FIELD OF INVENTION
The present invention relates to a mouth dissolving (through oral delivery system) nutraceutical
tablet and meth.od for preparing the same. More particularly the present invention relates to a
mouth dissolving nutraceutical tablet and method for preparing mouth dissolving nutraceutical
tablet from the extract of Beet root and hawthorn berry herbs with Sodium nitrite, Ascorbic acid,
Mecobalamin and L Citrulline. Nutraceutical tablet is meant to increase or restore Nitric oxide
level in the body. This is natural way to increase nitric oxide in the hody. Nitric oxide is an
impmtant signaling molecule, which is required for healthy blood circulation.
BACKGROUND OF THE INVENTION
The mammalian biosynthesis of nitric oxide (NO) discovered in the 1980's for its roles in the
immune, cardiovascular and nervous systems established a startling new paradigm in the history
of l:dlular signaling mechanisms. Nitric oxide is a gas that occurs naturally throughout the body.
NO is a powerful signaling molecule that causes the walls of blood vessels to relax, improving
circulation. Its production by vascular endothelium is particularly important in the regulation
ofblood flow. Any decrease in NO production or loss in generation ofNO due to dysfunctional
vascular endothelium is a very likely cause of heart disease. Experimental and clinical studies
also demonstrate that insufficient nitric oxide production. is associated with major cardiovascular
risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and atherosclerosis.
llnf0rtunately, the ability to generate nitric oxide decreases with age resulting in increased risk
of heart and vascular disease.
Prior to this discovery, NO was widely recognized as a toxic molecule: a common air pollutant, a
constituent of cigarette smoke, and a toxic gas, which appears in the exhaust of motor cars and
jet airplanes, causes acid rain, and destroys the ozone layer. Thus, it was essentially
inconceivable that cells would intentionally produce a toxic gas as part of normal physiology.
Referring to Figure 1, NO is now recognized as one ofthe most important signaling molecules in
the body, and is involved in virtually every organ system where it is responsible for modulating
an astonishing variety of effects. The primary targets for NO are metals and thiols. NO can bind
to soluble gualylylcyclase (sGC) and cause an increase in second messenger cGMP, and mediate
a number of physiological functions. This pathway was considered the basis of NO based
signaling until it was recognized that NO elicited a number of physiological and biological
effects that were not dependent upon cGMP. It is now recognized that NO can react directly with
thiyl radicals to form nitrosothiols or other reaction products of NO i.e., riitrite, N20 3, N20 4,
which can post-translationally modify thiols to affect protein structure and function. NO has been
shown to be involved in and affect practically every organ system in the body. One can then
imagine a host of diseases or conditions and multi-systemic symptoms may be caused or affected
by the body's dysregulation of NO production/signaling (referring to Figure 1). Maintaining NO
homeostasis is critical for optimal health and disease prevention. Developing novel NO based
diagnostics and therapies is central to better patient care, especially in the geriatric patient.
The first pathway to be discovered for the endogenO!JS production of NO was involving Larginine,
from a group of enzymes call nitriC oxide synthase (NOS). NOS enzymes produce NO
by catalyzing a five electron oxidation of the guanidine nitrogen of L-arginine. Oxidation of Larginme
to L-citrulline occurs via two successive mono-oxygenation reactions producing NOhydroxy-
L-arginine as an intermediate. Two moles of 0 2 and 1.5 moles of nicotinamide adenine
2
dinucleotide phosphate (NADPH) are consumed per mole of NO formed. NOS enzymes are the
only enzymes known to simultaneously require multiple bound cotactors/prosthetic groups:
flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, glutathione, NADPH,
tetrahydrobiopterin (Bfu) and Ca2+ -calmodulin. There are three isoforms of NOS, the genetic
se~uence of each residing on three distinct chromosomes. One type is constitutive,
Ca +/calmodulin dependent and releases NO for short time periods in response to receptor or
physical stimulation. NO released by this enzyme acts as a transduction mec.hanism underlying
several physiological responses. The other enzyme type is induced after activation of
macrophages, endothelial cells and a numher of other cells by cytokines and once expressed,
synthesizes NO for long periods of time. Furthermore, this enzyme is Ca2+ independent since
calmodulin is already bound to the enzyme, and its induction is inhibited by glucocorticoids.
Endothelial NOS (eNOS), neuronal NOS (nNOS) which are both constitutively express~..:d in
mammalian cells have now been well characterized in the cardiovascular system and nervous
system respectively, and an inducible NOS (iNOS) which was first believed to be expressed only
when activated by an immune response. Now it is appreciated that eNOS is found in other cells
and tissues besides the endothelium, iNOS is found constitutively in some tissues, and there are
inducible forms of both eNOS and nNOS, adding confusion to the nomenclature as it was first
described. In an attempt to clarify the nomenclature, the three different isoforms are now
commonly referred to as NOSI, NOSH, and NOSIII for neuronal, inducible and endothelial
isoforms, respectively, based on the order in which they were first purified and cloned.
For years, scientists and physicians have investigated L-arginine supplementation as a means to
enhance NO production. This strategy has been shown to work effectively in young healthy
individuals with functional endothelium or in older patients with high levels of asymmetric
C:imethyl L-arginine (ADMA) where the supplemental L-arginine could outcompete this natural
inhibitor of NO production. Patients with endothelial dysfunction, however, by definition, are
unable to convert L-arginine to NO and, therefore, this strategy has failed in clinical trials.
Schulman et al. found that L-arginine, when added to standard post infarction therapies, did not
improve vascular stiffness measurements or ejection fraction and was associated with higher post
infarction mortality. L-arginine should not be recommended following acute myocardial
infarction (MI). However, there are also a number of studies showing benefit to patients taking
L-arginine just as many showing no benefit, no harm.
Understanding the complex and complicated reaction pathway for NOS mediated production of
NO from L-arginine helps us define the context for rational interventions. Using L-arginine
supplementation therapy alone may not be effective due. to oxidative stress in geriatric patients
rP,sulting in constittJtive NOS uncoupling by redox-based post translational modifications.
Supplementing L-arginine with anti-oxidants to prevent oxidation of reduced co-factors such as
BH4, might prevent NOS uncoupling and lead to better results. In a study by Taddei et al., the
role of oxidative stress on NO availability and endothelial dysfunction was examined in both
younger and older aged populations. They found that NO availability was profoundly restored in
older patients when oxidative stress is removed by antioxidants such as vitamin C. In older
individuals (age> 60 years) characterized by a profound alteration in NO availability, vitamin C
not only enhanced the response to the endothelial agonist but also restoreq the inhibitory effect
of L-NMMA on vasodilation to acetylcholine. Although, it is demonstrated that anti-oxidant
supplementation can be very beneficial for those experiencing oxidative stress and endothelial
3
dysfunction, it showed no benefit in younger (age < 60) or healthy individuals with no
endothelial dysfunction. Collectively, the literature suggests that strategies to enhance NO
production through L·arginine supple mentation are equivocal at best.
Although the L-arginine-NO pathway was the first to be discovered, it does not necessarily mean
it is the primary pathway for the endogenous production of NO. In fact nitrogen cycling in
bacteria and production of NO as an intermediate in denitrification may be one of the most
primitive pathways known, dating back to the Archaean era. The now recognized human nitratenitrite-
nitric oxide pathway that still relies on bacteria may be a redundant system for
overcoming the body's inability to make NO from T .-arginine. It appears that we havt: at least two
systems tor affecting NO production/homeostasis. The first is through the classical L-arginineNO
pathway. This is a complex and complicated five-electron oxidation of L-arginine und if any
of the co-factors become limiting, then NO production from NOS shuts down, and in many
cases, NOS produces superoxide instead. The enzymatic production of NO normally proceeds
very efficiently. However, in disease characterized by oxidative stress where e.ssential NOS
cofactors become oxidized, NOS uncoupling, or conditions of hypoxia where oxygen is limiting,
this process can no longer maintain NO production. This process is illustrated in Figure 2.
';'her~fore, one can argue saliently that there has to be an alternate route for NO production. It is
highly unlikely that nature devised such a sophisticated mechanism of NO production as a sole
source of a critical molecule. This alternate route involves the provision of nitrate and nitrite
reductively recycled to NO (Figure 3). The two-electron reduction of nitrate to nitrite occurs
through symbiosis with facultative anaerobic bacteria that reside in the crypts of our tongue.
