Unique combination of beetroot and hawthorn berry promotes nitric oxide production and reduces triglycerides in humans.
Article Type: Report
Subject: Triglycerides (Physiological aspects)
Triglycerides (Research)
Nitric oxide (Physiological aspects)
Nitric oxide (Research)
Cardiovascular diseases (Research)
Cardiovascular diseases (Care and treatment)
Hawthorns (Health aspects)
Hawthorns (Research)
Authors: Zand, Janet
Bryan, Nathan S.
Pub Date: 05/01/2011
Publication: Name: Townsend Letter Publisher: The Townsend Letter Group Audience: General; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 The Townsend Letter Group ISSN: 1940-5464
Issue: Date: May, 2011 Source Issue: 334
Topic: Event Code: 310 Science & research
Product: Product Code: 2813772 Nitric Oxide NAICS Code: 32512 Industrial Gas Manufacturing SIC Code: 2813 Industrial gases
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 255389355
Full Text: Keywords: nitric oxide, dietary supplement, triglycerides, cardiovascular disease

Abstract

There is an emerging paradigm that certain foods promote nitric oxide production from the stepwise reduction of nitrate to nitrite to nitric oxide, providing an endothelium-independent source of bioactive nitric oxide. We hypothesize that a unique formulation containing nitrate-rich beetroot along with hawthorn berry shown to have a robust nitrite reductase activity would improve nitric oxide status in humans and modify cardiovascular risk factors. The trial was conducted at the Houston Institute for Clinical Research in Houston, Texas. Inclusion criteria for this double-blind, placebo-controlled study were patients over 40 years old with 3 or more of the following cardiovascular risk factors: hypertension, obesity, hyperlipidemia, smoking, sedentary, family history of cardiovascular disease, and diabetes. Subjects were instructed to take either the NO active supplement called Neo40 Daily or placebo twice daily on an empty stomach for 30 days. Patients' taking Neo40 Daily twice a day for 30 days led to a significant increase in both plasma nitrite (p < 0.01) and nitrate (p < 0.0001), indicating an increase in systemic NO availability. There was a statistically significant reduction in 72% of patients with elevated triglycerides (> 150mg/dL) after 30 days compared with their starting levels prior to taking Neo40 Daily (168 [+ or -] 17 mg/dL vs. 232 [+ or -] 19 mg/dL; p = 0.02). The strategy of formulating a combination of natural products and botanicals chosen specifically for their nitric oxide activity as in Neo40 Daily shows promise in restoring NO homeostasis in human subjects at risk for cardiovascular disease for use as a dietary supplement.

Introduction

Nitric oxide (NO) is one of the most important signaling molecules in the body. (1) Although NO is involved in virtually every organ system within the body, it is known primarily for maintaining normal blood pressure and blood flow to tissues and protecting the cardiovascular system from insult and injury. A deficiency in NO production or availability is a hallmark of numerous disease conditions. Experimental and clinical studies provide evidence that defect of endothelial NO function, or endothelial dysfunction, is not only associated with all major cardiovascular risk factors such as hyperlipidemia, diabetes, hypertension, smoking and severity of atherosclerosis, but also has a profound predictive value for future atherosclerotic disease progression. (2-5)

It has been previously demonstrated that plasma nitrite reflects acute changes in endothelial nitric oxide synthase (NOS) activity in various mammals and thus may provide an accurate measurement of patients at risk for cardiovascular events. (6) A report by Kleinbongard et al. demonstrated that plasma nitrite levels progressively decrease with increasing cardiovascular risk load indicative of a reduction in the production of NO. (7) Risk factors considered include age, hypertension, smoking, and hypercholesterolemia.

