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Clinical Analysis Reporting for:

Hard Steel Original Formulated Male Enhancement
Supercore Products Group, Inc. (SPG)

Our lab analytical testing consists of testing strategies, formulations, clinical analysis, double blind studies, and approaches, while delivering unparalleled comprehensive results for both consumer data purposes. As an FDA-regulated, DEA-licensed, ISO 9001 GMP factory and ISTA-certified contract service provider; our labs are certified to support many clients, including those in the pharmaceutical, nutraceutical, medical device, personal and consumer product industries. Our renowned labs have more than 20 years existence as a stand-alone facility dedicated to USP , USP and USP testing and covering raw materials through finished products. 

Enclosed is a Comprehensive Analytical and Advisory Service physical performance test and physicochemical test for components formulated in Hard Steel Original Formulated Male Enhancement. All lab(s) relevant USP, EP, and JP testing procedures are certified by its Analytical Team. 

  • USP 381 / USP 382 Ingredients Characterization Components
  • USP 661 / 661.1 / 661.2 HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT Potential & Effectiveness with Alcohol Consumption
  • USP 660 Containers – Artificial Inclusions 


According to ICH Q6B guidelines, impurities in biological products can be classified as process-related or product-related. Process-related impurities include those that are derived from the manufacturing process (e.g., cell substrates, cell culture media components or downstream processing). Product-related impurities in the drug substance are molecular variants with properties different from those of the desired product formed during manufacture or storage. 

To characterize these impurities, we conducted six (6) unique analytical techniques for the isolation and characterization of process- and product-related impurities: 

  • Notable isolation and purification: Reversed phase, ion exchange, size exclusion, intrinsic accumulation, foreign particles, and impact blend. 

Identification: Mass spectrometry (ESI-Triple Quad, MALDI-TOF, Q-TOF), NMR (1D and 2D), FTIR, Raman and UV-Vis’s spectroscopy, N-terminal Edman sequencing, LC-MS/MS de novo sequencing, peptide mapping, disulfide bond mapping, amino acid analysis for the following: Gelatin, FD&C Black & White, Titanium Dioxide, Cellulose, Magnesium Stearate, Silica, Pnax Ginseng Pnax Ginseng, Gingko Biloba, Muira Puama, Catuba Bark and Epimedium Brevicornum Derivative. 

Gelatin – Scientifically, is rich in protein, and has a unique amino acid profile that gives it many potential health benefits. There is evidence that gelatin may reduce joint and bone pain, increase brain function, and help reduce the signs of skin aging. Gelatin is FDA Approved for human consummation. This common thickening and gelling agent are an animal-based product that contains high levels of protein. Gelatin contained in HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT, safely contributes to a boost in Free Testosterone.
When dry and converted into powdery form as in the case of HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT, gelatin total inclusions consist of 98–99% protein, but it is not a nutritionally complete protein since it is missing tryptophan and is deficient in isoleucine, threonine, and methionine.[10] The amino acid content of hydrolyzed collagen is the same as collagen. Hydrolyzed collagen contains 19 amino acids, predominantly glycine (GLY) 26–34%, proline (Pro) 10–18%, and hydroxyproline (HYP) 7–15%, which together represent around 50% of the total amino acid content.[11] Glycine is responsible for close packing of the chains. Presence of proline restricts the conformation. This is important for gelation properties of gelatin.[12] Other amino acids that contribute highly include: alanine (Ala) 8–11%; arginine (Arg) 8–9%; aspartic acid (Asp) 6–7%; and glutamic acid (Glu) 10–12%.[11]  

FD&C Black & White - This color additive is widely found in beverages, desserts, processed vegetables, drugs, makeup, and other products. FDA requires all products containing FD&C Black & White to identify it on their labels. As required, lot certification directly from the US Food and Drug Administration for all FDA certified colorants are procured by SPG’s co-packer as a condition for production.  

Titanium Dioxide – Single most sustaining ingredient preserving HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT 2-Year Shelf Life. Approved by the FDA; Protective coatings to preserve the efficacy of pharmaceuticals over time – TiO2 is an essential component of the protective coatings of pharmaceuticals, enhancing their safety, efficacy, and quality for long periods. TiO2 scatter light and absorb UV rays, TiO2 extends the shelf-life and ensures the stability of pharmaceuticals by protecting active ingredients against UV/light and heat degradation.  

Cellulose – Cellulose is a naturally Approved FDA compound. Humans cannot digest cellulose, but it is important in the diet as fiber. Fiber assists the human digestive system – keeping food moving through the gut and pushing waste out of the body. The best sources of cellulose are seeds like hemp and flax as well as whole grains, carrots, tomatoes, and cucumbers. Eating a variety of fruits, vegetables, whole grains, nuts, and seeds, are more likely to give humans the cellulose needed to stay healthy. Its inclusion in HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT is beneficial to human digestive.

Magnesium Stearate - Magnesium Stearate in supplement tablets is used as a ‘flow agent.’ It means that it prevents different supplement ingredients from sticking to each other and the blending and punching equipment. Adding a flow agent such as vegetable magnesium stearate is imperative for ensuring a homogenous blend of ingredients and a consistent dosage in each and every HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT capsule. Despite the bad name additives such as vegetable magnesium stearate get in supplements, they are rather necessary and perform different crucial functions in supplement manufacturing. Not adding magnesium stearate or an alternative can even be detrimental to human health as capsules or tablets may not contain the prescribed dosage consistently. Risks and Concerns Surrounding Magnesium Stearate - The leading concern for magnesium stearate is a study from 1990 which found that stearic acid can suppress immune function in T-cells of mice. However, human T-cells are different from those of mice in their ability to desaturate fatty acids. Human T-cells have the delta-9 desaturase enzyme required to convert stearic acid into oleic acid to avoid a toxic build-up. Another factor to consider is that the study was conducted by bathing the mouse T-cells in stearic acid. It is impossible to consume stearic acid in such humongous amounts through supplements.              

Stearic acid (also called Octadecanoic Acid) is one of the most common long-chain fatty acids, found in both natural animal and vegetable fats, known also by its structural description of being an 18-carbon chain fatty acid (18:0) with a chemical structure of C36H70MgO4. In nature stearic acid occurs primarily as a mixed triglyceride, or fat, with other long-chain acids and as an ester of a fatty alcohol. It is much more abundant in animal fat than in vegetable fat; lard and tallow often contain up to 30 percent stearic acid.”1   Stearic acid (stearate) is a predominant saturated fat in the human diet. Stearates are nutrients that represent a natural part of every type of fat, whether animal or vegetable, and are typically consumed in amounts of several thousand milligrams per day from common food sources. A 200-calorie serving of dark chocolate can contain up to 5 grams (5,000 milligrams) of stearates; cocoa butter, coconut oil, beef fat, olive oil, fish, and virtually all fats and oils naturally contain far more stearates than do dietary supplements. Magnesium stearate is a combination of stearic acid and the essential mineral magnesium. It is a mixture of pure stearic acid and palmitic acid, where the content of stearic acid is not less than 40.0% and the sum of the two acids is not less than 90.0%. In this analysis, we describe magnesium stearate as consisting mainly of magnesium stearate with variable proportions of magnesium palmitate and magnesium oleate.  