Nitrite reduction to NO can occur in a much simpler mechanism than nitrate. The 1-electron
reduction of nitrite can occur by ferrous heme proteins (or any redox active metal) through the
following reaction: N02
- + Fe(II) + H+ - NO+ Fe(III) + OH-.
This is the same biologically active NO as that produced by NOS, with nitrite rather than Larginine
as the precursor and is a relatively inefficient process. Much of the recent focus on
nitrite physiology is due to its ability to be reduced to· NO during ischemic or hypoxic events.
Nitri!e reductase activity in mammalian tissues has been linked to the· m,itochondrial electron
transport system, protonation, deoxyhemoglobin, and xanthine oxidase. Therefore, for this
reaction to occur, the tissues or biological compartment must have a sufficient pool of nitrite
stored. Since plasma nitrite is a direct measure ofNOS activity, a compromised NOS system can
also affect downstream nitrite production and metabolism, which can perhaps exacerbate any
condition associated with decreased NO bioavailability. Considerable published data support the
notion that exogenous nitrite contributes to whole body NO production: NO produced from
nitrite in the upper intestine is up to 10,000 times the concentrations that occur in tissues from
enzymatic synthesis, nitrite can act as a circulating NO donor, and nitrite can itself perform many
actions previously attributable to NO without the intermediacy of NO. Experiments in primates
revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm. Moreover,
inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in
vivo in sheep.Topical application of nitrite improves skin infections and ulcerations.
Replenishing nitrate and nitrite through dietary means may then act as a protective measure to
compensate for insufficient NOS activity under conditions of hypoxia or in a number of
conditions characterized by NO insufficiency. Since a substantial portion of steady state nitrite
4
concentrations in blood and tissue are derived from dietary sources, modulation of nitrite and/or
nitrate intake may provide a first line of defense for conditions associated with NO insufficiency.
The recognition of this mammalian nitrogen cycle has led researchers to explore the role of
dietary nitrate and nitrite in physiological processes that are known to be regulated by NO.
Nitrite can transiently form nitrosothiols (RSNOs) under both normoxic and hypoxic conditions
and a recent study by Bryan et a!. demonstrates that steady state concentrations of tissue nitrite
and nitroso are affected by changes in dietary nitrite and nitrate (collectively, NOx) intake.
Furthermore, enriching dietary intake of nitrite and nitrate translates into significantly less injury
from heart attack. Previous studies demonstrated that nitrite therapy given intravt:nously prior to
reperfusion protects against hepatic and myoc.ardial ischemia/repcrfusion (I/R) injury.
Additionally, oral nitrite has also been shown to reverse L-NAME induced hypertension and
serve as an alternate source of NO in viv9. These results have since been conobon.tlt:d in
humans. ln tact, it has been reported that dietary nitrate reduces blood pressure in healthy
volunteers. Commercial development of nitrite and nitrate enriched dietary supplements has been
shown to impact important cardiovascular· risk factors in the aging population leading to a
reduction in triglycerides and restoration of NO homeostasis. Furthermore, in the stomach,
nitrite-derived NO seems to play an important role in host defense and in regulation of gastric
mucosal integrity. However, this is pH dependent. Since stomach acid production declines with
age and many patients are prescribed proton pump inhibitors, this pathway may be disrupted in
the geriatric patient causing additional problems with maintaining NO horne.ostasis.
Nitrite and nitrate therapy may then offer an all natural, over the counter and cost effective
regimen for conditions associated with NO insufficiency. This has the potential to provide the
basis for new preventive or therapeutic strategies and new dietary guidelines for optimal health.
From a public health perspective, we may be able to make better recommendations on diet and
dramatically affect the incidence and severity of cardiovascular disease and the subsequent
clinical events.
The major pathway for NO metabolism is the stepwise oxidation to nitrite and nitrate. For years,
both nitrite (N02
) and nitrate (N03
) have been used as surrogate markers ofNO production in
biological tissues, but there have not been any new developments in the use of NO biomarkers in
the cliniGal setting for diagnostic or prognostic utility. In fact, NO status is still not part of the
standard blood chemistry routinely used for diagnostic purposes. This is simply unacceptable
given the critical nature of NO in many disease processes and new technologies should be
developed. The only true measure of endothelial NO production (endothelial function) is through
tlow mediated dilatation (FMD). FMD is a non-invasive ultrasound-based method where arterial
diameter is measured in response to an increase in shear stress, which causes release ofNO from
the endothelium and consequent endothelium dependent dilatation. FMD has been shown to
conelate with invasive measures of endothelial function, as well as with the presence and
severity of the major traditional vascular risk factors. Nitrite and nitrate have recently been
shown to be biomarkers for cardiovascular and other diseases from both diagnostic and
therapeutic aspects. However, it is not known if levels of NOx correlate with FMD. In addition
to blood, urinary levels of NOx provide a means to assess systemic NO production in vivo, or
renal handling of these anions which may be compromist:d in the geriatric patient. A report by
Kleinbongard et a!. demonstrated that plasma nitrite levels in humans progressively decrease
with increasing cardiovascular risk load. Risk factors included age, hypertension, smoking, and
5
hypercholesterolemia, conditions all known to reduce the bioavailability of NO. Although a
correlation exists in the. plasma, it is not known whether the situation is mirrored in the heart or
other tissue of interest in specific disease. The recent recognition of a human nitrogen cycle
whereby nitrate and nitrite are reduced to NO by an enterosalivary circulation of nitrate now
opens up the potential for using saliva as a potential biomarker for NO status in certain diseases.
Aging and hypertension are well-documented cardiovascular risk factors. Most of the functional
and structural vascular alterations that lead to cardiovascular complications are similar in aging
and hypertension. Moreover, these vascular changes associated with essential hypertension are
generally considered to be an accelerated form of the changes. seen with aging. When we are
young and healthy, the endothelial production of NO through L-arginine is efficient and
sufficient; however, as we age we lose our ability to synthesize endothelial1.lt:rived NO. Most of
the works on the activity of NO in cells and tissues agree that the bioavailability or the
generation of NOS derived NO decreases with aging. It has been proposed that superoxide can
scavenge NO to form peroxynitrite and thereby reduce its effective concentrations in cells. It has
~1so been reported that there is decreased NOS expres~ion with aging both in constitutive and
inducible isoforms .. Berkowitz etal.observed the upregulation of arginase (an enzyme that
degrades· the natural substrate for NOS, L-arginine) in aged blood vessels and the corresponding
modulation of NOS activity. Taddei et al. have shown that there is a gradual decline in
endothelial function due to aging with greater than 50% loss in endothelial function in the oldest
age group tested as measured by forearm blood flow assays. Egashiraet al. reported more
dramatic findings in the coronary circulation of aging adults whereby there was a loss of 75% of
endothelium-derived nitric oxide in 70-80 year old patients compared to young, healthy 20 year
olds. Vita et al. demonstrated that increasing age was one predictor of abnormal endotheliumdependent
vasodilation in atherosclerotic human epicardial coronary arteries. Gerhard et al.
concluded from their 1996 study that age was the most significant predictor of endotheliumdependent
vasodilator responses by multiple stepwise regression analysis. Collectively, these
important findings illustrate that endothelium-dependent vasodilation in resistance vessels
declines progressively with increasing age. This is illustrated in Figure 4. This abnormality is
present in healthy adults who have no other cardiovascular risk factors, such as diabetes,
hypertension, or hypercholesterolemia. Most of these studies found that impairment of
endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast,
endothelium-independent vasodilation does not change· significantly with aging, demonstrating
that the responsiveness to NO does not change only the ability to generate it. These observations
enable us to conclude that reduced availability of endothelium-derived NO occurs as we age, and
to speculate that this abnormality may create an environment that is conducive to atherogenesis
and other vascular disorders, including Alzheimers disease. It appears that aging interrupts NO
signaling at every conceivable level, from production to inactivation. Given that NO is a
necessary molecule for maintenance of health and prevention of disease, restoration of NO
homeostasis may provide a new treatment modality for age and age related disease.
Aging is considered the single largest risk factor related to cardiovascular related diseases and
deaths. Cardia-protection decreases with increasing age and is attributed to a decline in NO. The
lack of NO production can lead to hypertension, atherosclerosis, peripheral artery disease, heart
failure, and thrombosis leading to heart attack and stroke, especially in geriatrics. Remarkably,
6
all of these conditions have been shown to be positively affected by dietary nitrite and nitrate
interventions.