The original discovery for the production of NO is through the oxidation of the semiessential amino acid L-arginine by NOS. NOS enzymes produce NO by catalyzing a five-electron oxidation of the guanidino nitrogen of L-arginine. Remarkably, though, strategies to restore NO homeostasis by supplementing L-arginine and antioxidants have consistently failed in clinical trials. This is due to the fact that the L-arginine-NO pathway is dysfunctional in patients with endothelial dysfunction. In fact, it is now known that feeding the nitric oxide pathway through L-arginine supplementation may not be effective and can actually be detrimental in at-risk patients. (8) Interventions to enhance NO production with organic nitrates such as nitroglycerin have been equivocal in human trials. Early entry therapy with nitrates does not significantly improve survival in myocardial infarction but increases the beneficial effects of the angiotensin converting enzyme (ACE) inhibitor enalapril by 50%. (9) Short-term experimental and clinical investigations suggest that nitrate tolerance induced by nitroglycerin is associated with toxic effects in the vasculature. (10) Chronic and long-term organic nitrate therapy has been associated with reduced survival when used in patients with coronary artery disease. (11), (12) The observed endothelial dysfunction induced by a continuous treatment with nitroglycerin may be an additional risk for patients who receive continuous nitroglycerin to treat conditions such as unstable angina and acute heart failure. (9) As such, all current strategies have failed, so there is a large unmet need for safe and effective NO-based interventions or strategies.

Despite NO's known and accepted importance in human physiology, there have been no hallmark therapeutic breakthroughs or effective strategies developed to enhance or restore NO homeostasis in humans at risk for cardiovascular disease. Developing such strategies or technologies to restore and replete NO availability, both through restoration of endothelial NO production and in an endothelium-independent manner, is of paramount importance and could save millions of lives worldwide and lessen the burden on the health-care system.

There is an emerging paradigm whereby functionalities of certain foods and diets may confer nitric oxide activity. (13), (14) Nitrate in the diet (primarily from green leafy vegetables or beetroot) is reabsorbed in the proximal intestines and concentrated in the salivary glands. It was known from the literature that the salivary glands extract nitrate from plasma, but the reason for this active process was not explained. (15) This active process leads to levels of salivary nitrate that are 10- to 20-fold higher than in plasma. Oral facultative anaerobic bacteria residing mainly in the crypts of the tongue then reduce nitrate to nitrite by the action of nitrate reductase enzymes. (16), (17) These bacteria use nitrate as an alternative electron acceptor to gain ATP in the absence of oxygen. Approximately 25% of ingested nitrate is secreted in saliva, where some 20% (or approximately 5% to 8% of the nitrate intake) is converted to nitrite by commensal bacteria on the tongue. (18) This bacterial nitrate reduction results in salivary levels of nitrite that are 1000-fold higher than those found in plasma. (19) When nitrite-rich saliva meets the acidic gastric juice, nitrite is protonated to form nitrous acid ([HNO.sub.2]), which then decomposes to NO and a variety of other nitrogen oxides that are then transported throughout the body. (18), (20) This nitrate-nitrite-NO pathway has been shown in both animals and humans to reduce blood pressure, restore endothelial function, protect from myocardial ischemia-reperfusion injury, prevent microvascular inflammation, and reduce triglycerides and C-reactive protein. (21-25)

Therefore there exists a pathway for increasing NO bioavailability within the body through supplementing NO-rich or NO-active food components that contain adequate amounts of nitrate and/or nitrite and antioxidants to facilitate reduction to NO and to inhibit any unwanted nitrosation reactions. Utilizing a unique formulation from intellectual property whereby this pathway is optimized with natural products developed out of the University of Texas Health Science Center in Houston, Neogenis Laboratories has the exclusive license to commercialize the technology. The all-natural formulation, called Neo40 Daily, provides an innovative delivery system for generating NO in an endothelium-dependent and -independent manner. The formula is based on a proprietary blend of NO-active herbs that act to replenish and restore NO production in the human body by exploiting the nitrate-nitrite-NO pathway. (26) Our hypothesis, based on a number of preclinical and clinical studies, is that utilizing a nitrate source such as beetroot along with an effective nitrite reductase as found in hawthorn would enhance NO production and modify cardiovascular biomarkers of risk. This formulation has been recently tested by an uninterested third party in a double-blind, placebo-controlled clinical trial to modify biomarkers of cardiovascular risk and increase steady state levels of NO biomarkers in human subjects over 40 years old. The information gained from this study will advance our understanding of human nutrition by providing a mechanistic explanation for the health benefits of certain foods, primarily nitrate-rich green leafy vegetables or beets by their ability to generate nitric oxide in the proper context.

