FOR PROVIDING HEALTH BENEFITS

Field of the Invention

[001 ] The present invention relates to an inflammatory and oxidative stress response suppressant and in particular to a composition, mixture, method of treatment for assisting in suppressing inflammatory and oxidative stress responses in cytokine- activated human coronary artery endothelial cells.

[002] The invention has been developed primarily for use in/with a medical sports drink supplement and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

Background of the Invention

[003] Chronic inflammation and oxidative stress underlies the pathogenesis of a number of lifestyle diseases including the major heart disease, atherosclerosis, and type-2 diabetes. Atherosclerosis is the major cause of mortality and morbidity in Western societies, with death rates exceeding those caused from any cancer.

[004] Atherosclerosis is a precursor for cardiovascular disease and can begin at an early age. Traditional risk factors that predispose a person to development of atherosclerosis include smoking tobacco, dietary intake, age, high blood pressure, physical activity and family history.

[005] Atherosclerosis is characterised by hardening and narrowing of blood vessels which transport oxygen rich blood to organs and other parts of the body, associated with a progressive build-up of plaque over a period of time which restricts flow of oxygen-rich blood.

[006] It has been suggested that formation of plaque is initiated when the innermost layer of cells (endothelial cells) of blood vessels is damaged, for instance, by high blood pressure, low-density lipoprotein (LDL cholesterol) or smoking. Impaired endothelial cells initiate increased LDL permeability into the artery wall, platelet aggregation, leukocyte adhesion, and generation of inflammatory cytokines. Once damage occurs, white blood cells stream in to digest the LDL, and with passage of time, accumulation of: i. LDL, it. white blood cells;

iti, other debris in the blood stream including calcium and other crystallised material,

forms a plaque in the artery wall. The degree of endothelial dysfunction is believed to predict future cardiovascular events such as heart attack and stroke. Given the large number of people suffering from diseases/disorders of the vascular system, there is considerable interest in finding therapeutic ways to prevent and treat these diseases.

[007] In a healthy artery, endothelial cells are known to regulate vascular tone and structure, and provide anticoagulant, antiplatelet aggregation, and smooth muscle cell properties. The vascular tone is thought to be maintained by release of various dilator and constrictor substances. One particular vasodilator produced by healthy endothelial cells is nitric oxide (hereafter NO). NO is biosynthesized endogenously from L-arginine, oxygen, and NADPH by the enzyme endothelial nitric oxide synthase (eNOS). NO produced, leaves the endothelial cells and stimulates activity of guanylate cyclase in adjoining smooth muscle cells. Activation of guanylate cyclase increases the level of cyclic guanosine monophosphate (cGMP) and causes the smooth ceil to relax, thus dilating the vessel and increasing the blood flow. The endothelium of blood vessels uses NO to signal the surrounding smooth muscle to relax, resulting in vasodilation and increasing blood flow, providing more oxygen to reach the brain, heart, and other important organs.

[008] Production of NO is therefore important to assist increase blood flow to tissues or organs requiring oxygen and nutrients. Damage to the endothelium is symptomatic of decreased production of NO.

[009] If a healthy diet is consumed, there is usually more than ample L-arginine present for conversion to NO. However, conversion of L-arginine is limited by the rate of expression of the enzyme eNOS in the vascular endothelial cells. Therefore, eNOS represents a rate limiting step in nitric oxide production because at any one time the enzyme is present in rate limiting amounts.

[0010] NO has the ability to dilate blood vessels. Dilated blood vessels allow more efficient nutrient delivery to active muscles and other tissues. More efficient blood supply will also allow higher levels of oxygen to be transported at a quicker rate to our tissues, including active muscles. [001 1 ] NO is important during exercise because it is able to boost the function of mitochondria in cells, including those in skeletal muscle cells. Mitochondria are the energy generating units in cells. The substrate that goes into mitochondria are simple sugars such as glucose and these are converted into the energy unit, ATP.

[0012] Nitric oxide boosts the speed at which glucose is converted to ATP and it facilitates the entry of glucose into the cell. The faster glucose can be taken up by cells, the faster it can be converted into ATP.

[0013] Nitric Oxide is a natural stimulant of muscle growth and repair. This will benefit the active athlete increasing strength, endurance and recovery. Some in-vitro studies have shown that NO is useful in vasodilatiion of blood vessels.

[0014] A source of NO stimulant can be derived from pine bark extract (PBE). Previously this PBE was generated in liquid. Generally now this PBE is obtained and provided in particulate form. Application of such PBE is usually in in-vitro format or in topical format although some ingested forms are known.

[0015] Generally the quantities of such particulate matter is very low quantities such as the dietary supplement of patent document SK500882012 (U1 ), which contains 0.03 g of maritime pine bark extract, 0.19425 g of microcrystalline cellulose, 0.001 15 g of colloidal silica, 0.0023 g of magnesium stearate and 0.0023 g of crosscarmellose sodium.

[0016] Also in patent document CN101780181 , one embodiment of the present invention, a broad spectrum of nitric oxide radical and health products, daily amounts by weight was prepared as follows: L- 4-6 g of arginine, L- citrulline 200-1000 mg , 1 -25 grams of cooked soy flour, Bi Luozhi (pine bark extract) 80-100 mg, 100-150 mg of grape seed extract, ginkgo biloba extract EGB761 100-150 mg, 500 mg of curcumin, vitamin C 500 mg Vitamin E 200-400 mg, β- 20-30 mg carotene, folic acid (containing leucovorin) 800 micrograms, 100 micrograms of selenium.