Hypertension, along with aging is a well-known cardiovascular risk factor that leads to
functional and structural alterations in the heart and vasculature. Vascular changes associated
with hypertension, such as endothelial dysfunction, are an accelerated form of the type of
changes seen with aging. Additionally, the presence of acetyl c.holine alongside a NO synthase
i'lhibitot (L-NMMA) was tested for NO availability in the vasculature. A noteworthy finding is
that after the age of 60 years old, the inhibiting effect of L-NMMA on response to acetylcholine
was extremely weak, suggesting that N() availability· is completely l:umpromised in older
populations. These results indicate that essential hypertension is characterized by an age-related
reduction of endothelial function by mechanisms that ~'~pp.;-ar to be c;imilnr to tho~c observed hi
older normotensive individuals. NO based therapies can reduce blood pressure. Transdermal
nitroglycerin has been shown to reduce blood pressure in patients with recent stroke. Dietary
intervention with nitrite and nitrate hn3 been ~huwu io modestly reduce blood pressure in humans
showing remarkable efficacy using this approach.
Atherosclerosis is the major source of morbidity and mortality in the· developed world. The
magnitude of this problem is profound, as atherosclerosis claims more lives than all types of
cancer combined and the economic costs are considerable. Reduced NO availability is a hallmark
of atherosclerosis. The endothelium-derived NO plays a crucial role in regulating a wide
spectrum of functions in the cardiovascular system, including vasorelaxation, inhibition of
leukocyte-endothelial adhesion, vascular smooth muscle cell (SMC) migration and proliferation,
as well as platelet aggregation. The concept of endothelial dysfunction arises from variations in
blood flow observed in patients with atherosclerosis compared with healthy subjects. In healthy
subjects, activation of eNOS causes vasodilation in both muscular conduit vessels and resistance
arterioles. In contra~t, in subjects with atherosclerosis, similar stimulation yields attenuated
vasodilation in peripheral vessels and causes paradoxical vasoconstriction in coronary arteries,
thus indicating a decrease in the production and/or bioavailability of NO. Interestingly,
endothelial dysfunction can be demonstrated in patients with risk factors for atherosclerosis in
the absence of atherosclerosis itself. These observations lend credence to the concept that
endothelial dysfunction is integral to the d·evelopment and progression of disease. Impaired
endothelium may abnormally reduce vascular perfusion, produce factors that decrease plaque
stability, and augment the thrombotic response to plaque rupture. There are a number of studies
showing· that insufficient NO production from the endothelium is associated with all major
cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension,· smoking and severity
of atherosclerosis, and importantly also has a profound predictive value for the future
atherosclerotic disease progression. Augmentation ofNO or restoration of NOS function seems a
logical means to inhibit atherosclerosis. Absence of eNOS in apoE-knockout mice accelerates
atherosclerosis that is not caused by hypertension. Paradoxically, however, overexpression of
endothelial NOS accelerates lesion formation in apoE-deficient mice demonstrating that
enhanced NOS derived NO may not always be beneficial. Supplementation with
tetrahydrobiopterin (BH4) reduced the lesion size to those seen in Apo E knockout mice
revealing the requirement of enzyme cofactors. Even with BH4 supplementation there was still
l'i.J effect on lesion development. These studies demonstrate the complexity of endothelium
derived NO in the setting of atherosclerosis but clearly illustrate the dysfunctional eNOS/NO
7
path~'.'ay as an early marker or a common mechanism for various cardiovascular disorders and
therefore provides an ideal target for therapeutic or preventive intervention including alternative
NOS independent sources of NO. Stokes et al. have demonstrated that supplementing nitrite in
the drinking water inhibits the adhesion and emigration of leukocytes to the vascular
endothelium, one of the earliest events of atherogenesis suggesting this nitrate-nitrite-NO
pathway may be useful in preventing chronic vascular disease.
Thrombosis affects nearly 1 million patients in the United States annually. Out of those million,
nearly 300,000 are reported as thrombosis related deaths. -\ majority of current treatment options
are centered on preventative anticoagulants, such as warfarin but with risk of bleeding. NO
inhibits platelet activation, adhesion, and aggregation by influencing several signaling pathways,
including activation of sGC anci . incr~;>asing intraoollular cGMP, iuhlbitlon of
phosphatidylinositol~3 kinase; and inhibition of capacitativecation influx and agonist-dependent
increases in intracellular calcium. These molecular events lead to an impairment of platelet
activation, adhesion, secretion, fibrinogen-binding to glycoprotein lib/lila, and ultimately,
aggregation. In addition, NO promotes platelet disaggregation. In conjunction with its
vasorelaxing actions, these anti-platelet effects of NO maintain blood fluidity and tissue
perfusion. A constitutive NO synthase has been found in both human platelets and
megakaryocytic cells and this isoform is active. Using ·an NO-selective microelectrode adapted
to a platdet aggregometer, Free,dman et al. recently showed that this platelet-derived NO not
only modestly modulates platelet activation to strong and weak agonists but, more importantly,
markedly inhibits platelet recruitment to the growing platelet thrombus. NO, derived both from
the endothelial cell and the platelet, modulates platelet activation, adhesion, and aggregate
formation, thereby serving as an important deterrent to platelet-mediated arterial thrombosis. NO
insufficiency, either through reduced production or oxidative inactivation leads to thrombotic
events.Efforts to restore the normal vascular redox balance and/or to restore normal NO
availability may provide one therapeutic avenue for reducing platelet-dependent arterial
thrombosis in older patients. In fact, recent studies have shown that dietary nitrate can inhibit
platelet aggregation again demonstrating this dietary approach to replete NO may provide first
line of defense for NO insufficiency.
The most feared disease of the geriatric population is Alzheimer's disease (AD). In the United
States, around 5.4 million people live with AD, a type of dementia. Patients with AD lose brain
function, resulting in problems with language, perception ·and memory. AD can start before age
60 (early onset) or after age 60 (late onset). The risk for AD increases as a person ages and that
rising risk is being seen as the baby boomers start turning 65 years old. Out of every eight baby
boomers; one will get AD after she turns 65 years old; at age 85, that risk grows to one in two.
With the 65 and over population in the United States expected to double by 2030, there may be
up to 16 million people with AD by 2050; there may be almost one million new AD cases
diagnosed each year. Each year in the United States, more than 800,000 people die from this
neurological disease. It is the sixth leading cause of death, with the number of deaths rising 66
percent from 2000 to 2008.
There is becoming a clear and convincing association with AD and NO. Decreased levels of
NOx has been detected in patients with different forms of dementia especially AD. The exact
etiology of sporadic AD is unclear, but it is interesting that cardiovascular risk factors including
8
hypertension, hypercholesterolemia, diabetes mellitus, aging, and sedentary lifestyle are
associated with higher incidence of AD. Th~ link between cardiovascular risk factors and AD
has yet to be identified; however, a common feature is endothelial dysfunction, specifically,
decreased bioavailability of NO. The pathogenesis of Alzheimer's disease is closely associated
with the accumulation of amyloid-~ (A~) peptides, which eventually form neuronal deposits
known as senile plaques on the outside surface of the neurons and lead to neuron death. AD is
characterized by progressive loss of neurons, cognitive decline, and two Jdining
histopathologies: extracellular amyloid plaques and intracellular tangles composed primarily of
AP peptide and hyperphosphorylated tau, respectively. Furthermore, AD is often accompanied
by cerebrovascular dysfunction, as well as amyloid deposition within the cerebral vessels, termed
cerebral amyloid angiopathy. NO in the brain can be produced either by iNOS in microglia and
astrocytes, or by constitutive NOS in neurons and endothelial cc.::lls (nNOS and eNOS). A large
body of evidence suggests that the NO produced by neuronal and endothelial constitutive NOS is
responsible for neuroprotection during AP-induced cell death, while NO production in the case
of iNOS activation plays a neurotoxic role due to the inflammatory response caused by the over
generation of other reactive nitrogen species from NO.A decrease in nNOS and an increase in
hippocampal iNOS have been demons trated in aged rats, suggesting the dual roles and
complexity of NO signaling in the brain and during AD. In mice, higher levels of constitutive
NO 1-'roduced by NOS protects beta-amyloid transgenic mice from developing most typical
human symptoms of AD.The protective role of NO in AD pathogenesis has been linked to
NO/sOC/cOMP/Protein Kinase 0 (PKO) signaling cascades. Treatment with NO donors and
cOMP analogues suppresses cell death, and increasing intracellular ·cOMP levels prevents
inflammatory responses in brain cells. Moreover, the use ofthe NO donors, sOC stimulators, and
cOMP-analogs reverses learning and memory impairment through PKO activation, in part by
reestablishing the enhancement of the transcription factor cAMP-responsive element binding
protein (CREB), which is phosphorylated during long term potentiation. It has also been shown
that NO modulates expression and processing of amyloid beta precursor protein.