Methods and Materials

Patient Enrollment

The trial was conducted at an Institutional Review Board-approved site at the Houston Institute for Clinical Research in Houston, Texas. Our inclusion criteria for this double-blind, placebo-controlled study were patients over age 40 with 3 or more of the following cardiovascular risk factors: hypertension, obesity, hyperlipidemia, smoking, sedentary lifestyle, family history of cardiovascular disease (CVD), and diabetes. Patients were screened to ensure that they met the inclusion criteria, informed of the study protocol, and signed a consent form. Subjects' ages ranged from 42 to 79 with a median age of 56, with 63% male and 37% female. Patients were excluded if they were taking organic nitrates for angina or nebivolol as a beta blocker. Our objective was to investigate if increasing NO through the nitrate-nitrite-NO pathway could modify known risk factors for cardiovascular disease such as triglycerides and C-reactive protein. Our preclinical studies in mice revealed that restoring NO homeostasis through this pathway could reduce triglycerides and C-reactive protein. (24) We began by enrolling only patients with elevated triglycerides (>150mg/dL) and at least 2 of the other risk factors from above. Subjects were instructed to take either the Neo40 Daily or placebo twice daily on an empty stomach for 30 days. In addition, patients were asked to maintain their normal dietary pattern and physical activity levels. After 30 days, the patients returned to the clinic for a blood draw and were asked to complete a survey. Patients were randomized using a randomization table to receive Neo40 Daily or placebo and asked to fast for 12 hours prior to all blood draws. Blood samples were sent to LabCorp for the following analysis: complete blood count (CBC) with differential/platelet; complete metabolic panel--14; lipid panel; bilirubin, total/direct, serum; phosphorus, serum as well as C-reactive protein. Separate blood samples were collected and analyzed for nitrite and nitrate at Dr. Bryan's laboratory with a well-validated and sensitive HPLC system. (27) A total of 30 patients have been enrolled to date, which include 23 on Neo40 and 7 receiving the placebo. The randomization schedule was intentionally and preferentially weighted to the Neo40 group in the first 30 patients. For the sample size calculations, we assumed an alpha of 0.05, power of 0.80, and a two-tailed t-test. The software package used was Stata Statistical Software, Release 10 (College Station, Texas, 2007). Sample size was calculated based on the steady state plasma nitrite levels and fasting serum triglycerides. Power analysis based on preliminary data revealed that 25 subjects would be sufficient for significant changes in plasma nitrite levels and the ability to determine a 20% reduction in triglycerides. Both the clinical coordinator and data analyst were blinded to the study. All investigators completed and passed an institutional course on Health Insurance Portability and Accountability Act (HIPAA).

Product Formulation

By screening over 100 herbs and botanicals as well as desiccated food products, we identified beetroot as containing the highest nitrate content of any tested. This finding is consistent with others, as beetroot is routinely used as a source of nitrate in experimental and human studies. (21), (23), (28) Commensal bacteria are responsible for the first reductive step of nitrate to nitrite. The reduction of nitrite to NO is a very inefficient process and occurs primarily under low-oxygen conditions. (29) Our most recent data on traditional Chinese medicines revealed that some herbs have a robust capacity to reduce nitrite to NO in an oxygen-independent manner. (30) We reported that gua lou and borneol contained the highest nitrite reductase of the traditional Chinese herbs, with 133 pmol NO/mg/min and 875 pmol NO/mg/min respectively.