[0017] PBE is known to be provided in a particulate form which is often added to fruit juice for consumption. However, PBE is only sparingly soluble in solution, and in the solid particulate form PBE is not particularly suitable for absorption to an extent that in order to obtain a desired effect in-vitro, a very substantial quantity of PBE is required.

[0018] In particular it is advantageous to have liquid formulations which contain the PBE in solution and which are convenient and easily administered by the oral route. However, it has been difficult to provide liquid formulations containing PBE due to the highly insoluble nature of the compound. A further issue with oral dosage forms is that while absorption is most desirable from the digestive tract, passage of active through the stomach causes decomposition before absorption can take place. These and other limitations are amplified given the characteristics and cost of PBE, and the desire to provide an oral liquid dosage form which can withstand stomach environment, present sufficient quantity for absorption in the digestive tract, and reduce costs for a consumer.

[0019] By developing suitable liquid formulations, the active is more likely to be absorbed. With commercial form of PBE being provided in a solid particulate, which is added to fruit juice for example, the dose rate is increased to make up for the less efficient transport and availability for absorption through skin tissues in the digestive tract.

One object of the invention therefore is to provide a stable liquid PBE solution

containing an effective amount of PBE which is suitable for oral administration to warm blooded animals, or one which at least provides the public with a useful choice.

[0020] The present invention seeks to provide an inflammatory and oxidative stress response suppressant, which will overcome or substantially ameliorate at least one or more of the deficiencies of the prior art, or to at least provide an alternative.

[0021 ] It is to be understood that, if any prior art information is referred to herein, such reference does not constitute an admission that the information forms part of the common general knowledge in the art, in Australia or any other country.

Summary of the Invention

[0022] The invention relates to liquid ingestible compositions and formulations of Pine Bark Extract (PBE) for providing health benefits, the composition and formulation including:

an effective amount of pine bark extract as active ingredient;

an effective amount of a suspension agent for suspending the active ingredient in a solution; and

an effective amount of a micelle forming component for improving transfer of PBE; wherein the liquid composition and formulation increase bioavailability efficacy of the active constituents of Pine Bark Extract (PBE) for absorption at the small intestine via the stomach to achieve improved rate of production of eNOS. [0023] The compositions and formulations of the invention provide a synergistic effect such that an effective lower dosage of PBE can be administered in a liquid form to substantially overcome the rate limiting step of production of eNOS.

[0024] The compositions and formulations provide a health benefit by increasing the bioavailability of the active constituents of Pine Bark Extract (PBE). Preferably the compositions and formulations are with regard to PBE formed from French Maritime Pine Bark of the form Pinus Pinaster.

[0025] The compositions and formulations of the invention can use an effective amount of PBE in order to provide an ingestible liquid food product that aids promotion of production of Nitric Oxide (NO).

[0026] This aided promotion of production of Nitric Oxide (NO) can aid the users vascular system including one or more of: a) reducing vascular inflammatory responses; b) improving blood oxygenation transfer by the vasodilation of blood vessels; and c) improving vascular damage recovery such as capillary repair.

[0027] This aided promotion of production of Nitric Oxide (NO) can aid the effectiveness of the user's physiological systems following general exercise including to promote recovery and repair of the user's tissues, muscles, blood vessels and other sporting damage repair.

[0028] An effective amount of the Pine Bark Extract (PBE) when provided in the increased bioavailable form is preferably is in the range of greater than 130 milligrams wt/vol.

[0029] An effective amount of PBE is preferably substantially in the range of 100 milligram wt/vol. to 400 milligrams wt/vol. More preferably the range of PBE is substantially in the range of 130 milligram wt/vol. to 260 milligrams wt/vol.

[0030] The bioavailability of the active constituents of Pine Bark Extract (PBE) from a comminuted particulate form is provided by the provision of the PBE in solution.

[0031 ] In particular the bioavailability is improved by providing the solution of PBE in a micelle forming component to allow transport through to the small intestine.

[0032] The micelle format is preferably provided by the use of Aloe Vera in liquid form to provide a coating like effect to allow transport through to the small intestine by surviving passage through the stomach and allowing absorption in the small intestine. [0033] The micelle format is preferably aided by the use of honey.

[0034] The bioavailability of the active constituents of Pine Bark Extract (PBE) can be improved by addition of a suspension agent to increase the quantity PBE held in suspension.

[0035] The present composition and formulations provide a substantial and unexpected improved effectiveness in delivery and absorption efficacy of PBE. The particular compositions provide a synergistic effect such that a small amount of PBE in the range of 0.1 % wt/vol to 0.4 % wt/vol in a fluid delivery form can present sufficient concentration of an effective amount of pine bark extract through the stomach for absorption in the small intestine.

[0036] The invention also relates to methods for treatment and prophylaxis of diseases or disorders of the vascular system including artherosclerosis and/or NO- responsive diseases and conditions by the oral administration of a liquid dosage composition comprising an active component derived from pine bark extract and delivery components comprising micelle forming component and suspension agent in combination with pharmaceutically acceptable carriers.

[0037] A first aspect provides a method for improving bioavailability of pine bark extract in an oral dosage form comprising providing a predetermined amount of suitable pine bark extract for a given dose volume, suspending the pine bark extract in a solution of predetermined volume of an effective amount of a suspending agent, and including a micelle forming constituent in effective amount to solubilise particulate or aggregate PBE, wherein said oral dosage form effectively solubilises and transports the pine bark extract to the intestine for absorption.