However, an accumulation of AP inhibits the NO signaling pathway and therefore may suppress
the protective effects of endogenous NO in the brain. Chronic administration of fibrillar AP
c;ecroses the expression of sOC in cultured rat astrocytes, desensitizing them to treatment with
sodium nitroprusside. Acute AP administration blocks NO-induced vasoactivity in rats [and
inhibits NO-stimulated phosphorylation of CREB. In postmortem temporal cortex from a series
of AD patients there was reduced NO responsive sOC providing the first evidence for a loss of
NO responsive sOC activity in AD brain.
Alternatively, current research suggests S-nitroslyl~tion, may be responsible for cOMP
independent mechanisms of NO and can contribute to neurotoxicity in neurogenerative diseases
such as AD. Consequently, NO does not always protect against disease and may help facilitate
neurode generative disorders through nitrosative stress and dysregulation of production. A
common theme in many neurodegenerative disorders is the finding of abnormal aggregates of
misfolded proteins. Recent findings have implied that NO-related species may significantly
prtir.ipate in the process of protein misfolding through protein S-nitrosylation under
degenerative conditions. Qu et al. demonstrate that Cdk5 activity, a cyclin dependent kinase
responsible for neuronal functions, is regulated by S-nitroslylation. They found significantly
increased S-nitrosylated Cdk5 (SNO-Cdk5) levels in postmortem human brain tissues of patients
9
with AD compared to control brain tissue. Significantly, SNO-Cdk5 was not detectable in
control human brains, thus indicating that measurable levels of SNO-Cdk5 are representative of a
diseased state. Additionally, researchers reported that formation of SNO-Cdk5 contributes to
NMDA-induced spine loss, neuronal damage, and to AP-induced loss of synaptic spines. In
conclusion, the SNO-Cdk5 mediated pathway may contribute to the path agenesis of AD and
serve as a unique therapeutic for restoring spine damage in AD and other neurodegenerative
diseases. ·
Collectively, the literature demonstrates a critical role for NO in the development of AD. It
appears that normal and sufficient NO production/availability can modulate and inhibit the
expression and formation of AP but once AP becomes present it further compromises NO
activity. This crel'ltes a perpetual system ofNO insufficiency and a feed furwurd mechanism that
may accelerate AD progression. The NO pathway (both cGMP and through S-nitrosylation) may
be an important therapeutic target in preventing and treating mild cognitive impairment, as well
as AD. In fact, a high nitrate diet has been shown to increase regional cerebral blood flow to the
frontal lobe in older patients. ·
It appears that the inability to produce sufficient NO under the right preclinical conditions
enhances the risk for a number of diseases that plague the older population. If true, then there
exist an opportunity to intervene early during this process, implement strategies to restore NO
homeostasis, and, perhaps, delay or prevent the onset and progression of certain diseases. This
gradual loss ofNO activity with age can be sped up or slowed down based on individual lifestyle
and diet. Adopting healthy habits such as a good diet and exercise can prolong the precipitous
·•· drop :n NO production with age. To the contrary a poor diet along with physical inactivity can
accelerate the process and lead to a faster decline in NO production at a younger age.
Therapeutic strategies directed at improving endothelial function or providing an alternative
source of NO should be the primary focus because they may reduce the incidence of
atherosclerosis or other diseases that occur with aging, even perhaps AD.
The role of diet in the prevention and control of morbidity and premature mortality due to noncommunicable
diseases has been well established by vast population-based epidemio logical
studies carried out during the last decade. NO is essential for maintaining normal blood pressure,
preventing adhesion· of blood cells to the endothelium, and preventing platelet aggr_egation; it
may, therefore, be argued that this single abnormality, the inability to generate NO, puts us at
risk for diseases that plague us later in life, such as atherosclerosis, myocardial infarction, stroke,
end neripheral vascular disease. Are dietary and nutritional strategies utilizing nitrite and/or
nitrate to restore NO homeostasis the best approach? More clinical trials are needed to define the
context for risks vs. benefit. Although modestly increased associations between consumption of
foods containing nitrite and nitrate and certain cancers .have been reported in some prospective
epidemiologic studies, findings across studies have been largely inconsistent and equivocal and
thus the overall burden of proof remains inconclusive. As with any therapy or treatment regimen,
a risk benefit evaluation should be considered and understood for certain persons or patient
populations. However, given the emerging data on the growing number of benefits from diets
and foods enriched in nitrite and nitrate, we predict the benefits will far outweigh any risks. What
remains clear is developing strategies and new technologies designed to restore NO availability
is essential for inhibiting the progression of certain common chronic diseases.
10
NO is formed from L-arginine via the enzyme nitric oxide synthase or synthetase (NOS). These
enzymes convert L ·- arginine into L -citrulline, producing NO in the process. Oxygen and
different co factors such as FAD, FMN, heme and tetrahydrobiopterine are necessary which
influence NO synthesis by this rection.
The dysfunctional nitric oxide synthase (NOS) nitric oxide pathway is considered an early
marker for various cardiovascular disorders. Decreased bioavailability of endothelial nitric oxide
plays a crucial role in the development and progre~sion of a number of human diseases.
Endothelial dysfunction results from decreased nitric oxide production or increased degradation
of nitric· oxide. ln certain aspects endothelial dysfunction-can be defined as the inability to -generate
NO. Endothelial dysfunction is a physiological dysfunction of normal biochemical
processes carried out by the-encinth~liutl1, the eells that line tho inner gurface ufall uluuU vessels
including arteries and veins (as well as the innermost lining of the heart and lymphatics).
Endothelial dysfunction is associated with several cardiovascular disorders, including
atherosclerosis.
l'rior attempts to restore nitric oxide homeostasis have m~t significant challenges. L-arginine and
antioxidant supplements have consistently failed in clinical trials. It is known that NOS enzymes
produce nitric oxide by catalyzing a five electron oxidation of the guanidino nitrogen of Larginine.
While nitric oxide is produced through oxidation of the semi-essential amino acid Larginine
by NOS, the L-arginine-nitric oxide pathway is dysfunctional in patients with
endothelial dysfunction. Thus, feeding the nitric oxide pathway through L-arginine
supplementation is potentially both ineffective and detrimental through the production of
superoxide instead of nitric oxide.
Prior attempts to enhance nitric oxide production with organic nitrates such as nitroglycerin have
faced challenges. Early entry therapy with organic nitrates do not significantly improve survival
in myocardial infarction but increases the beneficial effects of the Angiotensin Converting
I:nzyme (ACE)-inhibitor enalapril by 50%. Certain short-term experimental and clinical
investigations suggest that nitrate . tolerance induced by nitroglycerin is ~ssociated with toxic
effects in the vasculature. Chronic and long-term organic nitrate therapy has been associated with
reduced survival when used in patients with coronary artery disease. 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. . ·
Previous studies have shown that nitrites widen blood vessels, but US researchers writing in
Nitric Oxide: Biology and Chemistry, the peer-reviewed journal of the Nitric Oxide Society, say
theirs was the first to find that nitrites also increase blood flow to the brain.
Beetroot contains high concentrations of nitrates, which are converted into nitrites by bacteria in
the mouth. Nitrites help open blood vessels in the body, increasing blood flow and oxygen to
places lacking in oxygen.Beetroot is a dark red vegetable with an acquired taste which has been
getting a lot of coverage in the news. It has been linked with better stamina, improved blood
flood and lower blood pressure
11
,
Most beetroot is round and red, but yellow, white and stripy versions are available.The beetroot
taste is described as sweet, earthy and tender to eat. It is grown in the ground and is related to
turnips, swedes· and sugar beet. It contains potassium, magnesium, iron, vitamins A, B6 and C,
folic acid, carbohydrates, protein, antioxidants and soluble fiber.
Researchers have known for some time that juice may help lower blood pressure, but in 2010
UK researchers revealed that nitrate is the special ingredient in beetroot which lowers blood
pressure and may help to fight heart disease.
In a Queen Mary University of London study, healthy participants had-to drink a glass··of --·beetroot
juice while others had a dummy (placebo) drink. Others took nitrate tablets.Blood
pressure was lowered within 24 hours in people who took nitrate tablets and those who drank.
beetrootjuice.The researchers admitted to BootsWebMD that beetroot juice is a love it or hate it
kind of drink, but found people in the study didn't mind it so much when they were drinking it
every day.People with very high blood pressure can end up being on multiple tablets, so a more
natural approach could prove popular if the initial research results are confirmed.The study was
funded by the British Heart Foundation and is . published online in the American Heart
Association journal Hypertension.