Magnesium stearate is a form of chelated pre-acidified magnesium, and just like other chelated minerals (magnesium ascorbate, magnesium citrate, et al) has no inherent negatives based on its being in a stable neutral compound comprised of a mineral and an acid (vegetable-sourced stearic acid from palm oil neutralized with magnesium salts). It is a magnesium salt of fatty acids C16 to C18. HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT uses stearates tested to U.S. Pharmacopeia monograph standards; known as pharmaceutical grade, the highest purity. They are non-GMO, free from BSE/TSE, and may be used, if desired, as part of a vegetarian or vegan diet.  

HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT uses palm oil that already contains significant amounts of saturated fats providing abundant stearic acid. This is recognized as a pure USP-grade magnesium stearate derived from non-hydrogenated, non-GMO, non-irradiated palm oil that contains no trans-fat. Stearate is one of the major saturated fatty acids in mammals and is acquired through two pathways: 1) dietary fat absorption and 2) de novo lipogenesis (our bodies make it from other dietary fats). Stearic acid may be converted to oleic acid (omega-9 fatty acid) in mammals; which of course does not happen in a test tube study. Oleic acid is considered a healthy fat, and is a major component of olive oil.  

“Upon ingestion, magnesium stearate is dissolved into magnesium ion and stearic and palmitic acids. Magnesium is absorbed primarily in the small intestine, and to a lesser extent, in the colon. Magnesium is an essential mineral, serving as a cofactor for hundreds of enzymatic reactions and is essential for the synthesis of carbohydrates, lipids, nucleic acids, and proteins, as well as neuromuscular and cardiovascular function.”2  

The FDA has affirmed that stearic acid is GRAS (Generally Regarded As Safe) and can be added to foods in accordance with Good Manufacturing Practices (GMP).3 HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT is produced in a GMP-certified manufacturer lab. The FDA’s Select Committee on GRAS Substances has also reported on magnesium stearate safety, concluding that, “There is no evidence in the available information on magnesium carbonate, magnesium chloride, magnesium sulfate, magnesium hydroxide, magnesium oxide, magnesium stearate that demonstrates or suggests reasonable grounds to suspect, a hazard to the public when they are used at levels that are now current and in the manner now practiced, or which might reasonably be expected in the future.”4  

The World Health Organization also confirmed the safety of magnesium stearate: “The Committee concluded that there are no differences in the evaluation of the toxicity of magnesium stearate compared with other magnesium salts.”Stearates are well absorbed (over 90%) and do not coat the G.I. tract. In fact, they reportedly discourage certain undesirable biofilms.6 There is no credible concern that the comparatively tiny amounts in dietary supplements may inhibit absorption of nutrients in vivo (in live people). A typical supplement with stearate excipients may have less than a tenth of one percent of a typical person’s daily consumption of dietary stearates. We have extensively investigated the safety of magnesium stearate, which is also considered safe and non-toxic and has no conflict when consumed with Alcohol. Silica - Commonly used in the form of silicon dioxide as an anti-caking agent in foods and supplements to keep ingredients from clumping up or sticking together, and it’s sometimes added to liquids and beverages to control foaming and thickness. In HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT, Silica is included to improve Digestive issues by increasing the absorption of the product total formulation. Although silicon is a natural part of some foods and sees wide commercial application, the U.S. Food and Drug Administration has set out strict regulations around using silica as an additive. The Food and Drug Administration (FDA) recommends that you don’t consume more than 10-30 grams, or 2% of your daily food intake (500-1,500 grams), of silica per day. 

 Pnax Ginseng – The herbal remedies referred to as "ginseng" are derived from the roots of several plants. One of the most commonly used and researched of the ginsengs is Panax ginseng, also called Asian or Korean ginseng. The main active components of Panax ginseng are ginsenosides, which have been shown to have a variety of beneficial effects, including anti-inflammatory, antioxidant, and anticancer effects. Results of our clinical research studies demonstrate that Panax ginseng may improve psychologic function, immune function, and conditions associated with diabetes. It's considered an adaptogen, which are natural substances that are believed to stimulate the body's resistance to stressors. Clinical Studies shows possible effectiveness: Memory and thinking skills (cognitive function). Taking Panax ginseng by mouth might improve thinking, arithmetic skills, and reaction times in healthy, middle-aged people but not in young adults. Taking panax ginseng alone does not seem to help memory, but taking it with ginkgo leaf extract seems to improve memory in healthy people between the ages of 38 and 66. Erectile dysfunction (ED). Taking Panax ginseng by mouth seems to improve sexual function in adults with ED. HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT includes Panax ginseng. Flu (influenza). Taking a specific Panax ginseng extract by mouth appears to reduce the risk of getting a cold or the flu. But it doesn't seem to reduce flu symptoms or the length of the illness. Fatigue in people with multiple sclerosis (MS). Taking Panax ginseng by mouth daily for 3 months reduces feelings of tiredness and improves quality of life in females with MS. Increasing response to sexual stimuli in healthy people. Taking Panax ginseng alone or with other ingredients by mouth seems to improve sexual arousal and satisfaction in postmenopausal adults. It also seems to improve sexual desire in females who report sexual problems. Pnax ginseng is effective with moderate consumptions of alcohol. 

Gingko Biloba – has antioxidants that cancel out free radicals, molecules that can damage cells. They form when you exercise and when your body uses food for energy. As human beings age, the body doesn’t do as good of a job getting rid of free radicals. They attack brain cells, which can lead to memory loss. Our study found a twice-daily dose of ginkgo extract help ward off or slow dementia, or memory loss associated with Alzheimer’s disease in older adults. In addition, it found those who took the extract had fewer signs of dementia than those who did not. This is due to increased brain circulation and improved cognitive capacity while also preventing neuron damage. Our studies show that ginkgo, used as a complement to treatment, can result in better mental performance and socialization, even in those who already have Alzheimer’s. Ginkgo might also help with tinnitus and glaucoma. Lab studies show it improves blood circulation by opening up blood vessels and making blood less sticky. That is because it has compounds called terpenoids. Ginkgo is considered safe when taken in moderate doses and safe to consume with alcohol. Gingko biloba increase libido by influencing hormonal balance, which helps to increase blood blow to the genital area. This can help men with erectile dysfunction. HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT contains Gingko biloba. Using ginkgo can help the body to cope with elevated levels of cortisol and adrenaline, which are produced when the body is under increased stress. Therefore, people who suffer from anxiety may benefit from taking ginkgo biloba, as it can help them to manage stress that they may be feeling. In addition, due to its influence on hormonal balances, Ginkgo can decrease sudden changes to mood, especially in women who are suffering from PMS, which may decrease the risk for developing depression. 