[0038] A further aspect provides a method for one or more of:

(a) decreasing cytokine-induced inflammatory and oxidative stress responses in human coronary artery endothelial cells,

(b) increasing production of eNOS in human coronary artery endothelial cells,

(c) increasing plasma concentration of NO, and concomitant vasodilation of blood vessels,

(d) promoting or improving muscle, ligament or tendon mass, repair, form or function, or

(e) treating a disease or disorder of the vascular system and/or NO-responsive disease and condition, the method comprising administering to a subject an effective amount of a composition or formulation in a liquid (aqueous or non-aqueous) dosage form having a particulate form of pine bark extract present in an amount of about 0.1 % wt/vol to 0.4% wt/vol; an effective amount of a micelle forming component such as aloe vera; and a suspension agent selected from from sorbitol, syrup, methyl cellulose, gelatin,

hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents.

[0039] In a further aspect the invention provides a liquid composition for treatment of disease or disorder of the vascular system and/or NO-responsive disease and condition, increasing blood flow supply in blood vessels, increasing production of eNOS in human coronary artery endothelial cells, increasing plasma concentration of NO, and concomitant vasodilation of blood vessels, or promoting or improving muscle, ligament or tendon mass, repair, form or function, the composition comprising an oral dosage form comprising a predetermined amount of suitable pine bark extract for a given dose volume, a suspension agent for suspending the pine bark extract in a solution of predetermined volume, and a micelle forming constituent, wherein said oral dosage form effectively solubilises and transports the pine bark extract to the intestine for absorption.

[0040] In another aspect the invention relates to a liquid composition or pharmaceutical, or a liquid food additive or dietary supplement for oral delivery comprising pine bark extract and delivery components to provide an improved liquid bioavailable oral dosage which transports the pine bark extract to the small intestine for absorption. The composition can optionally contain an additional therapeutic or beneficial-to-health agent, or can be administered in combination with another therapeutic or beneficial-to-health agent.

[0041 ] Packaged products containing the above-mentioned compositions and a label and/or instructions for use for prevention or treatment of diseases/disorders of the vascular system and/or NO-responsive diseases and conditions are also within the scope of the invention.

[0042] In another aspect, the invention relates to methods for preventing or treating a disease or disorder of the vascular system and/or NO-responsive disease and condition in a mammal, such as a human, by oral administration of an effective amount of a composition including pine bark extract as an active ingredient, suspension agents, and micellular forming substances capable of enveloping active particulates so as to improve bioavailablity of the active for absorption.

[0043] According to a further aspect of the present invention, there is provided a composition containing an inflammatory and oxidative stress response suppressant in a form for oral delivery whereby an effective amount of the active ingredient pine bark extract is presented for absorption in a composition further including a micelle component and a suspension agent, such that the rate of expression of eNOS by endothelial cells of blood vessels is significantly increased for immediate conversion of L-arginine to NO in the endothelium of blood vessels such as arteries.

[0044] The oral composition of the invention achieves improved bioavailability and bioabsorption of pine bark extract by providing a form which substantially withstands decomposition in stomach acid environment, and both delivers and activates the active agent to the small intestine for absorption. The increased rate of production of eNOS addresses the rate limitation of insufficient enzyme for conversion of L-arginine. Accordingly, with this imbalance substantially corrected, NO is produced by the endothelium at a rate which enables consistent vasodilation hence improved flow of oxygenated blood to inflammatory tissues.

[0045] In one form the oral delivery composition can be a liquid composition containing an effective amount of pine bark extract in suspension and including a micellular envelope. Alternatively, the oral delivery composition can be provided in a powdered form or tablet for oral administration, or can be added to a solvent for oral delivery.

[0046] The present applicant has shown that the oral delivery composition of the invention comprising the inflammatory and oxidative stress response suppressant provides a beneficial means for improving the amount and rate of synthesis of eNOS so that NO can be generated more rapidly.

[0047] According to a second aspect of the present invention, an inflammatory and oxidative stress response suppressant is provided by increased bioavailability in the body of Nitric Oxide. In one embodiment there is described a composition for substantially assisting reducing and aiding muscle recovery following exercise, the composition for oral administration including an effective amount of pine bark extract, a suspension agent for suspending pine bark extract in aqueous solution, an acid such as acetic acid or citric acid, and micellular generating agent capable of solubilizing the extract for transport through digestive tract, wherein the composition is taken orally in an effective dose for increasing rate of eNOS expression in endothelial cells of blood vessels such as arteries for increased blood flow and transport of O2 and nutrients to aid tissue recovery.

[0048] Applicants have shown that the invention of an inflammatory and oxidative stress response suppressant provides the benefit of unique natural beverage supplement for consumers that result in increased levels of general health and wellbeing. It introduces a new category of product function for the human body. It has both a health benefit and a wellbeing benefit as well as being an athlete offering for all sports people including elite athletes.

[0049] The invention aims to reduce tissue inflammation, generate nitric oxide to expand blood vessels, which then delivers increased oxygen and nutrient levels to the muscles, removes toxic waste from the body faster, reduces DVT onset and increases internal antioxidant production via an epigenetic function in the body at the cellular level.

[0050] In a related aspect of the present invention, there is described a food supplement composition for assisting in increased rate of production of eNOS

comprising:

an effective amount of pine bark extract as active ingredient;

an effective amount of a suspension agent for suspending the active ingredient in a solution;

an effective amount of a micellular forming component; and

an effective amount of a food acid for obtaining an acidic composition.