Drinking beetroot juice increases blood flow to the brain in older people, which may be able to
. fight !he progression of dementia, a 2010 study suggested.Beetroot contains high concentrations
of nitrates, which are converted into nitrites by bacteria in the mouth. Nitrites help open blood
vessels in the body, increasing blood flow and oxygen to places lacking in oxygen.
There are some interesting side effects of eating too much beetroot. It can turn urine pink, which
can be mistaken for blood in the urine. ·
If you get kidney stones because of too much calcium, you may be advised to cut down on
oxalates in your diet. Beetroot is just one food which contains oxalates, which prevent calcium
from being absorbed by your body allowing it to build up as stones in the kidney.
The British Dietetic Association says beetroot contains flavonoids called anthocyanins which are
responsible for the deep pigments. Anthocyanins, the BDA says, can help with recovery from the
stress of exercise during training and competition as well as helping to couriter the effects of
pollution on the. body.
Hawthorn is a plant. The leaves, berries, and flowers of hawthorn are used to make
medicine.Hawthom is used for diseases of the heart and blood vessels such as congestive heart
failure (CHF), chest pain, and irregular heartbeat. It is also used to treat both low blood pressure
and high blood pressure, "hardening of the arteries" (atherosclerosis), and high cholesterol. So
far, research suggests that hawthorn might be effective in treating congestive heart failure, but
there hasn't been enough research on other heart-related. uses to know if it is effective for them.
Research suggests that hawthorn· can lower cholesterol, low density lipoprotein (LDL, or "bad
chole~terol"), and triglycerides (fats in the blood). It seems to lower accumulation of fats in the
liver and the aorta (the largest artery in the body, located near the heart). Hawthorn fruit extract
12
may lower cholesterol by increasing the excretion of bile, reducing the formation of cholesterol,
and enhancing the receptors for LDLs. lt also seems to have antioxidant activity.
L-citrulline is a naturally occurring amino acid found in food, such as watermelons, and also
made in the body. Our bodies change L-citrulline into another amino acid called L-arginine and
also to nitric oxide. L-citrulline might help increase the supply of ingredients the body needs to
making certain proteins. It might also help open up veins and arteries to improve bluud flow and
reduce blood pressure.
Vitamin Bl2, or cobalamin, is essential to a wide variety of critical body functions. We need
B12 for nervous system health, cardiovascular health, energy production and longevity. Getting
optimal amounts ofthis important nutrient can do great things for our· minds and bodies, but
unfortunately, many people aren't even getting the bare minimum.Vitamiil B12 is an essential
nutrient for the human body. In its active form, it is a methyl group donor that is involved in
energy production, homocysteine metabolism, nerve function and neurotransmitter production.
B12 works with folate to create red blood cells and the protective coating around nerve cells, to
convert food to energy and to synthesize and repair DNA. ·
B12 deficiency is not a symptom experienced only by the sick, malnourished and elderly. On the
contrary, it affects millions of Americans,_ even those with healthy diets and lifestyles. A recent
study conducted by Tufts University suggests that B12 deficiency and insufficiency affect 64%
of people between the ages of 26 and 83, and that suboprimal levels are equally as common at
every age. Clinical deficiency (serum levels below 148 pmoi/L) affected 9%, or nearly one in ten
people, out of 2,999 subjects. These dangerously low levels are known to cause nerve damage,
cognitive decline, loss of memory, fatigue, and anemia, among other things. An additional 16%
of subjects were verging on deficient (levels below 185 pmoi/L), and 39% had levels in the lownormal
range (below 258 pmoi/L). According to a recent study published in the September 2008
issue of Neurology, B 12 levels below 308pmoi/L are associated with an increased risk of brain
shrinkage and cognitive decline. Together, these two studies suggest that 56% of the population
may experience neurological impairment as a result of low levels of B 12, without being
diagnosed as B 12 deficient.
One of vitamin B 12's many vital functions is the formation and maintenance of the outer layer of
nerves, called the myelin sheath. The myelin sheath is the fatty insulation around nerve cells that
protects neurons and allows them to properly conduct signals. Integrity of the myelin sheath is
essential for healthy brain function and is dependent upon methylation reactions that require
B12. B12 also ·influences the production of neurotransmitters that control memory, thinking,
motor function and moods.
B 12 deficiency affects the nervous system first, by damaging the myelin sheath and then the
entire nerve cell. This damage can result in a host of neurological conditions including multiple
sclerosis, neuropathy, impaired coordination. and loss of vision, hearing and memory. Animal
studies have revealed that the methyl form of vitamin B12 (methylcobalamin) promotes the
regeneration of nerve cells. Limited human trials suggest a role for B 12 in slowing the
progression of neurodegenerative disease and lessening pain associated with neuropathy.
13
B12 deficiency can also result in elevated blood levels of the amino acid metabolite
homocysteine. Numerous studies have cited a correlation between elevated homocysteine and
Alzheimer's, dementia, depression and overall cognitive decline.
Vitamin B 12 is needed to create red blood cells and to convert homocysteine to methionine. Both
of these functions are critical to the health 'of the cardiovascular system. Deficiencies of B 12 can
result in megaloblastic anemia, characterized by oversized, immature red blood cells, and buildup
of homocysteine in the blood, a risk-factor for heart disease.
B12 is essential for energy production in every cell ofthe body and has earned the nickname "the
energy vitamin." Supplying our bodies with adequate amounts of B12 helps our cells derive
energy from the proteins. fats and c::~rhohydrates we eat. B 12' s role in mclauulisrn Impacts the
function of every tissue in the body, including the musdes, the heart and the brain.
Vitamin D12 is essential for the synthesis and repair of DNA and it supports healthy gene
expression. Gene expression is the process by which our genetic material is used to generate
biochemicals within the body, and it influences whether we will live long, healthy lives or will
suffer from disease. Nutrients that affect gene expression have the ability to "tum on" genes that
promote health while "turning off' those that undermine health. Though B 12 does not directly
regulate gene expression, it plays an important role in the production and recycling of S-adenosyl
methionine (SAMe), a biochemical that reacts with DNA to turn genes on or off. This reaction,
called methylation, is a natural reaction within our bodies that supports healthy gene expression.
Therefore, supplementing with B 12 supports the healthy replication and function of our genetic
material.
Vitamin B 12 is a product of bacterial fermentation and is found exclusively in animal products.
Beef, poultry, fish, cheese and eggs are all good sources of B 12. Although an inactive analogue
of vitamin B12 is found in some plants like algae, this form is not used by our bodies.
Absorption of B12 in the small intestine requires the help of a glycoprotein known as intrinsic
factor. Intrinsic factor, secreted by the parietal cells in the stomach, has a low affinity for inactive
B12 l:lnalogues found in plant foods. Ifthese inactive analogues are absorbed into the body, they
are taken to the liver and then excreted in the feces and urine.
The following categories, would likely to be benefited from supplementation:
~ A senior - an estimated 40% of seniors have low stomach aciditY (atrophic gastritis),
which is linked to impaired absorption of protein-bound vitamin Bl2, found in food. The
unbound B 12 found in supplements is generally better absorbed.
~ A vegetarian or vegan - vegetarians often do not consume adequate food sources of B 12
and vegans do not consume any food sources ofB12 (plant sources are analogues and are
not absorbed); studies have shown that up to 50% of vegetarians and 80% of vegans are
deficient.
~ A pregnant- pregnancy increases the demand for B12 to create more blood cells and to
nourish the baby's growing nervous system; B12 deficiency during pregnancy has been
linked with neural tube defects.
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..
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~ A man with low sperm count or low motility - B12 supports healthy sperm cell
replication. When given to men without B12 deficiency, it has reportedly increased sperm
count and motility.
~ An acid blocker- Use of proton pump inhibitors has been linked with B 12 depletion.
~ A person taking metformin - research has shown a correlation between metformin & B 12
deficiency. As dosage and duration increased, so did risk ofB12 deficiency.
~ A person havingGI problems (H, pylori, stomach acid imbalances, Crohn's Disease,
Gastric or Duodenal Ulcers, etc.)- these complications interfere with absorption; studies
have found low B 12 levels in individuals with stomach and/or intestinal problems.