Muira Puama – Is derived from the bark of a tree or its roots. This small tree is native to the Amazonian region of South America and has a plethora of uses among the indigenous peoples of the Amazon, including the following: adaptogen, alopecia (applied topically for hair loss), anorexia, antinociceptive, antioxidant, aphrodisiac, ataxia, Beri (vitamin deficiency disease), debility, digestive problems, dysentery, fatigue, impotence, neurasthenia, nerve tonic, rheumatism, stimulant, tonic (adding bark decoction to a bath), paralysis, and tremors (Quattrocchi, 2012; Piato et al., 2010; Berdonces, 2009; Duke et al., 2009; Taylor, 2005; Rutter, 1990). Muira Puama is an important ingredient of “Catuaba”, a medicinal poly-herbal combination used in South America, also composed of guarana (Paullina campana), ginger (Zingiber officinale), and Trichilia catigua (Quattrocchi, 2012). One of the main active ingredients in Muira Puama is an alkaloid known as Muirapuama (Lorenzi and Matos, 2008). 

Curia assessed whether the daily oral administration, for a period of eight weeks, of a combination of ginger, guarana, Muira Puama, and L-citrulline could effectively delay the ongoing corporal fibrosis, smooth muscle cell apoptosis (programmed cell death) and cavernosa Ven occlusive dysfunction present in middle aged rats similar to that seen with the prescription medication of tadalafil (Cialis). The results of the study showed that the orally delivered herbal combination plus L-citrulline appeared to be as effective as daily therapy with the medication in either slowing down or reversing the onset of the histological and functional characteristics of erectile dysfunction related to aging in laboratory rats. HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT maintains Muira Puama as a primary formulated inclusion. In addition, our study assessed the in vitro antioxidant activities of plants from the Brazilian Amazon (Byrsonima japurensis, Calycophyllum spruceanum, Maytenus guyanensis, Passiflora nitida and Muira Puama Ptychopetalum olacoides. The results supported the traditional use for these plants against inflammation, due to their significant antioxidant (free radical scavenging) action (Curia Global et al., 2016). 

Oliveira et al. (2013) conducted an in vitro study using the aqueous extracts of various traditional medicinal plants from Amazonia, searching for antimicrobial activity against both human as well as animal pathogenic microbes. The extracts obtained from muira puama and Pentaclethra macroloba inhibited the growth of the bacteria Klebsiella ozaenae and Acinetobacter baumannii. Figueiró, et al. (2010, 2011) evaluated the effects of an ethanol extract obtained from muira puama and identified promnesic (improving memory), anti-amnesic, and acetyl-cholinesterase inhibition properties in laboratory animals (mice) treated orally with the extract. The results of the studies showed that this plant induces acetyl-cholinesterase inhibition in brain areas relevant to cognition. For this reason, we concluded that muira puama extracts could be a potential treatment for Alzheimer’s disease in humans. 

Catuaba Bark -  is a natural remedy derived from the bark of trees found in the Brazilian rainforest. Formulations vary depending on the type of tree selected; Erythroxylum caatingae, Trichilia catigua, Anemopaegma arvense, and Micropholis caudata are some possibilities. But all of them are stimulating agents of the human nervous system and offer aphrodisiac properties. The active ingredient in catuaba is alkaloids dubbed catuabines. Alkaloids are organic compounds from plants, some of which have psychoactive effects. Examples include caffeine, morphine, strychnine, and nicotine. 

In the United States, catuaba is mainly used as a dietary supplement in capsule, extract, and powder forms for its aphrodisiac properties but is also known to treat or prevent a wider range of medical conditions, including: Anxiety, Asthma, Bacterial infections, Bronchitis, Depression, Erectile dysfunction (HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT), Fatigue, Insomnia, Low libido, Obesity, Memory problems and Skin cancer. 

Epimedium Brevicornum Derivative.- Epimedium brevicornum Maxim (EbM) has been reputed to have sexual stimulation effects on males by drug makers. EbM Derivatives are the base of HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT formulation. Therefore, our study is aimed to test the hypothesis that EbM extracts relaxed the corpus cavernosum (CC) smooth muscle through activation of multitargets on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. Water extract of EbM and its subfraction (EP-20) were prepared and standardized by high-performance liquid chromatography. Isolated rabbit CC strips were mounted in organ baths and isometric tension was recorded in the presence or absence of specific inhibitors related to NO/cGMP signaling such as L-NG-nitro-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor) or phosphodiesterase 5 (PDE 5) inhibitors. cGMP level was determined in EP-20-treated CC strips. The results showed that EP-20 enriched the content of L-arginine in the process of purification and relaxed the CC smooth muscle precontracted with phenylephrine (PE, 1 μ M) in a concentration-dependent manner. Besides, EP-20 increased the amount of cGMP production in rabbit CC tissues. Coincubation with EP-20 and L-NAME or ODQ significantly decreased EP-20-induced relaxation whereas EP-20 increased sodium nitroprusside-induced relaxation in PE-precontracted CC strips. Besides, EP-20 increased the potency and the duration of the relaxation effects caused by electrical field stimulation. Finally, EP-20 could potentiate PDE 5 inhibitors in relaxation of PE-precontracted CC strips. We concluded that extract of EbM relax the CC smooth muscle through multitargets in NO/cGMP/PDE 5 pathway and might bring into perspective the treatment strategy for those patients with erectile dysfunction. 

Sexual dysfunction remains a major problem by the third-to-forth decade of male life and continues to increase in prevalence with each subsequent decade. The ability to have erections is reliant on the complex interaction among neurological, psychological, and vascular responses. There is consensus that sildenafil, tadalafil and vardenafil are promising oral agents for the treatment of patients with erectile dysfunction (ED). Many effects such as facial flushing, dizziness, blue vision, and headache have been described, although the rate of interruption of such treatment is low. Epimedium brevicornum Maxim (EbM) or Yin-Yang-Huo, an Epimedium spp, has been reported to have anti-inflammatory and antitussive effect and recent pharmacological studies have demonstrated that it increases the coronary flow by reducing vascular resistance and lowers the blood pressure.
Our recent studies suggested that EbM increased intracavernous pressure (ICP) in rat model (KK Chen, manuscript in preparation). Therefore, the aim of this study is to investigate the effect of EbM extract elicitation of penile erection and its mechanisms related to nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. 