[0051 ] In a preferred embodiment, the pH is of high acidity such as about 4.

[0052] The food supplement can be added to food including drink, food and the like in a predetermined dosage.

[0053] In a preferred embodiment, the composition for substantially assisting recovery of muscle tissue following inflammatory response includes an oral dosage form comprising: a) an amount of between about 0.1 % w/v and 0.4% w/v pine bark extract as active ingredient; b) an amount of about between about 1 % and 6% w/v of a suspension agent for suspending the active ingredient in a solution; c) an amount of about 0.1 % w/v to 0.3% w/v of a micellular forming component; d) an amount of about 1 % to 4% w/v of a food acid; and e) balance of a pharmaceutically acceptable carrier. wherein the composition is administered orally in a predetermined dosage amount for substantially increasing the rate of production of eNOS in endothelial cells of blood vessels for improved O ₂ blood transport to affected tissues.

[0054] Applicants have shown that an effective dosage form according to the aforementioned composition substantially inhibits inflammatory responses.

[0055] In a further preferred embodiment, the pine bark extract is provided in particulate form and undergoes a comminute process. These compounds are generally hydrophobic, and while known to have been tested in-vitro with fruit juices, the bioavailability is a rate limitation hence usually large dosages are usually required.

[0056] Suspending agents for liquid preparations for example, can be selected from sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol.

[0057] Preferably the suspending agent can be selected from a range including honey, glycerine, polyethylene glycol and agents that are generally miscible with water and reduce liquid air interfacial tension.

[0058] The food acid can be selected from a range of acids generally regarded as safe for consumption including acetic acid, citric acid, ascorbic acid and the like.

[0059] The micellular forming component can be selected from a range of components including but not limited to aloe extract, surfactants including polysorbate, pluronics and poloxamers. Preferably the micellular forming component is aloe extract.

[0060] The composition of the invention can further include flavouring or coloring agents, natural or artificial sweeteners, and preservatives such as sodium benzoate, and potassium sorbate, methyl or proply p-hydroxybenzoate or sorbic acid for enhancing oral acceptability and storage.

[0061 ] In a preferred form, the composition is provided in a liquid composition. The pharmaceutical acceptable carrier can be selected from any suitable carrier including water. [0062] Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or with another suitable vehicle before use.

[0063] In one aspect, the composition for substantially assisting recovery of muscle tissue following inflammatory response includes an oral dosage form comprising: a) an amount of about 0.1 % w/v to 0.4% wt/vol of pine bark extract; b) an amount of about 4% of glycerine; c) an amount of about 0.175% w/v of aloe extract; d) an amount of about 2% w/v of acetic acid; e) an amount of about 0.3% sodium chloride; f) an amount of about 2% flavour; g) an amount of about 0.1 % stevia extract; h) an amount of about 0.04% sodium benzoate; i) an amount of about 0.04% potassium sorbate; and j) water

[0064] In an alternative embodiment, the composition can include an oral dosage form comprising: a) an amount of about 0.1 % wt/vol to 0.4% wt/vol pine bark extract (as Pine Bark Extract) ; b) an amount of about 10% honey; c) an amount of about 2% vinegar; d) an amount of about 0.175% aloe extract; e) an amount of about 2% flavour; f) an amount of about 0.3% sodium chloride; g) an amount of about 0.04% sodium benzoate; h) an amount of about 0.04% potassium sorbate; and i) a volume of water

[0065] In an even further preferred embodiment there is provided a prophylactic treatment for prevention and/or treatment of artherosclerosis in a subject, comprising orally administering a predetermined dosage at predetermined intervals of a composition comprising: a) an amount of between about 0.1 % w/v and 0.2% w/v pine bark extract as active ingredient; b) an amount of about between about 1 % and 6% w/v of a suspension agent for suspending the active ingredient in a solution; c) an amount of about 0.1 % w/v to 0.3% w/v of a micellular forming component; d) an amount of about 1 % to 4% w/v of a food acid; and e) a volume water.

[0066] The pharmaceutical compositions for oral administration according to the present invention are prepared by methods known per se and the administration thereof is by known methods of oral administration.

[0067] A method of assisting tissue recovery from inflammatory response in endothelium of blood vessels including administering an effective dose of a composition comprising an amount of between about 0.1 % w/v and 0.2% w/v pine bark extract as active ingredient; a) an amount of about between about 1 % and 6% w/v of a suspension agent for suspending the active ingredient in a solution; b) an amount of about 0.1 % w/v to 0.3% w/v of a micellular forming component; c) an amount of about 1 % to 4% w/v of a food acid; and d) balance of a pharmaceutically acceptable carrier.

[0068] In a further related aspect of the invention, there is described a drink composition for assisting recovery of inflammatory tissue response following exercise can include an oral dosage form comprising: a) an amount of about 0.26% pine bark extract; b) an amount of about 10% honey; c) an amount of about 2% vinegar; d) an amount of about 0.175% aloe extract; e) an amount of about 2% flavour; f) an amount of about 0.3% sodium chloride; g) an amount of about 0.04% sodium benzoate; h) an amount of about 0.04% potassium sorbate; and i) a volume of water

[0069] An amount effective to treat the disorder hereinbefore described depends on the usual factors such as the nature and severity thereof and the weight of the subject.

[0070] For oral administration, it is preferred that the active ingredient be administered in the form of a unit-dose composition. Such compositions are prepared by admixture and are suitably adapted for oral administration in the form of tablets, capsules, oral liquid preparations, powders, granules, etc.