We have since tested over 100 herbs and botanicals for their ability to reduce nitrite to NO. We found hawthorn berry to have the highest nitrite reductase activity of any botanical tested, and its activity is unaffected by oxygen (activity = 434,369 pmol NO/mg/min or 500 times higher than the Chinese herbs). The hawthorn also contains 5% polyphenols, which also help facilitate nitrite reduction while inhibiting nitrosation reactions. (31) The formulation also contains FDA-approved and generally regarded as safe (GRAS) amounts of sodium nitrite for use as a preservative as well as a substrate for NO production. We have discovered a specific product ratio with the highest NO activity. This proprietary formulation with precise ratio of ingredients has been submitted as an unpublished patent application through the University of Texas Health Science Center at Houston. We had these ingredients sourced and formulated into a unique quick-dissolving lozenge by a good manufacturing practice (GMP)-certified facility (Solara Inc., Miami, Florida). All ingredients were tested for microbiology and heavy metals. The Neo40 Daily product ingredients list and packaging was submitted to FDA Office of Compliance by Nleogenis Labs Inc. for use as a dietary supplement (registration # 3008524085). All ingredients are listed on the FDA GRAS list; therefore no investigational new drug (IND) application is required. The product label is shown in Figure 1.

Nitrite Reductase Activity

Nitrite reductase activity was quantified by adding 100 mg of the Neo40 product into a reaction vessel containing 5 ml of PBS, purged with nitrogen or medical grade air (21% oxygen) at 37 [degrees]C. The area under the curve was integrated to quantify the total amount of NO generated.

Results

To test the NO activity of our rationally designed combination in its final product formulation, we injected 100 mg of the Neo40 product into a buffered solution (pH 7.4) connected to an ozone-based chemiluminescent analyzer (EcoPhysics) and monitored the NO release profile. Immediately upon dissolving, the Neo40 Daily produced NO with a sustained release. We stopped the analysis after 100 minutes. The NO release kinetics is shown in Figure 2A. In this in vitro system, the Neo40 Daily has a half-life of roughly 1 hour with respect to NO release. Based on this information, we then tested the pharmacokinetics of this formulation when administered to human subjects. Study participants were asked to place the lozenge in the mouth and let dissolve. Baseline venous blood was drawn and then sampled every 5 minutes for 1 hour. The Neo40 Daily is designed to act as a quick-dissolving lozenge that melts in your mouth and enriches your saliva. When allowed to dissolve in the mouth, this leads to a slow and steady rise in plasma nitrite of humans as shown in figure 2B. These data indicate that the NO activity of the lozenge is absorbed and transported through the blood as nitrite.

[FIGURE 2 OMITTED]

To demonstrate proof of concept that Neo40 Daily could restore systemic NO levels over a period of 30 days, we enrolled patients with known cardiovascular risk factors, over age 40, and with hyperlipidemia. Patients' blood was analyzed at baseline for screening and inclusion. If subjects met inclusion criteria, they were administered placebo or Neo40 Daily. After 30 days, subjects returned for follow-up labs for plasma levels of nitrite and nitrate, which are markers of NO production. It has been reported that normal plasma nitrite levels in healthy human volunteers is 250-500nM. (32), (33) Taking Neo40 Daily twice a day for 30 days led to a modest but significant increase in both plasma nitrite (p < 0.01) and nitrate (p < 0.0001), indicating an increase in systemic NO availability. Results are shown in Figure 3 below. There was no significant change in the plasma levels of nitrite and nitrate from the placebo group (data not shown).

[FIGURE 3 OMITTED]

Smoking, diabetes, high blood pressure, high cholesterol and triglycerides, sedentary lifestyle, obesity, and family history of cardiovascular disease are all established risk factors for developing cardiovascular disease. Blood values can reveal levels of cholesterol and triglycerides as well as systemic inflammation based on C-reactive protein. We compared levels of cholesterol, triglycerides, and C-reactive protein of the patients taking the Neo40 Daily product at baseline and after 30 days on the product. There was no significant change in total cholesterol after 30 days on Neo40 Daily or placebo (data not shown). Our preclinical data in mice indicated that restoring NO through the nitrate-nitrite-NO pathway could reduce triglycerides. (24) Therefore we only enrolled patients with triglycerides greater than 150mg/dL. Of these patients with baseline triglycerides greater than 150mg/dL, 72% of the subjects on Neo40 Daily experienced a reduction in triglycerides after 30 days. The individual changes in triglycerides are shown in Figure 4A (p. 67). There was a statistically significant 27% reduction in all patients' triglycerides after 30 days compared with their starting levels prior to taking Neo40 (168 [+ or -] 17mg/dL vs. 232 [+ or -] 19mg/dl; p = 0.02). These data are illustrated in Figure 4B.