~ A postmenopausal woman: Bone health is a concern for all of us, but particularly for
postmenopausal women, who are most likely to suffer from bone loss and osteoporosis.
B12 plays ~n important role in bone health via its rok in homocysteine metabolism.
Women with homocysteine levels above 15 IJ.mol/L were found to have a 96% higher
risk of low bone density than women with levels·below 91J.moi/L.
~ To improve memory, mental clarity, mood or energy levels- B12 supports all of these
bodily functions.
Cyanocobalamin and methylcobalamin are the two most common chemical forms of B12.
Cyanocobalamin is B12 (cobalamin) bound to a cyanide molecule while methylcobalamin is B12
bound to a methyl group. Both forms are produced synthetically. Methylcobalamin is considered
an active form because it does not need to be converted in the body before it can function as B12.
Cyanocobalamin is not an active form because it must be converted to methylcobalamin before it
c:1n function as B12. Another form of B12, called dibencozide (also known as
adenosylcobalamin),·is another active form ofB 12.
B 12 comes in sublingual, pill and liquid form. Sublingual form (a tablet that dissolves under the
tongue) is a popular method of oral administration because the B12 is absorbed directly into the
bloodstream through the mouth, and does not require intrinsic factor. This may be beneficial for
individuals with impaired digestion or inadequate intrinsic factor.
Although many medical doctors give B12 injections, oral administration is equally as effective,
even in the absence of intrinsic factor. Research has established that, in individuals with
pernicious anemia (B12 deficiency symptom), the average absorption rate of cyanocobalamin is
1.2 percent for a wide range of doses. This means that the average person taking a daily dose of
2.50 icrograms would absorb 3 micrograms. Since the daily turnover rate of B12 is about 2
micrograms, 250 meg daily would be sufficient for many people. However, there is little risk in
taking higher doses, up to 2,000 micrograms daily, and many people benefit greatly from
amounts as high as 5,000 micrograms per day or higher. Higher doses are generally more
effective at replenishing diminished B12 stores rapidly.
Several medical tests are available to assess the levels ofB12. These include:
Serum Bl21evels-the most common, but often inadequate test.
~ Holotranscobalamin II--can detect B12 deficiency in its earliest stages-low levels
indicate that the B 12 stores are diminishing.
~ Serum levels or Urinary excretion of methylmalonic acid-elevated levels indicate that
cellular insufficiency or deficiency has already manifested.
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~ Serum levels of homocysteine-elevated levels indicate that cellular insufficiency or
deficiency has already manifested.
Limitations oftesting: Serum Bl2 levels alone are inadequate to diagnose B12 deficiency and
insufficiency. In the original Framingham Study, more than half of seniors with cellular B12
deficiency had serum B12levels considered normal. This finding can be attributed to 2 factors:
1. The clinical ranges for "normal" serum B 12 in the United Slates are much too broad.
While levels between 200 pmol/L and 300 pmol/L are considered normal in the U.S., individuals
with levels this low often have obvious B 12 deficiency symptoms. In Japan and some European
countries, the clinical cutoff for deficiency is betwet:n 370 and 407 pmol/L
2. A serum B12 test measures all circulating B12, but about 80% of this B12 is unavailable
to most cells. A holotranscobalamin II test, on the olht:r hand, measures the remaining 20% of
circulating cobalamin that is attached to a carrier molecule, enabling it to reach the body's cells.
Levels ofholotranscobalamin can be low in individuals with normal serum B12-a situation that
results in cellular deficiency. Methylmalonic acid and homocysteine are also indicators of
cellu;ar deficiency. These three functional metabolite· tests are very important for accurately
assessing B 12 levels in the body. ·
The NHLBI Study: The Promise of New Medical Uses for Sodium Nitrite for Heart Attack and
Organ Damage suggest that: Sodium nitrite, a naturally occurring chemical and common meat
preservative, is only used medically to treat cyanide poisoning. But if the results of a new animal
study hold up under further research in people, the chemical may one day be used to protect and
preserve tissue and organ function after heart attack, high risk abdominal surgery, and organ
transplantation.
The new study was conducted by scientists with the National Heart, Lung, and Blood Institute
(NHLBI) ofthe National Institutes ofHealth (NIH) in collaboration with investigators supported
by the National Institute of Diabetes and Digestive and Kidney Diseases at Louisiana State
Univ<>:rsity Health Sciences Center and published in The Journal of Clinical Investigation.
The scientists found that low concentrations ·of sodium nitrite had a strong protective effectpreventing
cell death in the hearts and livers of mice undergoing experimental heart attack and
liver injury. In the heart study, nitrite reduced the size. of the area of dead tissue known as an
infarct by 67 percent compared to control animals given nitrate, another nitrogen compound.
This potent protective effect was observed at concentrations of nitrite in blood that were only
slightly higher than the physiological normal levels in blood.
The study, Jed by David Lefer, Ph.D., of Louisiana State University Health Sciences Center in
Shreveport and Mark Gladwin, M.D., Head of the Vascular Therapeutics Section of the
NHLBI's Cardiovascular Branch, follows another study conducted by the NIH research team
that found that infusions of sodium nitrite into the human circulation leads to the production of
nitric oxide (NO), a strong blood vessel dilating molecule that increases blood flow. The
conversion of nitrite .to NO will occur only in tissue or blood that is very low in oxygen. It was
this finding that triggered the team's interest in sodium nitrite as a treatment/preventive for the
tissue damage and cell death that can occur in conjunction with organ transplantation, heart
attack, and treatment of a heart attack.
16
In both the liver and heart components of the current study, the research team compared the
effects of both lower and higher concentrations of nitrite versus control' treatments of saline or
nitrate, a chemical compound that is related to nitrite but cannot convert to NO. in the blood.
Surprisingly, they found that only low concentrations of nitrite provided protection against
injury.
The investigators are currently studying the mechanism for the protective effect of sodium nitrite
r.nd they believe it is related in some way to the conversion of nitrite to nitric oxide.
"The remarkable thing about nitrite is that it is only converted to nitric oxide in the organs and·
tissues with the .lowest oxygen levels, allowing for targeted NO delivery - and thus improved
blood flow - to tis.•me~ under gtrecs. Moro re3cnrch is neeucu lu look at the effectiveness of
nitrite in various organs and disease states in humans," said NHLBI's Gladwin who is also an
investigator in the Critical Care Medicine Department, NIH Clinical Center.
Gladwin is currently studying the use of sodium nitrite as a way to help adults with sickle cell
disease. It is hoped that this treatment will reverse the effect of decreased blood flow due to the
patients'. "sickled" blood cells. Patients with sickle cell disease have abnormal hemoglobin
molecules in their red blood cells. The molecules damage the red cells, causing them to change
into a crescent or sickle shape and stick to blood vessel walls. This can lead to· narrowed, or
blocked, blood vessels leading to pain, damage, and anemia.
Further studies either underway or in planning translate the new findings to humans. These
studies evaluate sodium nitrite's effect on heart attacks, kidney failure, solid organ
transplantation, cerebral vasospasm (a complication of a ruptured aneurysm leading to reduced
blood flow and possible stroke), and high blood pressure in the lungs in babies.
One nutrient that can positively affect nitric oxide synthesis is vitamin C, or ascorbic acid. A
study published in 2003 in "Circulation Research" found that vitamin C increased the activity of
the enzyme nitric oxide synthase, the protein that produces nitric oxide. The vitamin may support
nitric oxide production and help to maintain the levels of the compound within your body.
Attempts targeting delivery of nitric oxide to precise cellular locations have also faced
challenges. The most widely known and effective means for targeted delivery to the pulmonary
circulation is inhaled nitric oxide, which requires specialized inhaling equipment. Biomaterials
for sustained release of nitric oxide for topical applications for wound healing, infections, etc. are
still in development. Nanoparticle delivery of nitric oxide is still emerging, particularly in cancer
biology. NO-eluting stents or nitric oxide-coating of orthopedic implants for preventing biofilm
growth and infection is also still in development. Phosphodiesterase inhibitors, such as sildenafil,
do not directly affect nitric oxide production but act through affecting the downstream second
messenger of nitric oxide, cyclic guanosine monophosphate ( cGMP).
Oral Formulation Concerns an oral disintegrating tablet (ODT) is a solid oral dosage form that
disint~::grates and dissolves in the mouth without water within 1 minute or less. In the literature,
ODT:; also are identified as rapidly-dissolving tablets, orally disintegrating, quick-dissolve,
orodisperse, mouth-dissolving, fast-melt, and rapid-disintegrating tablets. ODTs were designed
17
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tor C:-iildren and the elderly or for any individual that has difficulty with swallowing, especially
entire tablets or capsules, commonly referred as dysphagia. With an ODT tablet, all of the
components will liquefy in the mouth and then the individual swallows the liquid.