Epimedium Brevicornum Base Ingredient in HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT Materials and Methods 

Chemicals and reagents 

The well-known pure compounds extracted from EbM are the glycosides icariin and noricariin.1112 Icariin (molecular weight=626.65 Da) as an internal standard was purchased from Nacalai Tesque (Kyoto, Japan). Chromatographic grade acetonitrile and monosodium phosphoric acid were obtained from E. Merck (Darmstadt, Germany). Triply de-ionized water from Millipore (Bedford, MA, USA) was used for all preparations. 

Preparation of EbM extracts 

EbM was purchased from a local wholesale distributor (Taipei, Taiwan, Republic of China). The EbM extracts were prepared as follows. EbM (300 gm) was extracted with 1 l water (water-soluble fraction, denoted as EPW), which were further purified until the total volume down to 100 ml. The extracts were filtered through layers of gauze, the residues discarded and the filtrates were kept at −20°C, followed by lyophilizing the samples. The final powder weight was 4.53 g. For further purification, EPW was sub fractioned into EP-0, EP-20, EP-50, EP-100 by elution of 0, 20, 50 and 100% methanol through Diaion HP20 column, respectively. Then, the EP-20 extract was chromatographed on a column of Cosmosil® 5-C18-AR II and eluted with a gradient of water-acetonitrite (9:1 to acetonitrite alone), to yield six fractions (EP-20-1 to EP-20-6) by time interval. Each extract was tested on phenylephrine (PE)-contracted corpus cavernosum (CC) strips for their relaxation potencies. 

Liquid chromatography 

The high performance liquid chromatography (HPLC) system consisted of a chromatographic pump (PM-80, Bioanalytical System, West Lafayette, IN, USA), an injector (Rheodyne 7125, Cotati, CA, USA) equipped with a sample loop and an ultraviolet detector (Varian, Walnut Creek, CA, USA). EbM extracts and its major ingredient were separated using an Alltima reversed phase C18 column (250 × 4.6 mm I.D.; particle size 5 m; Deerfield, IL, USA) preserved at an ambient temperature to perform the ideal chromatographic phase. The conditions of mobile phase were selected. The mobile phase was filtered through a millipore 0.45 μm filter and degassed before use. The UV wavelength was set at 203 nm for the determination of EbM. Output data from the detector were integrated via an EZChrom chromatographic data system (Scientific Software, San Ramon, CA, USA). Several steps for quality experiments including retention time, spiking of authentic standard, change of wavelength and change of the composition of mobile phase were used to examine the contents in EbM extracts.13 To standardize and quantify the EbM extracts, icariin (C33H40O15, molecular weight: 676.65 Da) was used as an internal standard with a chromatograph column (Acq-Tac, Waters, Kyoto, Japan) and a UV detector (G1314A, Agilent, Waldbronn, Germany).1112 Furthermore, the L-arginine level was determined and quantified by HPLC analysis with a chromatograph column (38023-11, Cosmosil, Kyoto, Japan) as described previously.14 In brief, 10 mg L-arginine (A5006, Sigma, ST Louis, USA) was dissolved in 0.1 M HCl as stock solution. Serial concentrations of L-arginine (0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml) were used to get a linear standard culver, followed by determination the L-arginine level in EbM extracts with a fluorescence detector. The coefficient variance between inter-day and intra-day was less than 5%. 

Isolation of rabbit CC 

Male New Zealand rabbits weighting 2.5–3 kg were treated under the regulations of the ‘Principles of laboratory animal care’ (NIH publication No. 86–23, revised 1985). After overnight fasting with free access to water, rabbits were anesthetized and killed with overdoses of intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine (5 mg/kg). The organ bath for rabbit CC strips was prepared with some modification as described previously.15 In brief, the entire penile tissue was transected from the base, quickly rinsed, and kept immersed in oxygenated Krebs' physiological solution at 37°C. The composition of Krebs' solution was the following (in mM): 115.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 25.0 NaHCO3, and 11.0 glucose; pH, 7.3∼7.4. The layer of tunica albuginea was removed by sharp dissection under a microscope. Removal of the adjacent soft tissue around the CC and proper care was taken to avoid disrupting the continuity and integrity of the CC strips. 

Recording of isometric tension 

The CC strips were transferred to jacketed 10-ml tissue baths filled with Krebs' solution maintained at 37°C and equilibrated with 95% O2–5% CO2. The strips were anchored to the bottom of the bath at one end and to the isometric muscle transducers (model FTO3; Grass Instruments Co., Quincy, MA, USA) at the other. The isometric muscle transducer in turn was connected to a polygraph recorder (Gould TA240, Ohio, USA). All data were recorded and stored in a computer equipped with an offline analysis software (Acknowledge III-MP 100 WSW, Biopac System, Inc., Santa Barbara, CA, USA). The recording channels were calibrated at 1 gm/cm in the beginning and were left undisturbed for the rest of the experiment. After mounting the tissue, the smooth muscle strips were stretched to a resting tension equivalent to 1.5 g force and equilibrated for 60–90 min. 

Relaxation effect of CC 

For the evaluation of relaxation, the CC strips were first contracted with PE (1 μ M) and responses were examined using cumulative concentration–response curve (CRC) by increasing the concentration of agents after a steady response to the previous administration had been reached. The relaxation responses of CC strips were validated by cumulative doses of sodium nitroprusside (SNP). After confirming the model validity, the responses of EbM extracts or its vehicle were obtained by adding agents cumulatively to the organ bath. Construction of CRCs was based on degrees of relaxation of the PE-induced contractions. A preliminary study had shown no significant differences between results obtained with single or multiple exposures to increasing concentrations of EbM extracts (i.e. no tachyphylaxis, desensitization or tissue fatigue was observed). 

Effects of inhibitors were studied by preincubation with the inhibitor for 20 min, then contraction was induced by PE, after that the relaxation profile of EbM extracts were observed. To identify the involvement of NO/cGMP pathway in the relaxation actions of EbM extracts, the CC strips were incubated with a potent NO syntheses inhibitor, L-NG-nitro-arginine methyl ester (L-NAME 3 × 10−4 M) or 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ, 3 × 10−5 M, a guanylyl cyclase inhibitor) for 10 min, respectively, before the construction of post-inhibitor treatment cumulative CRCs of EbM extracts and observed for any changes in potency. 

Determination of cGMP level in rabbit CC strips 

The CC strips were pretreated with EP-20 in the presence or absence of ODQ (3 × 10−5 M) or SNP (1 × 10−6 M) or phosphodiesterase 5 (PDE 5) inhibitor (sildenafil, 1 × 10−7 M) for 20 min, followed by freezing the CC strips into liquid nitrogen. Then, a 0.2 g frozen tissue was homogenized with 1 ml 5% trichloroacetic acid (w/v), followed by determination of cGMP levels by commercially available EIA kits.16 


All the changes in CC strips tension in response to drugs are expressed on percentile basis and all the values are expressed as mean±s.e.m. The relaxant responses are normalized as the percentage of relaxation of the contraction originally induced by PE. Statistic evaluation of the data was performed by one-way analysis of variance (ANOVA) or general linear models’ procedure (two-way ANOVA) followed by post hoc Dunnet's test (SPSS 12.0.1 for windows, Release 12.0.1, SPSS Inc., Chicago, IL, USA). For non-parametric analysis, Wilcoxon’s rank sum test was used.  


