weight is that the increased mount-ing behavior, blood flow, and other demands of increased
sex drive experienced by the rats treated with VigRX createdan increased energy need, thus increasing the rate of glycogenolysis in the liver. Further investigation of this hypothesis
is needed. However, the decrease in liver and kidney sizes
were not found to be dose dependent. This, coupled with therelatively small number of test animals, makes it difficult todetermine whether this finding was due to a treatment effectof VigRX or spontaneous findings in individual test animals.
Unlike the 14-day study, the 12-week study on VigRX
found no significant effect on the sexual behavior of malerats. However, this study did corroborate the earlier findingsof increased sperm concentration, width of erect penis (butnot 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,while the 14-day study found 30 mg/kg/day to be the more
effective dose, perhaps it is the lower dose that could account for the decreased treatment response in the 12-week
study.

APIs=

active pharmaceutical ingredients

ALT=

alanine aminotransferase

AST=

aspartate aminotransferase

ATP=adenosine triphosphate

The 12-week study did not show the decreases in liverand kidney weights found in the 14-day study, and histopathological examination revealed no abnormalities in any organs. This may lend further credence to the hypothesis thatthe initial findings in the shorter study were sporadic and nottreatment related.

BUN=

blood urea nitrogen

CNS=

central nervous system

eNOS

=

endothelial nitric-oxide synthase

ELISA

=

enzyme-linked-immunosorbent
assay

ESI

=

electrospray ionization

g

=

gram

HPLC

=

high performance liquid chromatography

HPLC=
PDA-MSnhigh performance liquid chromatographyphotodiode array-mass spectrometry

HRP=horseradish peroxidase

IC50

=

50% inhibitory concentration

ICP=

intracavernous pressure

LC-MS

=

liquid chromatography-mass spectrometry

LSD=

least significant differenceMBS=myosin–binding subunitmg=

milligrammL=

millilitermm=

millimetermM=millimolarMS=

mass spectrometryng=

nanogram

nm=

nanometerNO=

nitric oxide

NSAIDs=non-steroidal anti-inflammatory drugs

PDA=

photodiode array

PDE-5

=

phosphodiesterase type 5

PPCT

=

polypropylene centrifuge tube

ppm=

parts per millionrpm=

revolutions per minuteUV=

ultraviolet

Two significant hematological findings were noted in the
12-week study. The first was an increase in testosterone,
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 thelong-term effects of VigRX on prostate health. Recent longterm studies in humans have questioned whether elevatedtestosterone levels contribute to adverse prostate health or an
increased risk of cancer, hence the clinical significance ofelevated testosterone remains unknown [28,29]. A retrospective analysis of numerous studies found no causal relationship between testosterone replacement and prostate cancer orheart disease risk, nor did another study that reviewed decades of research find compelling evidence that higher thannormal testosterone levels increased the risk of prostate cancer or cardiovascular disease [28,29].
The second statistically significant hematological finding
was a decrease in lymphocyte production. However, because
no other clinically significant findings accompanied this result, including histopathological abnormalities, the decreasein lymphocytes may be an incidental finding, not related tothe treatment article. No other blood parameter measured
differed significantly from control.
5. CONCLUSION
Overall this research provides scientific evidence in support of the purity, safety, and potential efficacy of this herbal
formulation in supporting male sexual health. Inhibition of
the enzyme Rho-kinase, and increases in serum testosterone
are two possible mechanisms of action for this formula. It is
likely that additional mechanisms play a role in the efficacy
as seen in the 14-day and 12-week rat studies, which require
further investigation. Research exploring other physiologicaleffects, as well as human clinical trials, are needed to better
understand the role VigRX may play in supporting and/or
enhancing male sexual function. Additional in vivo toxicology studies would be beneficial to more fully evaluate thesafety of long-term consumption of this product.
CONFLICT OF INTEREST STATEMENTAIBMR Life Sciences, Inc. was hired as an independent
consulting agency to design and implement studies on
VigRX. None of the authors have any financial interest in
the product.
ABBREVIATIONS
L=

microliter

m=micrometer

ANOVA

=

analysis of varianceserologic

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© Smitasiri et al.; Licensee Bentham Open.
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License(http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided thework is properly cited.