However, there are a number of disadvantages and complexities associated with the formulation
development and scale-up process of ODTs, including drug loading, taste masking,friability,
high facility and manufacturing costs, and stability of the finished product.Poor friability is the
most frequent problem found in ODTs. For a compressible tablet to dissolveinstantly, it may be
quite friable. However, making the tablet harder and less friable maynegatively impact the fast
disintegration and dissolution time. Generally, an ideal ODT musthave a balance between
durability, friability and speed of disintegration/dissolution.
Accordingly, there exists a large unmet need to recapitulate nitric oxide homestatis in the body
using an effective delivery system. Oral disintegrating tablets were previously
consideredunsuitable because they dissolve in the mouth, where oral dispersion of nitric oxide
wasperceived as ineffective. Oral disintegrating tablets are designed for children, the elderly
andindividuals with difficulty swallowing. Complexities challenge the formulation,
developmentand scale-up process of oral disintegrating tablets, including drug loading, taste
masking,friability, high facility and manufacturing costs~ and stability of~he finished product.
Regular tablets and capsule do not allow the proper reaction to take place and minimize
nitricoxide generation. Capsules pass through the gastrointestinal tract and release their contents
in thestomach. However, many people taking proton pump inhibitors or who may have
achlorhydriafor other reasons may not experience the same benefit since low pH in the stomach
i~ required togenerate NO from nitrite. Nitrosative chemistry occurring in the stomach also has
the potential toform potentially carcinogenic N-nitrosamines. By slowly titrating the saliva one
can avoid theburst ofnitrosative chemistry that would occur at once in the stomach.
SUMMARY OF THE INVENTION
The present invention solves the above mentioned problems, restores the nitric oxide
homeostasis in an endothelium-independent manner through a safe and effective oral delivery
system. The present invention restores physiological levels of nitric oxide in the body thereby
treating or preventing disease. The present invention overcomes the challenges faced in the prior
art by delivering bioactive nitric oxide sources to targeted locations, including the mouth where
such sources may be reacted to produce nitric oxide which is absorbed in the mouth and then
circulated in the body. The nutraceutical tablet is meant to increase or restore Nitric oxide level
in the body. This is natural way to increase nitric oxide in the body. Nitric oxide is an important
signaiing molecule, which is required for healthy blood circulation. A nutraceutical composition
comprising of Vitamin C, Hawthorn Berry, Beetroot, L- Citruline, Sodium Nitrite,
Methylcobalamin and CO Q 10 in an oral disintegrating tablet form.
BRIEF DESCRIPTION OF THE DRAWINGS
An embodiment of the present invention is described in greater detail below and more fully
appreciated as the same becomes better understood when considered in conjunction with the
accompanying drawi~gs:
18
FIGURE 1 shows NO as one of the most important signaling molecules in the body, and its
involvement in every organ system where it is responsible for modulating an astonishingvariety
of effects.
FIGURE 2 shows the classical L-arginine-NO pathway. It explains the NO production and
biochemistry. There are a number of critical steps for the NOS production of NO from Larginine.
Under healthy conditions (top), enzymatic function proceeds normally.
FIGURE 3 shows the two pathways for endogenous nitric oxide (NO) production. The Larginine
NO pathway can be enhanced through regular exercise, which becomes dysfunctional
with age. The dietary pathway through reduction of nitrate and nitrite is not affected by age.
FIGURE 4 shows the graphical representation of NO production in percentage in the body of
different age groups. It explains the consequences of NO insufficiency in the aging population
and also a comparison within population of different dietary and exercise habits.
FIGURE 5 shows the process flow diagram of different stages involving raw materials and
equipments in manufacturing ofthe nutraceutical composition.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
'i he term "Nutraceutical" refers to products that range from isolated nutrients, dietary
supplements and herbal products, specific diets and processed foods such as cereals, soups, and
beverages. Neuraceutical is regarded as the bio active sqbstance and the constituents are either of
known therapeutic activity or are chemically defined substance generally accepted to contribute
substanWtlly to the therapeutic activity ofthe drug.
The present invention is explained in detail below. This description is not intended to be a
detailed catalogue of all the different ways in which the invention may be implemented, or all
features that may be added to the instant invention. For example, features illustrated with respect
to one embodiment may be incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that embodiment. In addition, numerous
variations and additions to the various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure, which do not depart from the instant invention.
,, Genr~, the following specification is intended to illustrate some particular embodiments of the
invention, and not to exhaustively specify all permutations, combinations and variations thereof.
It is becoming increasingly clear that many diseases are characterized or associated with
perturbations in nitric oxide (NO) production/signaling. Therapeutics or strategies designed to
restore normal NO homeostasis will likely have broad application and utility in human health.
The production of nitric oxide is one of the most important biological processes in the body.
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.
Providing a rich source of nitrate either through direct supplementation with nitrate salts or
through nitrate rich foods such as beet root, for example, will increase . circulating levels of
nitrite. However, because of the inherent inefficiencies that exist for reducing nitrite to NO along
the physiological oxygen gradient, this step in the pathway must be enhanced to effectively
utilize nitrite to make NO. ·
19
The nutraceutical composition includes a novel daily regimen that can safely and effectively
restore nitric oxide levels as well as reduce blood biomarkers routinely used to assess patient
risks for developing cardiovascular disease.
The nutraceutical composition is distinct from nitri~ oxide production through endothelial
production using the L-arginine pathway. The nutraceutical composition utilizes dietary sources
of nitrite~ nitrate, anti- oxidants and vitamins.
The present nutraceutical composition comprising a nitrite salt preferably sodium nitrite wherein
,r:itrate source is preferahly Reet root extract, amino source preferably L-citrulline, vitamins
preferably, Vitamins C along with sweetening agents, antioxidants, disintegrants, lubricants,
col9rs andflavors.
The nutraceutical composition further comprising CoQ 10 wherein CoQlO stands for Coenzyme
QlO which is an oil-~oluble, vitamin-like substance and its primary function in cells is generating
energy.
As botanical source is generally acceptable and regarrled safe for both human and animal
consumption, the botanical source for nitrite_ reduction used here in the invention is Hawthorn
berry extract.
EXAMPLES
The following examples are included to demonstrate preferred embodiments ofthe inventions. It
should be appreciated by those of skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to function well in the practice of
the inventions, and thus can be considered to constitute preferred modes for its practice.
However, those of skill in the art should, in light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the inventions.
EXEMPLARY FORMULATIONS
EXAMPLE 1 (A): In an exemplary embodiment, the formulation of nutraceutical composition is
shown below:
r-_
S.No. Ingredients
1 Vitamin C
2 Mecobalamin 40% Overages
3 Beet root extract
4 Sodium Nitrite
5 L-Citrulline
6 Hawthorn Berry
7 CoQ 10
8 Pearlitol
9 Xylitol
10 Pharmatose DCL-15
II Citric acid anhydrous
20
r
12 Sucra1ose
13 Mixed Fruit (flavour)
14 Pineapple (flavour)
15 Banana (flavour)
16 Vanilla (flavour)
17 Croscarmellose sodium
18 Erythrosine lake
19 Aerosit·
20 Talc
---
21 Magnesium stearate
EXAMPLE l(B): In an exemplary embodiment, the formulation of nutraceutical composition is
shown below:
S.No. Ingredients
~ Vitamin C
2 Mecobalamin 40% Overages
3 Beet root extract
4 Sodium Nitrite
5 L-Citrulline
6 Hawthorn Berry
7 CoQ 10
8 Pearlitol
9 Xylitol
t--
10 Pharmatose DCL-15
11 Citric acid anhydrous
12 Sucralose
13 Mixed Fruit (flavour)
14 Pineapple (flavour)
15 Banana (flavour)
16 Vanilla (flavour)
17 Croscarmellose sodium
18 Erythrosine lake
l9 -
Aerosil
20 Talc
21 Magnesium stearate
EXAMPLE I (C): In an exemplary embodiment, the formulation of nutraceutical composition is
shown below: ·
21
S.No. Ingredients
l VitaminC
2 Mecobalamin 40% Overages
3 Beet root extract
4 Sodium Nitrite
5 L-Citrulline
6 Hawthorn Berry
7 CoQ 10
8 Pearlitol
9 Xylitol
10 Pharmatose DCL-15
11 Citric acid anhydrous
12 Sucralose
13 Mixed Fruit (flavour)
14 Pineapple (flavour)
15 Banana (flavour)
16 Vanilla (flavour)
17 Croscarmellose sodium
18 Erythrosine lake
19 Aerosil
20 Talc
21 Magnesium stearate
One key concept in the neutraceutical compostion is having an appropriate overage. The term
refers to the use of kinetic data on nutrient stability to calculate the amount of added nutrient so
that the anticipated level of the nutrient at the end of the product's shelf life is in accordance with
the level indicated on the label. In the present neutraceutical composition Mecobalamin overages
by40%.