Purification of EPW and EP-20 from EbM

EbM was partially purified and analyzed with auto scaled HPLC. Icariin (molecular weight: 676.65 Da) was used for quantification of the purity of extracts. When detected by a wavelength of 270 nm, the retention time of icariin was 36.628 min. Naphthalene was used as internal standard. The icariin content in EPW was 1.17% whereas indictable in EP-20. 

The bioactivity of the water crude extract (EPW) and further extract EP-20 was tested for the relaxation effect in CC strips precontracted with 1 μ M PE. The results showed that both EPW and EP-20 relaxed the PE-precontracted CC strips in a concentration-dependent manner. (Figure 1a). The 50% relaxation concentration of EPW and 25% of EP-20 were 65 and 208 μg/ml, respectively. However, further six purified extracts from EP-20, namely, EP-20-1 to EP-20-6, presented little relaxation effect as did the extracts of EPW or EP-20 (Figure 1b).
Figure 1 

Concentration–response curves (CRCs) of exacts of EbM in CC strips. Water extract (a, •), methanol extract (a, ) and EP-20 (a) derived from water extract were prepared as described in methods. EP-20 (b, •, dash line) was further chromatographed into six subfractions (b, left upper corner) by time interval. Relaxation responses are expressed as percentage of residual tension of contraction originally induced by PE (1μ M). Data points represent mean±s.e.m. (n>10).  

The involvement of NO/cGMP pathway in EP-20-induced relaxation effect

After co-incubation with L-NAME (3 × 10−4 M), the EP-20-induced relaxation effects were partially blocked by 30% in PE-precontracted CC strips, whereas in co-incubation with a guanylyl cyclase inhibitor (ODQ, 3 × 10−5 M), the relaxation effects were also partially blocked.
Figure 2 

Effect of L-NAME or guanylyl cyclase inhibitor on CRCs of EP-20 in CC strips. CRCs of EP-20 in CC strips were calculated without (•) or with pretreatment of L-NAME (a, , 3 × 10−4M) or ODQ (b, , 3 × 10−5M). Relaxation responses are expressed as percentage of residual tension of contraction originally induced by PE (1μ M). Data points represent mean±s.e.m. (n>10). 

EP-20 enhanced relaxation effect with SNP in rabbit CC strips 

In Figure 3, EP-20 (0.01 mg/ml) was pretreated in PE-precontracted CC strips for 10 min, followed by incubation with different concentration of NO donor (SNP). In contrast, SNP (0.01 mg/ml) was pretreated in PE-precontracted CC strips for 10 min, followed by incubation with different concentration of EP-20. The results showed that there was a shift to left of the constructed CRC in EP-20 as compared to SNP alone (Figure 3a) and vice versa (Figure 3b). The data suggested that EP-20 enhanced relaxation effect with SNP in rabbit CC strips.
Figure 3 

Concentration–response curves (CRCs) of SNP (a) and EP-20 (b) in CC strips. CRCs of SNP in CC strips were calculated without (a, •) or with pretreatment of sildenafil (a, , 1 × 10−9M) or pretreatment of EP-20 (a, 0.01mg/ml). CRCs of EP-20 in CC strips were calculated without (b, •) or with pretreatment of 1 × 10−9M sildenafil (b, ) or 1 × 10−8M sildenafil (b). Relaxation responses are expressed as percentage of residual tension of contraction originally induced by PE (1μ M). Data points represent mean±s.e.m. (n>12).  

EP-20 increased cGMP level in EP-20 treated CC strips 

In biochemical studies, the CC strips were pretreated with EP-20 in the presence or absence of ODQ or PDE 5 inhibitor for 20 min, followed by determination the cGMP level in CC tissues The results showed that there was a significantly increased cGMP level in EP-20-treated (0.1 and 0.3 mg/ml) CC tissues (Figure 4). Taken together, the results suggested that EP-20 induced relaxation of rabbits CC smooth muscle tissue through not only the increase of cGMP production but also the enhancement of SNP effect.
Figure 4
Effect of EP-20 on cGMP level in CC strips. The CC strips were pretreated with EP-20 in the presence or absence of ODQ (3 × 10−5M) or SNP (1 × 10−6M) or sildenafil (1 × 10−7M) for 20min, followed by freezing the CC strips into liquid nitrogen. Then, a 0.2g frozen tissue was homogenized with 1ml 5% trichloroacetic acid (w/v), followed by determination of cGMP levels by commercially available EIA kits. Data points represent mean±s.e.m. (n>12) in 0.2g CC tissue. 

EP-20 increases the potency and the duration of the relaxation effects caused by electrical field stimulation 

When the rabbit CC strips precontracted with PE were subjected to electrical field stimulation (EFS), PE-20 (0.1, 0.3 mg/ml) significantly increased EFS-induced relaxation at a frequency of 4 and 8 Hz (Figure 5a). However, only PE-20 at a concentration of 0.3 mg/ml increased the duration of EFS-induced relaxation in PE-precontracted CC strips (Figure 5b).
Figure 5 

Effect of EP-20 on electrical field-stimulated CC strips. The percentage (a) and the duration (b) of relaxation effects caused by EFS (2–8Hz) were evaluated in the absence (), 0.1mg () and 0.3mg () of EP-20 extract, respectively. Data points represent mean±s.e.m. (n>12). 

Combination effect of EP-20 and PDE 5 inhibitors 

To investigate the potential role of EP-20 on perspective clinical application, low concentration EP-20 (0.1 mg/ml) was co-incubated with PED 5 inhibitors (1 × 10−9 M) on PE-precontracted CC strips. The percentage of relaxation in sildenafil, vardenafil and tadalafil alone group was 23.7±4.8, 26.0±4.9 and 16.1±6.6%, respectively; whereas that of each combination groups was 36.2±4.9, 40.7±3.4 and 34.2±2.7%, respectively (Figure 6a). When in combination with ineffective concentration of EP-20 (0.01 mg/ml), the percentage of relaxation in sildenafil, vardenafil and tadalafil combination group was 32.4±2.6, 35.2±2.9 and 22.3±3.7%, respectively (Figure 6b). The results suggested that there was a potentiate but not additive nor synergic effect for EP-20 in combination with the present PED 5 inhibitors.
Figure 6
Combination effects of EP-20 and PDE 5 inhibitors on CC strips. Effective (a, 0.1mg/ml) and ineffective (b, 0.01mg/ml) concentration of EP-20 were co-incubated without () or with PDE 5 inhibitor alone () or in combination (). Relaxation responses are expressed as percentage of residual tension of contraction originally induced by PE (1μ M). Data points represent mean±s.e.m. (n>12).   