[FIGURE 4 OMITTED]

Only 4 of our 18 patients taking Neo40 Daily had elevated C-reactive protein (> 4.9mg/dL) at baseline. Normal CRP levels are between 0.0 and 4.9 mg/L However, all 4 patients who started with elevated CRP experienced a reduction after 30 days on Neo40 Daily. There was anywhere from a 6% to 37% reduction in CRP in these 4 subjects. We will need to enroll more patients with elevated CRP in order reach any conclusions on CRP.

Upon completion of the study, participants were asked to complete a subjective questionnaire based on how they felt. The results do not include patients taking placebo. More than 50% of the respondents indicated that they had more energy and felt less anxious. Most strikingly, over 75% of the subjects indicated that they would continue taking the product if available. There was no adverse health effects reported.

Discussion

Nitric oxide (NO) is one of the most important signaling molecules produced within the body. Insufficient NO production is associated with a number of diseases, particularly cardiovascular disease. Neogenis Labs Inc. has developed a unique and novel formulation of natural products designed to exploit the nitrate-nitrite-NO pathway to restore NO homeostasis in adults over 40 years old, based on patent-pending technology developed out of the University of Texas Health Science Center at Houston. As we age, we lose our ability to produce NO, which is the earliest event that puts us at risk for a host of conditions such as atherosclerosis, heart attack, stroke, and even Alzheimer's disease later in life. (34), (35) By age 40, most adults produce only about 50% of the NO produced when they were 20. (34-36) The strategy of formulating a combination of natural products and botanicals chosen specifically for their nitric oxide activity shows enormous promise in restoring NO homeostasis in human subjects at risk for cardiovascular disease for use as a daily dietary supplement.

Despite NO's being recognized by the scientific and medical community as one of the most important molecules produced within the body and being named "Molecule of the Year" by Science in 1992 and a Nobel Prize in Physiology or Medicine awarded in 1998, there are currently only three products on the market directly related to NO: (1) organic nitrates such as nitroglycerin for the treatment of acute angina; (2) inhalative NO therapy for neonates for treatment of pulmonary hypertension due to underdeveloped lungs; (3) phosphodiesterase inhibitors such as sildenafil (Viagra). These classes of drugs do not directly affect NO production but act through affecting the downstream second messengers of NO known as cyclic guanosine monophosphate (cGMP). Therefore, there is an unmet need for safe and effective strategies to restore and enhance NO production, particularly in patients with endothelial dysfunction.

The first pathway to be discovered for the endogenous production of NO was through the five-electron oxidation of the guanidino nitrogen group of L-arginine (a semiessential amino acid) by a group of enzymes called nitric oxide synthase (NOS) localized to the vascular endothelium. For years, scientists and physicians have investigated L-arginine supplementation as a means to enhance NO production. However, patients with endothelial dysfunction, by definition, are unable to convert L-arginine to NO and therefore this strategy has failed in clinical trials. In fact, an article published in the journal of the American Medical Association in 2006 concluded that L-arginine, when added to standard postinfarction therapies, did not improve vascular stiffness measurements or ejection fraction and was associated with higher postinfarction mortality. (8) The Neo40 Daily technology uses a natural source of NO through the reduction of nitrate and nitrite to NO and overcomes the endothelium's inability to generate NO. This is the first-of-its-kind technology using natural products for NO activity in patients insufficient in NO production. Neo40 Daily provides a functional system to produce NO that is derived from natural products. There are numerous preclinical and some clinical trials under way that support this notion. (26), (37)