[0071 ] Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colorants, flavorings, and wetting agent.

[0072] Suitable fillers for use include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.

[0073] These solid oral compositions may be prepared by conventional methods of blending, filling or tabletting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art.

[0074] Oral formulations also include conventional sustained release formulations, such as tablets or granules having an enteric coating.

[0075] In one embodiment of the present invention there is disclosed a method of increasing the rate of production of eNOS by administering an effective amount a composition containing pine bark extract.

[0076] Inflammatory response is found to be significantly inhibited by treatment with a dosage form of the present invention. This has the benefit of assisting blood flow, aiding in the recovery of inflammatory response in muscle tissue following exercise, and reducing incidences of plaque initiation and thus artherosclerosis. Without being bound by theory, applicants postulate that significant improvements are achieved by the present invention because pine bark extract particulates are i) substantially maintained in suspension in an oral liquid dosage form, ii) micellular component aggregate to help transport the active compound to the intestine to be absorbed, and iii) the active component of pine bark extract is activated by the acid present in the formulation for increased bioavailability.

[0077] Effective amount of pine bark is seen to be based around the preferred amount of 0.26% wt/vol when used in the specific embodiments using the particular pine bark of pinus pinaster. However the effective amount was a range around this figure which provided a substantial and unexpected improvement in effectiveness.

[0078] By review in the particular pine bark of pinus pinaster, this has been clearly shown that the range of 0.1 % wt/vol to 0.4% wt/vol provides an effective amount particularly a synergistic effective amount in combination with the the composition and formulation including an effective amount of pine bark extract as active ingredient; an effective amount of a suspension agent for suspending the active ingredient in a solution; and an effective amount of a micellular forming component.

[0079] Any composition described herein may be used to practice the methods described in the present application. As used herein, "vascular disease or disorder" or "diseases or disorders of the vascular system" refers to any disease or disorder affecting the vascular system, including the heart and the brain. Examples of such conditions include atherosclerosis, thrombosis (including occlusive thrombosis), hypertension (e.g. primary, secondary and pulmonary hypertension), cardiovascular disease (CVD), coronary artery disease (CAD) (including myocardial ischemia, myocardial infarction, stable and unstable angina, acute occlusion or restenosis), diabetes (type I and type II) (e.g. vascular complications of diabetes), cognitive dysfunction or disorder and/or vascular circulation disorders (including those of the brain), heart attack, cerebrovascular disease.

[0080] An "NO-responsive disease or disorder" refers to a health condition which responds to treatment with NO. Examples of such conditions include, but are not limited to NO-mediated or NO-dependent diseases and disorders, in which the pathology of the disease/disorder is caused by abnormal functioning of the NO pathway. For example, the conditions include hypertension (e.g. primary, secondary and pulmonary hypertension), cardiovascular disease, coronary artery disease, diabetes (type I and type II) (e.g. vascular complications of diabetes), vascular circulation disorders (including those of the brain), heart attack, stroke, congestive heart failure, kidney failure, and renal disease. Because high blood pressure increases the risk of heart attack, stroke, congestive heart failure, and kidney failure, the compounds described above which cause vasorelaxation can be utilized, alone or in combination with other vascular (including cardiovascular)-protective agents, to prevent these conditions.

[0081 ] Other aspects of the invention are also disclosed. For example it can be seen that the invention provides an inflammatory and oxidative stress response suppressant

Brief Description of the Drawings

[0082] Notwithstanding any other forms which may fall within the scope of the present invention, a preferred embodiment / preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Fig. 1 is a schematic representation of pathway inhibition of inflammatory response by action of a dose of the composition of the present invention

Fig. 2 represents the results of (i) pre-treatment of HCAEC for 24-hours with plasma taken from subjects before dosage and three-hours following dosages of a supplement in accordance with a preferred embodiment of the present invention, and (ii) exposure of pre-treated HCAEC with inflammatory stimulus;

Fig. 3a to 3c represents the results of inflammation suppression of human coronary artery endothelial cells in terms of TNF-a induced expression of cell adhesion molecules VCAM-1 , ICAM-1 and ELAM expression following exposure to various concentration equivalents of a supplement in accordance with a preferred embodiment of the present invention

Fig. 4a and 4b represent the results of inflammation suppression of protein levels of VACAM-1 and ICAM-1 in HCEAC in the presence of inflammatory stimuli following treatment with a composition according to the present invention;

Figure 5 represent results of the effect of inventive composition on decreased activation of key mediator NFkB for expression of VCAM-1 , ICAM-1 and ELAM. Fig. 6 represents results following tests directed at determining the effect of the composition of the invention on decrease of reactive oxygen species (ROS) accumulation in HCEAC; Fig. 7a and 7b represents results following test of effect of composition of present invention on modulation of expression of genes encoding for the ROS generating enzyme, NOX4 and the ROS metabolizing enzyme SOD-1 ;

Fig. 8 represents test results of effect of composition of the present invention on athletes including runners and cyclists at predetermined intervals using plasma concentrations of inflammatory marker C Reactive Protein (CRP)

Description of Preferred Embodiments

[0083] It should be noted in the following description that like or the same reference numerals in different embodiments denote the same or similar features.

[0084] By way of example, the formulations and treatments which follow are not intended to be limiting, and are provided for illustrating the efficacy of the inventive concept on decreasing cytokine-induced inflammatory and oxidative stress response in vitro and in-vivo.