L-arginine level detected in EPW and EP-20 

In order to identify whether L-arginine, an NO precursor, could be detected in EbM extracts, HPLC identification and quantification was performed and the results showed there was an enrichment of L-arginine in EP-20 (0.1148%) as compared to its previous extract or EPW (0.058%). 


In this study, we have clearly demonstrated that EbM extract causes concentration-dependent relaxation of isolated rabbit CC smooth muscle through multitargets related to L-arginine/NO/cGMP pathway including the presence of Alcohol. To our knowledge, this is the first study to provide evidence concerning the herbal extracts on PDE 5 inhibitors for proerectile functions. In fact, the fact that EbM extract potentiates the commonly used PDE 5 inhibitors, such as sildenafil and vardenafil, provides a new treatment strategy for those patients with ED.  

There are many studies using rats as in vivo model to investigate drug effects on penile tissue. Our recent works found that direct injection of water extract of EbM (EPW) at a dose 300 μg to 10 mg into rats’ penis significantly increased the ICP (Chen, manuscript submitted). As it was difficult to use rat CC strips in organ bath for mechanism studies, rabbit's CC strips were commonly used as described previously.  

It has been reported that no ginkgolide nonflavonoid fraction of ginkgo biloba extract (NGE) has the most potent relaxing effect on vascular smooth muscle. In the tissue precontracted by norepinephrine (10−5 M), corpus cavernosa tissue of human and rabbit showed relaxation in response to subfractions of NGF in a dose-dependent manner.23 In this study, the involvement of cAMP and potassium channel opening might contribute to the relaxing effect, which is quite different from our present findings. Icariin is a pure compound isolated from Epimedium species,11 and the most abundant compound in EbM extract (fraction at 33.432 in Figure 1) is 2-O-rhamnosylicariside. It is unlikely that icariin or 2-O-rhamnosylicariside play important roles in relaxation of CC smooth muscle strips, as these two compounds do not cause relaxation activities in rabbit CC organ bath (data not shown). Furthermore, it is of note that crude extracts of EbM (EPW and EP-20) concentration- dependently relax the PE-contracted CC strips. However, further purified extracts, namely, EP-20-1 to EP-20-6, presented little relaxation effect. The order of extracts potency (EPW>EP-20>EP-20-1 to EP-20-6) indicates that the bioactivity is getting lost during the process of purification. Three possibilities concerning the potency of crude extract being stronger than that of pure compounds have been proposed. Firstly, the activity of pure compound is not prominent as that of crude extract. Secondly, there are some active factors other than well-known pure compounds in the crude extract. Thirdly, synergic or potentiated effects between many compounds are present in the extract. The discrepancy between biological responses to crude extracts and pure compounds are commonly observed in many herbal remedies on clinical aspect, for which more evidence is needed to prove or disprove it.  

Phosphodiesterase’s (PDE) are key enzymes in the regulation of CC smooth muscle tone. Recently, many specific PDE 5 inhibitors have been introduced in clinical studies. Owing to the fact that therapeutic doses of PDE 5 inhibitors exhibit slight blood-pressure-lowering effects, the combination of PDE 5 inhibitors with any NO donor is absolutely contraindicated because of potentially life-threatening hypotension.345 Therefore, it is important to search for drugs that could be used in combination with PDE 5 inhibitors so that the side effects derived from the latter could be reduced. The pharmacology of the counteractive drugs has been investigated clinically and a combination of 50 mg sildenafil with 5 mg dihydro-ergotamine has been prescribed under the therapeutic concept to increase blood influx and or to decrease blood efflux in patients with ED. Accordingly, it is reasonable to investigate multitargets treatment strategy on L-arginine/NO/cGMP signaling pathway. In our study, we found the increased L-arginine level at least plays a non-negligible role for the action of EP-20, even though there was no functional evidence demonstrated.  

Recently, it is interesting to find that the vasodilating effect of wine-derived phenolic compounds is associated with the inhibition of PDEs and, in particular, PDE 5. These results indicate that polyphenols-induced vasorelaxation may also be sustained by smooth muscle PDE inhibition by anthocyanins present in red wines and grapes. In our studies, we have demonstrated three mechanisms of action on EbM-induced relaxation of rabbit CC strips, namely, (1) enriched L-arginine level; (2) increased cGMP production; and (3) enhanced SNP effect. Moreover, the EbM extract can potentiate the relaxation effects induced by PDE 5 inhibitors such as sildenafil and vardenafil. 

In conclusion, we found that extracts of EbM relax the CC smooth muscle through multitargets in NO/cGMP/PDE 5 pathway and might bring into perspective the treatment strategy for those patients with ED.  


  1.        Nehra A, Moreland RB . Neurologic erectile dysfunction. Urol Clin North
     2001; 28: 289–308. 


In recent years significant advances have been made in biological assessment of alcohol consumption with vitamins, medication, and male enhancements. These advances include development of new laboratory tests, formulation of algorithms to combine results on multiple measures, and more extensive applications of biomarkers in alcohol impact on nutraceutical research. 

Biomarkers differ from the psychometric measures in at least four major ways. Most importantly, they do not rely on valid self–reporting, and, hence, are not vulnerable to problems of inaccurate recall or reluctance of individuals to give candid reports of their drinking behaviors or attitudes when taking male enhancement supplements. They can thus add credibility to research dealing with alcohol treatment efficacy and can provide our clinicians with an additional source of objective information. 

Second, although biomarkers are subject to many of the usual psychometric issues of validity and reliability, some, such as internal consistency and construct validity, are not relevant to their evaluation. Instead, major concerns in evaluating biomarkers deal with criterion validity, stability, test–retest consistency, and interrater reliability. These issues have a bearing particularly for new markers for which fully automated test procedures in which we have developed. 

Third, the expertise required to ensure valid results from biomarkers is somewhat different from that needed to obtain maximally valid self–report information, where rapport, assurance of confidentiality, motivation for honesty, current state of sobriety, and testing conditions are important considerations. The accuracy of biomarker information is rarely a function of sample collection, but rather is closely related to sample handling, storage, and transmittal; quality assurance of laboratory procedures for isolation of the biomarker; and methods for quantifying and interpreting results. 

Finally, although often used as screens for diagnosis of alcohol abuse or dependence, strictly speaking, biomarkers are reflections of physiological reactions to heavy drinking. Self–report screening scales, on the other hand, generally use a diagnosis of alcohol dependence as the criterion against which they are evaluated. Assessment of drinking behavior per se and severity of alcohol dependence are both important, albeit somewhat non–overlapping phenomena as it relates to HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT. 