A daily regimen that can safely and effectively restore NO levels as well as reduce blood biomarkers routinely used to assess patient risks for developing cardiovascular disease is novel with profound public health implications. Our preclinical data in mice fed a high-cholesterol diet revealed that nitrite-based interventions could reduce triglycerides after 9 weeks on a high-fat diet. (24) We were surprised to see a significant reduction in triglycerides in our human subjects after only 30 days. Recently the triglyceride-lowering effects seen here were corroborated in eNOS-deficient mice and in rats whereby 7 weeks of inorganic nitrate administration caused a significant reduction in triglycerides. (38) Furthermore, hawthorn extract has previously been shown to reduce triglycerides in animal models. (39) There are several medications that reduce triglycerides, including fibrates, such as fenofibrate (Lofibra, TriCor) and gemfibrozil (Lopid); niacin (Niaspan); and ethyl esters of omega-3 fatty acids known as Lovaza. Lovaza is a combination of long-chain polyunsaturated fatty acids, EPA and DHA, indicated as an adjunct to diet to reduce triglyceride (TG) levels in adult patients with severe ([greater than or equal to]500 mg/dL) hypertriglyceridemia. Lovaza is FDA-approved for patients with very high triglycerides (> 500mg/dL) and has been shown to reduce triglycerides by up to 43%. (40) However, the vast majority of Americans have elevated triglycerides greater than 150 but less than 500. Other than changing their diet, there is no effective regimen for this population. The results from this study suggest that Neo40 Daily can indirectly restore NO homeostasis and reduce triglycerides, thereby reducing the overall burden of risk for the development of cardiovascular disease.

Dietary supplementation of nitrite and nitrate in animals has shown to reverse endothelial dysfunction, suppress microvascular inflammation, and reduce levels of C-reactive protein in mice subjected to a high-cholesterol diet and protect from ischemia reperfusion injury. (24), (25), (41), (42) Most recently, feeding the nitrate-nitrite-NO pathway has been shown to reverse symptoms of metabolic syndrome. (38) This proof of concept has now been extended to humans supplemented with dietary sources of nitrate. Dietary nitrate has also been shown to reduce blood pressure, inhibit platelet aggregation, and restore endothelial function. (21-23) What is clearly emerging is that there are two pathways for NO production, one through endothelial production through the L-arginine pathway and one through dietary sources of nitrite, nitrate, and antioxidants. This is illustrated below in Figure 5. The L-arginine pathway becomes dysfunctional with age and we therefore need a backup system to compensate. Eating a diet rich in NO activity; that is, sufficient nitrite and nitrate along with antioxidants and botanicals to facilitate reduction to NO, can appear to overcome an insufficiency in endothelial-derived NO. This dietary pathway doesn't appear to be affected by age. However, overuse of antibiotics or antiseptic mouthwashes can affect this pathway by killing off the commensal bacteria that are essential for the first step of nitrate reduction to nitrite. Furthermore, use of proton pump inhibitors can decrease the acid secretions in the stomach, thereby affecting the acidic disproportionation of nitrite to NO. This dietary pathway is reliant upon recognizing foods that are rich in NO activity.

[FIGURE 5 OMITTED]

We believe that the inherent NO bioactivity of certain cardioprotective foods (such as green leafy vegetables) or diets (Mediterranean or DASH diet) is a delicate balance between nitrite and nitrate content as well as antioxidant capacity to facilitate reduction to NO and to inhibit any unwanted nitrosation reactions. The nitrate-nitrite-NO pathway is illustrated below in Figure 6. The two ways to promote NO production are shown. Providing a rich source of nitrate either through direct supplementation with nitrate salts or through nitrate-rich foods such as beetroot will increase circulating levels of nitrite. (19), (23), (43) However, due to the inherent inefficiencies that exist for reducing nitrite to NO along the physiological oxygen gradient, to effectively utilize nitrite to make NO, this step in the pathway must be enhanced. (29) One could accomplish this by supplying more nitrate to generate supraphysiological concentrations of nitrite, or one could introduce a robust nitrite reductase to more effectively reduce nitrite to NO along the physiological oxygen gradient in vivo. We have found hawthorn berry to serve such a role. This system uses physiological concentrations of nitrate and nitrite, supplied by the diet, to effectively generate NO through the stepwise reduction of nitrate and nitrite by supplying the necessary substrates and machinery to perform these steps. The ingredients in Neo40 Daily were selectively and intentionally included to affect each step of the nitrate-nitrite-NO pathway, providing a system for restoring nitric oxide homeostasis safely, effectively, and naturally.