[0085] Referring to figure 1 , there is shown a schematic representation illustrating effect of formulations and treatments according to the present invention to protect human coronary artery endothelial cells (HCAEC) against TNF-a induced inflammatory response and oxidative stress resulting from dysfunctional endothelial cells in an artery.

[0086] In particular figure 1 broadly shows a series of interdependent reaction pathways that follow a TNF-a induced 1 inflammatory response and activation of ROS sensitive transcription 2 in HCAEC 3. In a first pathway 4, TNF-a activates a key mediator for cell adhesion molecules, nuclear factor kappa B (NFkB) 5, in the endothelium 3. As a direct consequence of this activation, NFkB significantly increases the expression of cell adhesion molecules 6 (VCAM-1 , ICAM-1 and ELAM). In turn, cell adhesion molecules bind leukocytes including monocytes 7 which migrate into the blood vessel wall 8. Use of formulations and treatments of the invention 9 act in pathways A, B, C and D to suppress the production of cell adhesion molecules by intervention of activation of NFkB at (A), (B) and/or (C).

[0087] As indicated, when endothelial cells lining the lumen of an artery are activated and rendered dysfunctional, the cells express cell adhesion molecules VCAM- 1 , ICAM-1 and ELAM. Adhesion molecules VCAM-1 and ICAM-1 are regulated by NFkB following activation increasing expression of VCAM-1 , ICAM-1 . NFkB is activated when endothelial cells are exposed to inflammatory cytokines (TNF-a) via NFkB signaling pathway, and reactive oxygen species (ROS). Oxidative stress occurs from an imbalance caused by increased ROS in tissues and cells when enzymes that produce ROS are expressed in higher levels than enzymes that scavenge or metabolise ROS.

[0088] The invention in one aspect relates to the treatment of inflammatory response conditions and tissue recovery using compositions and formulations in accordance with the present invention. In a further aspect the invention relates to treatment of vascular disease such as artherosclerosis. In a further aspect, the invention relates to a composition for oral administration that increases bioavailability of the active constituent of pine bark extract for increase rate of production of eNOS.

[0089] TREATMENTS

[0090] The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage.

[0091 ] Thus, for example, the present method of "treating" a condition encompasses both prevention of the condition in a predisposed individual, treatment of the condition in a clinically symptomatic individual and treatment of a healthy individual for beneficial effect.

[0092] "Treating" as used herein covers any treatment of, repair or prevention of a condition in a mammal, particularly a human, and includes inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition and producing a beneficial effect.

[0093] "Prophylaxis" or "prophylactic" or "preventative" therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.

[0094] As used herein, "condition" refers to any deviation from normal health and includes a disease, disorder, defect or injury, such as injury caused by trauma, and deterioration due to inflammatory response. Conditions in which the oral composition administration is beneficial generally fall into the categories of those in which involve endothelium dysfunction and reduced expression of eNOS for production of Nitric Oxide.

[0095] Nitric oxide (NO) contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Humans with atherosclerosis, diabetes, or hypertension often show impaired NO pathways.

[0096] ORAL COMPOSITIONS

[0097] The oral compositions of the present invention include pine bark extract as the active constituent derived from commercially available Pine Bark Extract or Pine Bark Extract® These products are obtained as an extract from the bark of the French maritime pine (Pinus pinaster). It is rich in water-soluble polyphenol compounds including procyanidins, catechin, taxifolin and phenolcarbonic acids that have known antioxidant and anti-inflammatory activity. In-vitro experiments using Pine Bark Extract or Pine Bark Extract® suggest that they may influence endothelial function by stimulating endothelial nitric oxide synthase with the result of elevated nitric oxide levels.

[0098] Pine bark extract (Pine Bark Extract or Pine Bark Extract®) is known to be taken in a liquid form in water or has been introduced by mouth in fruit juice mixture. However, a particular drawback with conventional methods of administration is that bioavailability is limited by the poor ability of the pine bark extract to withstand stomach acidity, i.e. it decomposes rapidly in stomach acid, and its ability to reach the small intestine in sufficient for absorption is .

[0099] It appears that even if a sufficient amount of the pine extract could withstand passage through the stomach, the physical characteristics of the extract make it substantially incompatible with cells in the small intestine.

[00100] The compositions used in examples comprise a first composition for oral administration comprising:

about 0.26% w/v of pine bark extract;

about 4% of glycerine;

about 0.175% w/v of aloe extract;

about 2% w/v of acetic acid; about 0.3% sodium chloride;

about 2% flavour;

about 0.1 % stevia extract;

about 0.04% sodium benzoate;

about 0.04% potassium sorbate; and

balance of water

[00101 ] A second composition for oral administration comprises: about 0.26% pine bark extract (as Pine Bark Extract);

about 10% honey;

about 2% vinegar;

about 0.175% aloe extract;

about 2% flavour;

about 0.3% sodium chloride;

about 0.04% sodium benzoate;

about 0.04% potassium sorbate; and

balance to volume of water

[00102] ADMINISTRATION

[00103] The composition is administered in a pharmaceutical composition together with a pharmaceutically acceptable carrier for oral administration.

[00104] The composition can be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated and the subject. The composition can be administered orally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[00105] A typical daily dosage might range from about 6.25 uL/mL to up to 50 uL/mL or more.

[00106] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. [00107] The invention will now be described by way of reference only to the following non- limiting examples.