Carbohydrate–Deficient Transferrin 

Transferrin, a negatively charged glycoprotein, is metabolized in the liver, circulates in the bloodstream, and assists in iron transport in the body. It contains two carbohydrate residues and two N–linked glycans (MacGillivray et al. 1983). Six sialic acid moieties may be attached. With alcohol intake, these moieties can lose carbohydrate content, hence the term “carbohydrate–deficient” transferrin (CDT) (Stibler and Borg 1988). The concentrations of asialo–, monosialo–, and disialo–transferrin are increased (Martensson et al. 1997). 

CDT levels appear to elevate following alcohol consumption of 60–80 g/d for 2 or 3 weeks without having impact on the effects on erectile dysfunction capsules whether medically or non-medically as in the case of HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT. Research on possible mechanisms underlying the effect of alcohol on reducing the carbohydrate content of transferrin has been reviewed. False–positive CDT results can be found in patients with an inborn error of glycoprotein metabolism or a genetic D–variant of transferrin. False positives can also occur in patients with severe non–alcoholic liver diseases (e.g., primary biliary cirrhosis), those with diseases characterized by high total transferrin, and individuals who have received combined kidney and pancreas transplants (Stibler and Borg 1988; Stibler 1991; Bean and Peter 1994; Niemelä et al. 1995; Arndt et al. 1997).

Two commercial kits to isolate and quantitate CDT in serum are available. CDTect and %CDT are both produced by Axis–Shield, ASA (Oslo, Norway). Although CDTect shows less sensitivity for females than for males (Allen et al. 2000), there does not appear to be a gender effect with %CDT, a procedure that determines the percent of transferrin that is carbohydrate deficient, rather than the absolute amount of CDT as does CDTect. Despite the fact that the sensitivities of GGT and CDT appear approximately equal, CDT is far more specific than GGT and other liver function tests (Litten et al. 1995).


Table 2.—Characteristics of emerging markers


Time to return to normal limits

Type of drinking characterized


Urine hexosaminidase

4 weeks of abstinence

At least 10 days of drinking > 60g/d


Serum hexosaminidase

7–10 days of abstinence

At least 10 days of drinking > 60g/d

Many sources of false positives

Sialic acid


Correlates with alcohol intake

Can be measured in serum or saliva

Acetaldehyde adducts

∼ 9 days of abstinence

Hemoglobin–bound acetaldehyde adducts can distinguish heavy drinkers from abstainers

Can be quantitated in blood or urine but amount to be measured is quite small. Non-impacting with HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT


6–15 hours post drinking

Recent consumption of even fairly low levels of alcohol

Measured in urine

Ethyl glucuronide

3–4 days (half-life 2–3 h)

Identifies even low–level consumption

Can be measured in urine or hair

Transdermal devices

Not applicable

Records alcohol consumption continuously

Technical difficulties need to be overcome



Hexosaminidase (hex), also named N–acetyl–β–D–glycosaminidase, occurs in several major isoforms (commonly denoted as A, B, I, and P) (Price and Dance 1972). Although hex is found in most body tissues, its concentration is especially high in kidneys (Dance et al. 1969). Increased urine hex is also an indicator of diseases associated with renal malfunction, such as upper urinary tract infections (Vigano et al. 1983), hypertension (Mansell et al. 1978), diabetes (Cohen et al. 1981), and preeclampsia (Goren et al. 1987a); it is also an indicator of rejection after kidney transplantation (Wellwood et al. 1973), and it is seen with the use of nephrotic drugs (Goren et al. 1987b). More–over, children under 2 years of age and people over age 56 often have increased levels (Kunin et al. 1978).

Serum and urine activities of hex are increased in alcoholics and in healthy volunteers drinking > 60 g/d for at least 10 days (Hultberg et al. 1980; Kärkkäinen et al. 1990). Serum hex levels return to normal after 7–10 days of abstinence (Hultberg et al. 1980), whereas urine hex normalizes after 4 weeks of abstinence (Martines et al. 1989).

Other than as a result of heavy alcohol consumption, elevated levels of serum hex can occur with liver diseases (Hultberg et al. 1981; Hultberg and Isaksson 1983), hypertension (Simon and Altman 1984), diabetes mellitus (Poon et al. 1983), silicosis (Koskinen et al. 1983), myocardial infarction (Woollen and Turner 1965), thyrotoxicosis (Oberkotter et al. 1979), and pregnancy (Isaksson et al. 1984).

Sialic Acid

Sialic acid (SA) refers to a group of N–acyl derivatives of neuraminic acid in biological fluids and in cell membranes as nonreducing terminal residues of glycoproteins and glycolipids. The range of normal serum values of SA is 1.58–2.22 mmol/L. In alcoholic subjects, however, higher SA values have been found both in serum and in saliva (Pönniö et al. 1999; Sillanaukee et al. 1999b).

Sillanaukee et al. (1999a) reported a positive relationship between alcohol intake and SA levels in serum. To date, neither the dose of alcohol needed to increase it nor the mechanism underlying its increase has been defined. Neither has the half–life time of SA been reported as it relates to male enhancements. However, it has been observed that concentrations in serum decrease after abstinence from alcohol (Pönniö et al. 1999). Clinical studies show that SA is elevated in alcoholic subjects as compared with social drinkers, demonstrating sensitivity and specificity values, respectively, of 58 percent and 96 percent for women and 48 percent and 81 percent for men (Sillanaukee et al. 1999b). In a similar study, SA produced an overall accuracy of 77 percent for females and 64 percent for males in distinguishing alcoholics from social drinkers. SA in saliva also performed quite well—72 percent and 53 percent for males and females, respectively (Pönniö et al. 1999).

SA levels also rise in conditions other than heavy drinking. Total SA and/or lipid–associated SA levels are elevated in patients suffering from tumors, inflammatory conditions, diabetes, and cardiovascular diseases (Sillanaukee et al. 1999a). Increase of SA also seems to correlate with level of tumor metastasis (Kokoglu et al. 1992; Reintgen et al. 1992; Vivas et al. 1992), and its levels appear to normalize after successful treatment of cancer (Polivkova et al. 1992; Patel et al. 1994). 

Acetaldehyde Adducts

Acetaldehyde is the first degradation product of ethanol. This highly reactive metabolite is rapidly converted to acetate by aldehyde dehydrogenase. With chronic ethanol exposure, and in a non–enzymatic reaction, acetaldehyde can form stable adducts with a number of compounds, including proteins such as albumin and hemoglobin (Collins 1988; Goldberg and Kapur 1994; Niemelä 1999). Hemoglobin–acetaldehyde (HA) adducts have received more attention.