[FIGURE 6 OMITTED]

The role of diet in the prevention and control of morbidity and premature mortality due to noncommunicable diseases has been well established by the vast population-based epidemiological studies carried out during the last decade. (44) NO is essential for maintaining normal blood pressure, preventing adhesion of blood cells to the endothelium, and preventing platelet aggregation. It may be argued that this single event, the inability to generate NO, puts us at risk for diseases that plague us later in life, such as atherosclerosis, heart attack, stroke, peripheral vascular disease, and even Alzheimer's disease. Therefore, developing strategies and new technologies designed to restore NO availability in the body is essential for inhibiting the progression of certain diseases. We believe that we have identified key components in the dietary pathway to promote NO production that can make this pathway more effective and overcome endothelial-derived NO from L-arginine. Although these studies were simply designed to show proof of concept for restoring NO homeostasis through a dietary supplement, therefore modifying risk factors for cardiovascular disease, more studies are needed to more clearly define the role of Neo40 Daily for optimal health and nutrition. Future studies will determine if Neo40 Daily can help reduce the progression of cardiovascular disease and the incidence of heart attack and stroke.

Disclosures

N. S. Bryan has a financial interest in Neogenis Inc. His financial and research conflicts of interest in Neogenis Inc. His financial and research conflicts of interest are managed by UTHSCH Conflicts of Interest Management Plans., developed from and reviewed by the Research Conflicts of Interest Committee and approved by the executive vice president for research at the University of Texas Health Science Center at Houston. J. Zand has a financial interest in Neogenis Inc.

Acknowledgements

The authors would like to thank Drs. lose Kocca, Kathleen Zelman, Norm Hard, and Max Talbott for their helpful comments and guidance. We would also like to thank Franny Rack and Wendy Thurston at the Houston Institute for Clinical Research for their help and enthusiasm in recruiting and screening the subject for this study. This study was funded by Neogenis Labs Inc.

Notes

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(13.) Bryan NS, ed. Food, Nutrition and the Nitric Oxide Pathway: Biochemistry and Bioactivity. DesTech Publishing: Lancaster, PA; 2009:238.

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(16.) Ibid.

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(23.) Webb AJ et al. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrire. Hypertension. 2008;51(3):7841-790

(24.) Stokes KY et al. Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction. Am J Physiol Heart Circ Physiol. 2009;296(5):H1281-H1288.

(25.) Bryan NS et al. Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2007;104(48): 19144-19149.

(26.) Lundberg JO, Weitzberg E, Gladwin MT. The nitratenitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(8): 156-167.

(27.) Bryan N5, Grisham MB. Methods to detect nitric oxide and its metabolites in biological samples. Free Radic Biol Med. 2007;43(5):645-657.

(28.) Vanhatalo A et al. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am J Physiol Regul Integr Comp Physiol. 2010.

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(31.) Bartsch H et al Inhibition of nitrosation. Basic Life Sci. 1993. 61:27-44.

(32.) Kleinbongard P et al. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med. 2006;40(2):295-302.

(33.) Kleinbongard P et al Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals, free Radic Biol Med. 2003;35(7):79O-796.

(34.) Taddei S et al. Age-related reduction of NO availability" and oxidative stress in humans. Hypertension. 2001;38(2):274-279.

(35.) Egashira K et al. Effects of age on endothelium-dependent vasodilation of resistance coronary artery by acetylcholine in humans. Circulation. 1993;88(1):77-81.

(36.) Gerhard M et al. Aging progressively impairs endothelium-dependent vasodilation in forearm resistance vessels of humans. Hypertension. 1996; 27(4):849-53.

(37.) Lundberg JO et al. Nitrate and nitrite in biology, nutrition and therapeutics. Nat Chem Biol. 2009;5(12):865-869.

(38.) Carlstrom M et al. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Set USA, 2010.