[00108] The invention will now be described by way of reference only to the following non- limiting examples. EXAMPLES:

[00109] Example 1 : Determine the effect of composition on inflammatory stimulus TNF-a by measuring VCAM-1 levels:

[001 10] Methods and treatment

[001 1 1 ] Human subjects were requested to consume 100ml of an oral composition according to first composition (refer above). Blood sampling was taken and completed immediately before oral administration and at three hours after ingestion of the measured volume.

[001 12] Plasma was isolated from collected blood samples, and the plasma was used to treat HCAEC.

[001 13] HCAEC were treated for a period of 24 hours with the isolated plasma and the treated HCAEC were exposed to an inflammatory stimulus pro-inflammatory cytokine TNF-a.

[001 14] Figure 2 shows the extent of inflammation by monitoring cell adhesion molecule protein VCAM-1 levels. The results show a significant reduction in TNF-a expression.

[001 15] Example 2 : Determine the effect of AOD on cartilage repair by evaluating tissue formation:

[001 16] Methods

Cell culture and treatment

[001 17] Human coronary artery endothelial cells were cultured in standard laboratory conditions. HCAECs were pre-incubated with composition 1 (50, 25, 12.5, and 6.25 ul) for 3 hours before they were stimulated with the inflammatory cytokine, TNFalpha, for 1 (mRNA) or 3 (protein) hours.

[001 18] HCAEC were then assayed for cell adhesion molecules VCAM-1 , ICAM-1 and ELAM. HCAECs were exposed to increasing volumes of the composition for 3 hours. After 3 hours, HCAECs were activated with an inflammatory insult, TNFalpha for 1 hour. In HCAEC that were not exposed to the composition, TNFalpha increased VCAM-1 expression by 15-fold, ICAM-1 by 10-fold, and ELAM by 100-fold. The composition supplementation was able to suppress TNFalpha induced mRNA levels for all the CAMs. The composition dose of 25ul_/ml is equivalent of consuming 100ml of the composition. The results suggest that composition is having an anti-inflammatory effect in HCAECs.

[001 19] Figures 3a to 3c show that the composition of the invention decreases TNFalpha-induced VCAM-1 , ICAM-1 and ELAM expression in HCAECs.

[00120] Example 3 : Effect of composition on protein levels of VCAM-1 and

ICAM-1 in HCAEC:

[00121 ] Treatment

[00122] This example examines if CAM protein levels are decreased by a composition according to the invention in the presence of inflammatory stimuli. As in example 2 (above), HCAECs were exposed to composition 1 for 3 hours and then activated with TNFalpha for 3 hours.

[00123] Referring to figure 4, the composition was found to significantly decrease protein levels of both VCAM-1 and ICAM-1 (Figure 4, ELAM was not measured). Due to the half-life of membrane proteins, the effective suppression of the composition was not as great as for the mRNA, which has a much quicker turnover.

[00124] Example 4 : Effect of the composition on NFkB activation/expression and ROS levels

[00125] Treatment

[00126] The expression of VCAM-1 , ICAM-1 and ELAM are regulated by NFkB.

Example 4 determines if the composition 1 decreases the activation of NFkB, and ROS levels within TNF-a activated HCAECs.

[00127] NFkB is a redox sensitive protein. Increased ROS levels will therefore activate this regulatory factor. The next experiment explored whether the composition was able to decrease ROS accumulation in human coronary artery endothelial cells. To this end, HCAECs were exposed to the composition for 3 hours before loaded with the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH). The cells were then activated with TNFalpha for 15 minutes. DCFH can be rapidly oxidised to a highly fluorescent compound called dehydrodichlorofluorescin by various ROS and peroxynitrite and it was determined that after TNF-alpha activation, fluorescence intensity increased dramatically. This effect was significantly abrogated in HCAECs that were pre-exposed to the treatment.

[00128] As shown in figures 5 and 6, the composition decreases activation of the key mediator of inflammation NFkB, and decreases ROS levels within TNF-a activated HCAECs.

[00129] Example 5 : Effect of modulation on expression of genes encoding for the ROS generating enzyme, NOX4 and the ROS metabolising enzyme SOD-1

[00130] Treatment

Results shown in figures 6a and 6b indicate that the composition decreases NOX4 mRNA levels by a small but significant level. The composition was also shown to restore SOD-1 mRNA levels back to baseline.

[00131 ] Example 6 :

[00132] A laboratory study has shown that the composition 2 Supplement can reduce plasma concentrations of the inflammatory marker, acute phase reactant, C Reactive Protein (CRP). Athletes were asked to complete a long run (15— 20km+) or long ride (90— 160km+) on two separate occasions, 3— weeks apart.

[00133] Blood sampling was performed prior to exercise and within 15 minutes post- exercise. On one of the occasions, the composition supplement was ingested, prior to the training run or ride. On the other occasion a placebo supplement was ingested.

[00134] The athletes were blinded to the supplement that they ingested each time. ELISA tests showed that CRP levels were markedly decreased in the blood of the athletes post— training after the composition, whereas there was no effect of the placebo supplement (slight increase in CRP levels after exercise).

[00135] A decrease in CRP reflects a decrease in systemic inflammation caused by endurance exercise training. In turn, this may reduce muscle damage due to inflammation.

[00136] Results

[00137] The compositions decreases cytokine-induced inflammatory and oxidative stress responses in human coronary artery endothelial cells. The mechanism involves suppressing the activation of the key mediator of inflammation, NFkB, by decreasing the generation of ROS and thereby lowering the level of oxidative stress in HCAECs in response to inflammatory stimuli.