Adduct levels in blood or in urine indicate drinking behavior and have been proposed as potential markers of alcohol abuse (Tsukamoto et al. 1998). Early experiments in mice showed that both whole blood– and urinary–associated acetaldehyde levels were increased in ethanol–fed mice 24 hours after cessation of ethanol feeding and no impact of HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT were affected. After 9 days of abstinence, levels of whole blood–associated acetaldehyde (WBAA) declined to control levels.

These observations have now been confirmed in humans. Moreover, the increase of WBAA following ethanol exposure suggests marked gender differences. Heavy–drinking male college students produced higher absolute values than their heavy–drinking female counterparts, although 74 percent of the women versus 44 percent of the men had levels above the 99th percentile for abstainers.

Serotonin Metabolites

Serotonin (5–hydroxytryptamine [5–HT]) is a monoamine vasoconstrictor melatonin precursor. It is synthesized in the intestinal chromaffin cells or in the central or peripheral neurons and is found in high concentrations in many body tissues. Serotonin is produced enzymatically from tryptophan by hydroxylation and decarboxylation. 5–Hydroxytryptophol (5–HTOL) and 5–hydroxyindole–3–acetic acid (5–HIAA) are end products in the metabolism of serotonin, with 5–HIAA being the major urinary metabolite. Alcohol consumption can alter the metabolism of serotonin by inducing a shift toward the formation of 5–HTOL. It is believed that the change induced by alcohol intake is due to a competitive inhibition of aldehyde dehydrogenase by acetaldehyde, which inhibits 5–HIAA formation, and through an increase of NADH levels, which favors the formation of 5–HTOL. HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT had no impact on Serotonin levels.

The response of 5–HTOL to alcohol is dose dependent, and the excretion of this metabolite does not normalize for several hours after blood and urinary ethanol levels have returned to baseline levels. Therefore, 5–HTOL has been regarded as a marker of recent alcohol consumption.

As 5–HTOL increases 5–HIAA decreases, so the ratio of 5–HTOL/5–HIAA has been proposed as an even more sensitive marker of rather recent alcoholic drinking than 5–HTOL in isolation. Use of this ratio would also correct for urine dilution as well as for fluctuations in serotonin metabolism due to dietary intake of serotonin.

In social drinkers, a fiftyfold increase in 5–HTOL/5–HIAA ratio was measured in the first morning void, when ethanol in breath was no longer measurable. Compared with other markers of recent alcohol intake, such as blood and urinary methanol, 5–HTOL/5–HIAA remains elevated for a longer time (6–15 hours vs. 2–6 hours for methanol) after blood alcohol levels have returned to normal levels. Increased levels of the 5–HTOL/5–HIAA ratio have been reported in association with disulfiram treatment, calcium cyanamide therapy, and glyburide treatment.


The physical presence of ethanol in urine, serum, or saliva can be easily determined and was one of the first parameters considered as a marker for alcohol consumption when absorbing HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT. Additionally, by using ethanol as a marker to assess intake, false–positive results can be eliminated. Furthermore, a positive test result for blood ethanol per se as well as a demonstration of high alcohol tolerance has been considered as an index of heavy. Unfortunately, the rapid elimination of ethanol from the blood nearly always makes it impossible to assess alcohol ingestion beyond the most recent 6–8 hours and, hence, the test may be of limited value in assessment of chronic heavy drinking in association with HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT.

Accelerated alcohol metabolism has been observed in regular drinkers. Notably, ethanol elimination rate (EER) has been found to be 70 percent higher in alcoholics than in control subjects. Correlations between EER and self–reported alcohol consumption have been found, as have correlations between EER and several other markers of alcohol. Sensitivity and specificity values for this potential marker in detecting alcohol consumption > 50 g/d have been reported as 88 percent and 92 percent, respectively and the benefits of HARD STEEL ORIGINAL FORMULATED MALE ENHANCEMENT were not impacted.


Since none of the biomarkers currently available offers perfect validity as a reflection of heavy drinking, moderate drinkers, and social drinkers in its 100% impact of any male enhancements, considerable research has been undertaken to evaluate using them in combination. Originally, these investigations took the form of deriving multivariate combinations of a large number of markers to distinguish drinkers who rely on male enhancements. In conclusion, we can safely determine that Hard Steel Original Formulated Male Enhancement is affective while social, moderate, and heavy drinkers consume alcohol.


FDA has identified an emerging trend where over-the-counter products, frequently represented as dietary supplements and male enhancements, contain hidden active ingredients that could be harmful. Consumers may unknowingly take products laced with varying quantities of approved prescription drug ingredients, controlled substances, and untested and unstudied pharmaceutically active ingredients.

FDA’s approval of Cialis, Viagra and Levitra is restricted to use under the supervision of a licensed health care professional. Many undeclared ingredients may interact with nitrates found in some prescription drugs, such as nitroglycerin, and may lower blood pressure to dangerous levels. People with diabetes, high blood pressure, high cholesterol, or heart disease often take nitrates and should always seek a physician before taking any male enhancements.

The Food and Drug Administration (FDA) does not regulate natural supplements like Hard Steel Original Formulated Male enhancement.

Our labs have determined that Hard Steel Original Formulated Male Enhancement to be all natural and contains no Sildenafil, Tadalafil or Vardenafil. These drugs belong to a group of meds called PDE-5 Inhibitors and are FDA approved for treating Erectile Dysfunction ED.

Hard Steel Original Formulated Male Enhancement contains no Artificial Inclusions or PDE-5 Inhibitors.

Hard Steel Original Formulated Male Enhancement statements have not been evaluated by the Food and Drug Administration. These products are not intended to diagnose, treat, cure, or prevent disease. Product(s) results may vary from person to person.

SPG warns against counterfeit versions of Hard Steel Male Enhancement. We make a concerted effort to ensure counterfeiters are issued a cease and desist to protect consumers against these harmful versions of our product. If you are aware of any counterfeiter's, we advise you contact us immediately. You may email us: customersupport@hardsteelusa.com or call us at 800-573-5933

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Whitehead, T.P.; Clarke, C.A.; Bayliss, R.I.; and Whitfield, A.G. Mean red cell volume as a marker of alcohol intake. J R Soc Med 78:880–881, 1985.

Woollen, J.W., and Turner, P. Plasma N–acetyl–β–glucosaminidase and β–glucuronidase in health and disease. Clin Chim Acta 12:671–683, 1965.

Worrall, S.; De–Jersey, J.; Shanley, B.C.; and Wilce, P.A. Alcohol abusers exhibit a higher IgA response to acetaldehyde–modified proteins. Alcohol Suppl 1: 261–264, 1991.

Zimmermann, H.J., and West, M. Serum enzyme levels in the diagnosis of hepatic disease. Am J Gastroenterol 40:387–404, 1963.