(39.) Shanthi S et al. Hypolipidemic activity of tincture of Crataegus in rats. Indian J Biochem Biophys. 1994;31 (2): 143-146.

(40.) Sadovsky R, Kris-Etherton P. Prescription omega-3-acid ethyl esters for the treatment of very high triglycerides. Postgrad Med. 2009;121(4):145-153.

(41.) Bryan NS et at. Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice. Free Radic Biol Med. 2008;45(4):468-474.

(42.) Shiva S et at Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J Exp Med. 2007, 204(9):2089-2102.

(43.) L Sobko T et al. Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide. 2005;13(4):272-278.

(44.) World Health Organization. Report on Diet, Nutrition and the Prevention of Chronic Diseases. 2003.

Correspondence

Nathan S. Bryan, PhD

Center of Cell Signaling

Institute of Molecular Medicine

The University of Texas

Health Science Center at Houston

1825 Pressler St. 530C, Houston, Texas 77030

713-500-2439 office

713-500-2447 fax

Nathan.Bryan@uth.tmc.edu

Beetroot and Hawthorn Berry

Janet Zand, LAc, OMD, Dipl Ac, Dipl CH, was the cofounder and chairman of the board of Zand Herbal Formulas from 1978 to 2002. The company spent over 20 years as a market leader in the herbal industry. Dr. Zand has over 25 years of private practice experience in acupuncture, herbal medicine, homeopathy, and nutrition. She received a bachelor of arts degree from Sarah Lawrence College, and a doctor of Oriental medicine degree from California Acupuncture College. Dr. Zand is the author of Smart Medicine for a Healthier Child, A Parent's Guide to Medical Emergencies, and Smart Medicine for a Healthier Living (Avery Publishing, 1994, 1997, 1998). She holds licenses and memberships with the Commission for the Certification of National Acupuncturists, California Acupuncture Association, The National Council of Acupuncture Schools and Colleges, Occidental Institute Research Foundation, Oriental Healing Arts Institute, International Foundation of Homeopathy, National Center for Homeopathy, Herbal Research Foundation, and American Herbal Products Association. Dr. Zand currently serves on the Biomedical Board for the National Certification Commission for Acupuncture and Oriental Medicine.

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Dr. Nathan S. Bryan is an assistant professor of molecular medicine within the Brown Foundation Institute of Molecular Medicine, part of the School of Medicine at the University of Texas Health Science Center at Houston. He is also on faculty within the Department of Integrative Biology and Pharmacology and Graduate School of Biomedical Sciences at the UT Houston Medical School. Dr. Bryan earned his undergraduate bachelor of science degree hi biochemistry from the University of Texas at Austin and his doctoral degree from Louisiana State University School of Medicine in Shreveport, where he was the recipient of the Dean's Award for Excellence in Research. He pursued his postdoctoral training as a Kirschstein Fellow at Boston University School of Medicine in the Whitaker Cardiovascular Institute. Dr. Bryan's research is dedicated to providing a better understanding of the interactions of nitric oxide and related metabolites with their different biological targets at the molecular and cellular level and the significance of these reactions for physiology and pathophysiology. He has published a number of highly cited papers and authored or edited four books.

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by Janet Zand (1) and Nathan S. Bryan (1), (2)

(1) Neogenis Laboratories Inc.

(2) Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston
Supplement Facts

Serving Size: 1 Lozenge       Servings per container: 30

                         Amount Per Serving      % Daily Value

Vitamin C                      100 mg                167%

  (as magnesium ascorbate and ascorbic acid)

Vitamin B12                   1000 meg              16,667%

  (as methylcobalamin and cyanocobalamin)

Proprietary Nitric Oxide Blend 420 mg *

  for optimal Nitric Oxide activity [dagger]

  Beet Root Powder (root), Hawthorn Berry Extract (berry),

  L-Citrulline, Sodium Nitrite

* Daily Value not established.

Other ingredients: mannitol, modified cellulose, xylitol, natural
orange flavor, magnesium stearate, Stevia, silica.
Figure 1: Product label of Neo40 Daily
Gale Copyright: Copyright 2011 Gale, Cengage Learning. All rights reserved.