According to above mentioned embodiments the neutraceutical compostion is prepared in the
ranges as given b e1o w:
Ingriedients Best Mode Range
Lower Range (mg) Upper Range (mg)
Vitamin C 40mg 20 100
Hawthorn Berry 150mg 100 300
Beetroot 200mg 100 400
COQ 10 50mg 50 100
Methylcobalamin 750 meg Omcg 1000 meg
L- Citruline 70 mg 50 200
Sodium Nitrite 10mg 5 50
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The said neutraceutical composition is prepared bydispensing the ingredients at ambient
conditions followed by passing of Vitamin. C, Beet root extract, Sodium Nitrite, L-Citrulline,
Hawthorn Berry, Pearlitol, Xylitol, Pharmatose DCL-15, .Croscarmellose sodiumthrough
Mechanical sifter · #40SS screen, Mecobalamin, All Flavors, sucralose, Citric acid,
Aerosilthrough Mechanical sifter #60SS screen and Erythrosine lake colorthrough Mechanical
sifter #80SS screen.
Further the compression of the blend is performed in an octagonal blender for a time period of
10-15 minutes.After blending, this mixture is again mixed with Magnesium stearate and talc_,
which has been already been sifted through #60 SS screen. The final mixture is blended
compressed and packaged in moisture protective containers or films that offer exceptional barrier
properties against moisture and oxygen penetration.
According to present invention the critical step is the preparation of a nutraceutical composition
comprising of Vitamin C, Hawthorn Berry, Beetroot, L- Citruline, Sodium Nitrite and
Methylcobalamin in an oral disintegrating tablet form which is solved by the procedure disclosed
in the present invention.
Referring to figure 5, in one of the preferred embodiments with disClosed ingredients,
equipmentsand their respective amounts as disclosed in exemplary formulations, the
manufacturing process is disclosed thereof.
Method of manufacturing:
I. Weighing
Check weights of all active and inactive raw materials as per the manufacturing formula.
II. Sifting
Sift Vitamin C, Beet root extract, Sodium Nitrite, .L-Citrulline, Hawthorn Berry, Pearlitol,
Xylitol, Pharmatose DCL-15, Croscarmellose sodium through a mechanical sifter fitted with
#40 SS screen. Sift Mecobalamin, All Flavors, sucralose, citric acid anhydrous, Aerosil
through #60 SS screen separately. Sift erythrosine lake color through #80 SS screen. Collect
the sifted material in polythene lined drum and label accordingly.
III. Mixing
Load the sifted material of above step in Octagonal blender and mix for 10-15 minutes.
IV. Lubrication
Sift magnesium stearate and talc through 60# SS screen and add in. mixed material in
octagonal blender and mix for 5 minutes. Unload the mixed lubricated blend in polythene
lined drums: Intirpate QC department for in process sampling and bulk analysis.
Dehumidifiers required during whole process.
Compression stage:
Punch size 16 mm flat round punch
Upper punch : plain
Lower punch : plain
With corresponding dies
Description Light purple round biconvex plain on both sides uncoated tablet.
Weight 1445 mg
23
Thickness 5.5mm
Dosage- Initially 2 tab in a day in divided dosages
After 30 days 1 tab daily
Dosage should be adjusted as per Plasma nitrite or it can be checked by Nitrite testine strips.
NiLrite testing strips are works on griess reagent principle;they diagnose nitrite level from
plasma.
Saliva Nitric Oxide Test Strips are hand held strips that rapidly turn from white to deep pink
based on the amount of a surrogate marker for Nitric Oxide. Within seconds, you are e1ble to selfcheck
and assess your Nitric Oxide status by comparing the intensity of the color on the Nitric
Oxide Test strips to a color chart found on the Berkeley Test tube.
The Nitric Oxide test strip collects and measures the precursor and by-product of Nitric Oxide.
On one end of the strip is the saliva absorption pad. Place absorption pad ("saliva here" on strip)
on your tongue for 3-5 seconds to collect saliva. Fold the absorption pad against the test pad for
3-5 seconds. After pressing together, gently, release and watch the test pad change to a reddish
color within 45 seconds. If the pad remains white with a hue of light pink, you have low Nitric
Oxide levels; however, if the pad shows a deep pinkish-red, your levels are elevated. On the
outside of the Berkeley Test canister a color chart is found. Simply hold your Nitric Oxide test
strip to the color chart to assess your Nitric Oxide status.
Nitric Oxide Test Strips are based on the amount of a metabolite for Nitric Oxide. It is not a
diagnostic or genetic test. It simply increases after a few hours with the consumption of leafy
green foods. The metabolite, nitrite, is a well-accepted marker for Nitric Oxide and a stable
molecule that is both a source and by product of Nitric Oxide. Nitric Oxide is a critically
important molecule in our body that maintains blood vessel health. Nitric Oxide is not to be
confused· with nitrous oxide or nitric acid.
The neutraceutical compositions of the present invention have many possible usages such as,
Blood Circulation improvement, Improvement in lipid profile, Adjuvant therapy in mild to
moderate hypertension, Nutritional Supplementation during diabetes, Anti-ageing.
While at least some exemplary embodiment has been presented in the foregoing detailed
description of the invention, it should be appreciated that a vast number of variations exist. It
should also be appreciated that the exemplary embodiment are not intended to limit the scope,
applicability, or configuration of the invention in any way. It is being understood that various
changes may be made in the function and arrangement of elements described in an exemplary
embodiment without departing from the scope of the invention.

We claim:
I. A nutraceutical composition, comprising:
a nitrate source provided in an amount ranging from I 00 to 400 mg;
a nitrite source provided in an amount ranging from 05 to 50 mg;
L-citrulline provided in an amount ranging from 50 to 200 mg;
Vitamin C provided in an amount ranging from 20-100 mg;
Methylcobalamin provicied in an amount ranging from 0-1000 meg;
CO Q 10 provided in an amount ranging from 50-100 mg;
wherein the nutraceutical composition is in oral disintegrating tablet form.
2. The neutraceutical composition as claimed in claim 1, wherein the nitrite source is
prefer<1bly Sodium nitrite.
3. The neutraceutical composition as claimed in claim 1, wherein the nitrate source is
pref~::rably Beet root extract.
4. A nutraceutical composition as claimed in claim 1, wherein the nutraceutical composition
comprises of 40 mg of Vitamin C, 150 mg of Hawthorn Berry, 200 mg of Beetroot, 70
mg L- Citruline, 10 mg of Sodium Nitrite, 750 meg of Methylcobalamin and 50 mg of
COQ 10.
5. The neutraceutical composition as claimed in claim 1, wherein the neutraceutical
composition · comprises, Pearlitol, Xylitol, Sucralose, Pharmatose DCL-15,
Croscarmellose sodium, Citric acid anhydrous, Erythrosine lake, Aerosil, Talc,
Magnesium stearate and flavors.
6. A method for preparing the neutraceutical composition as claimed in claim I, comprising
the steps of,
Weighing of all active and inactive raw materials as per the manufacturing formula;
Sifting Vitamin C, Beet root extract, Sodium Nitrite, L-Citruiline, Hawthorn Berry,
Pearlitol, Xylitol, Pharmatose DCL-15, Croscarmellose sodium through a mechanical
· sifter fitted with #40 SS screen;
Sifting Mecobalamin, All Flavors, sucralose, citric acid anhydrous, Aerosil through
mechanical sifter #60 SS screen separately;
Sifting Erythrosine Lake color through mechanical sifter #80 SS screen;
Collecting the sifted material in polythene lined drum and label accordingly;
Loading the sifted material in Octagonal blender and mixing for 10-15 minutes;
Sifting magnesium stearate and talc through mechanical sifter 60# SS screen and adding
in mixed material in octagonal blender and mix for 5 minutes;
Unloading the mixed lubricated blend in polythene lined drums;
Compressing and packing the final mixture.