[00138] Inflammation and oxidative stress underlies a number of chronic diseases including atherosclerosis, liver inflammation associated with type 2 diabetes and obesity, endometriosis, and osteoarthritis. The laboratory findings for the composition of the invention suggest that this supplement may decrease the symptoms and causes associated with these chronic diseases.

[00139] General Characteristics

[00140] Pine bark extract (preferably from pinus pinaster) is known for antiinflammatory efficacy. Pine bark extract is conventionally taken in a liquid form in water or has been introduced by mouth in fruit juice mixture.

[00141 ] A particular drawback with conventional methods of administration is that bio- absorption is limited by the poor ability of the pine bark extract to withstand stomach acidity, i.e. the extract is decomposed by the stomach acid and is not available in a form for effective bio-absorption in the small intestine.

[00142] It appears that even if a sufficient amount of the pine extract could withstand passage through the stomach, the physical characteristics of the extract make it substantially incompatible with cells in the small intestine.

[00143] In another form the invention includes increasing bioavailability in the body of Nitric Oxide. The composition of the invention decreases cytokine-induced inflammatory and oxidative stress responses in human coronary artery endothelial cells. The mechanism involves suppressing the activation of the key mediator of inflammation, NFkB, by decreasing the generation of ROS and thereby lowering the level of oxidative stress in HCAECs in response to inflammatory stimuli.

[00144] Inflammation and oxidative stress underlies a number of chronic diseases including atherosclerosis, liver inflammation associated with type 2 diabetes and obesity, endometriosis, and osteoarthritis. The laboratory findings for the composition of the invention suggest that this supplement decreases the symptoms and causes associated with these chronic diseases by

(a) increasing bioavailability in the body of Nitric Oxide (b) effecting expression of eNOS to improve the amount and rate of synthesis of eNOS so that NO can be generated more rapidly in the user.

(c) Decreasing tissue inflammation

[00145] Nitric oxide (NO) contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Humans with atherosclerosis, diabetes, or hypertension often show impaired NO pathways.

[00146] Pine Bark Extract may influence endothelial function by stimulating endothelial nitric oxide synthase with the result of elevated nitric oxide levels. Bioavailability of pine bark extract is compromised by poor ability to withstand stomach acidity. Even if a sufficient amount of the pine extract could withstand passage through the stomach, the physical characteristics of the extract make it substantially incompatible with cells in the small intestine for bio-absorption.

[00147] Pine bark extract is therefore usually administered in a tablet or capsule dosage form. The present invention provides a liquid dosage form which is easy to ingest and transports an effective amount of PBE to the small intestine for absorption. The availability of PBE increases rate of production of enos such that concentration of enos is no longer a rate limitation in the production of NO from L-Arginine. Aloe surrounds/coats the pine bark extract especially when hits gut so great bioavailability

[00148] Vascular NO dilates all types of blood vessels by stimulating soluble guanylyl cyclase and increasing cyclic guanosine monophosphate (cGMP) in smooth muscle cells. NO released toward the vascular lumen is a potent inhibitor of platelet aggregation and adhesion. NO also can inhibit leukocyte adhesion to the vessel wall either by interfering with the ability of the leukocyte adhesion molecule CD1 1/CD18 to form an adhesive bond with the endothelial cell surface or by suppressing CD1 1 /CD18 expression on leukocytes.

[00149] White cell adherence is an early event in the development of atherosclerosis; therefore, NO may protect against the onset of atherogenesis. Furthermore, ΝΟ· has been shown to inhibit DNA synthesis, mitogenesis, and proliferation of vascular smooth muscle cells. The inhibition of platelet aggregation and adhesion protects smooth muscle from exposure to platelet-derived growth factor(s). Therefore, ΝΟ· also prevents a later step in atherogenesis, fibrous plaque formation. Based on the combination of those effects, endothelial NO* probably represents the most important antiatherogenic defense principle in the vasculature.

[00150] Impaired function of the endothelium, which is the innermost cell layer of the arteries, is an initial step in the development of atherosclerosis. The degree of endothelial dysfunction is believed to predict future cardiovascular events such as heart attack and stroke. Pine Bark Extract can influence endothelial function by stimulating endothelial nitric oxide synthase with the result of elevated nitric oxide levels.

Interpretation

Embodiments:

[00151 ] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[00152] Similarly it should be appreciated that in the above description of example embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description of Specific Embodiments are hereby expressly incorporated into this Detailed Description of Specific Embodiments, with each claim standing on its own as a separate embodiment of this invention.

[00153] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Different Instances of Objects

[00154] As used herein, unless otherwise specified the use of the ordinal adjectives "first", "second", "third", etc., to describe a common object, merely indicate that different instances of like objects are being referred to, and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

Specific Details

[00155] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Terminology

[00156] In describing the preferred embodiment of the invention illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar technical purpose. Terms such as "forward", "rearward", "radially", "peripherally", "upwardly", "downwardly", and the like are used as words of convenience to provide reference points and are not to be construed as limiting terms.

Comprising and Including

[00157] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. [00158] Any one of the terms: including or which includes or that includes as used herein is also an open term that also means including at least the elements/features that follow the term, but not excluding others. Thus, including is synonymous with and means comprising.

Scope of Invention

[00159] Thus, while there has been described what are believed to be the preferred embodiments of the invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the scope of the invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.

[00160] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Industrial Applicability

[00161 ] It is apparent from the above, that the arrangements described are applicable to the sports medicine and health industries.