阅读说明：本技术 代谢障碍的植物调节剂 (Plant modulators of metabolic disorders ) 是由 贾廷德·拉纳 凯莉·米切尔 于 2019-09-04 设计创作，主要内容包括：公开了也起到PPAR-γ激动剂作用的MMP-9的基于植物的抑制剂,以及此类基于植物的抑制剂/激动剂在调节代谢障碍中的用途。所述基于植物的抑制剂/激动剂至少是从越橘属(Vaccinium)的叶获得的提取物。(Plant-based inhibitors of MMP-9 that also function as PPAR-gamma agonists are disclosed, as well as the use of such plant-based inhibitors/agonists in the regulation of metabolic disorders. The plant-based inhibitor/agonist is at least an extract obtained from the leaves of the genus Vaccinium (Vaccinium).)

1. A composition comprising a plant extract of cranberry (Vaccinium macrocarpon) leaves, wherein the plant extract exhibits modulation of one or more metabolic disorders.

2. The composition of claim 1, wherein the plant extract is present in an amount of about 1.0 μ g/mL or more.

3. The composition of claim 2, wherein the plant extract is present in an amount of about 1.0 μ g/mL to about 2000.0 μ g/mL.

4. The composition of claim 1, wherein the composition further exhibits MMP-9 inhibition.

5. The composition of claim 4, wherein the plant extract is present in an amount of about 1.0 μ g/mL to about 2000.0 μ g/mL.

6. The composition of claim 1, wherein said composition further exhibits PPAR-gamma agonist activity.

7. The composition of claim 6, wherein the plant extract is present in an amount of about 50.0 μ g/mL to about 2000.0 μ g/mL.

8. A dietary supplement having modulating properties for one or more metabolic disorders comprising a therapeutically effective amount of a plant extract of cranberry leaves.

9. The dietary supplement of claim 8, wherein the plant extract of cranberry leaves is present in an amount of about 1.0 μ g/mL or greater.

10. A method of modulating one or more metabolic disorders in a subject, the method comprising administering a composition comprising a plant extract of cranberry leaves at a concentration of about 1.0 μ g/mL to about 2000.0 μ g/mL.

Technical Field

The present invention relates generally to MMP-9 inhibitors and PPAR-gamma agonists, and more particularly to plant-based or plant inhibitor, i.e., cranberry (cranberry) leaves, of MMP-9 that also functions as a PPAR-gamma agonist, and the use of such plant-based inhibitors/agonists in the modulation of one or more metabolic disorders.

Background

Under normal conditions, the synthesis and degradation of extracellular matrix ("ECM") is tightly regulated. While planned degradation of ECM is an important feature of tissue repair and remodeling, uncontrolled changes in ECM are associated with many diseases such as inflammation, cancer, and cardiovascular dysfunction. Of the cardiovascular diseases, myocardial infarction ("MI") is one of the most common cardiac disorders in the united states. It is associated with long-term complications and high mortality as a result of the development of post-myocardial infarction remodeling into congestive heart failure.

Matrix metalloproteinases ("MMPs") are key enzymes that play a critical role in cardiac ECM remodeling. MMPs are a class of structurally related zinc-dependent endopeptidases that degrade several components of the ECM, and their increased expression and/or activity is associated with a variety of different pathophysiological processes. In particular, MMP-9 (also known as gelatinase B) plays a major role in myocardial ECM remodeling. MMP-9 has been found to increase early after MI, and its levels positively correlate with the severity of heart failure. Thus, reducing the expression level and/or activity of MMP-9 may have a beneficial effect in cardiovascular health.

MMP-9 is also one of the enzymes involved in the degradation of the articular cartilage matrix. Cartilage is the major component of joint structure and is composed of chondrocytes embedded in a dense and highly organized ECM. The ECM is synthesized by chondrocytes and consists of a collagen network comprising mainly type II collagen, as well as glycosaminoglycans ("GAGs") and related proteoglycans. Collagen forms a fibrous network and provides tensile strength to the cartilage matrix, while aggrecan is the predominant cartilage proteoglycan that draws water into the matrix and makes it resistant to compression. As aggrecan breaks down, collagen degradation is a central feature of arthritis. Proinflammatory cytokines such as tumor necrosis factor alpha ("TNF-alpha"), interleukin 1 ("IL-1"), and IL-6 are known to play important roles in the degradation of cartilage matrix in articular cartilage by causing a cascade of events that stimulate the production of aggrecanase and matrix metalloproteases (e.g., MMP-9). The reduction of MMP-9 by the plant extract indicates the ability of the extract to contribute to a healthier joint structure by maintaining intact cartilage.

MMP-9 appears to be involved in the enzymatic processes of many pathological conditions. Cancer (breast, pancreatic, lung, bladder, colorectal, ovarian, prostate and brain cancer), periodontal disease (periodontitis and gingivitis), secondary complications of diabetes (plaque formation in atherosclerosis), delayed wound healing (venous leg ulcers), inflammatory bowel disease complications (crohn's disease), neuroinflammation (multiple sclerosis) and gastric ulcers are several of the numerous human diseases affected by the presence of this enzyme. Therefore, modulation of MMP-9 expression and/or activity is crucial for correcting many chronic and acute diseases.

Impaired insulin resistance and glucose tolerance are two key imbalances in the metabolic syndrome, closely related to abdominal obesity, hypertension and dyslipidemia. People affected by these disorders are at greater risk of cardiovascular disease, type II diabetes, chronic low-grade local tissue inflammation, and have increased susceptibility to other disease conditions such as fatty liver, sleep disorders, and cancer. Over the years, several anti-hyperglycemic products have been developed that address these challenges by increasing insulin secretion in a targeted manner, sensitizing tissues and organs to insulin, increasing glucose uptake and transport, and reducing carbohydrate absorption in the gut. Among these targets, for example, peroxisome proliferator-activated receptor gamma ("PPAR- γ") affects insulin sensitivity of peripheral tissues by controlling the expression of many factors secreted by adipose tissues, such as adiponectin, leptin, resistin, and tumor necrosis factor- α (TNF- α). PPAR- γ also directly upregulates the type 4 glucose transporter (Glut4) and thus regulates glucose homeostasis.

The PPARs are ligand-activated transcription factors that regulate the expression of target genes. Upon endogenous or exogenous agonist binding, PPAR receptors heterodimerize with Retinoid X Receptors (RXRs) and bind to PPAR response elements (PPREs) located in the promoter region of target genes, causing regulation of gene expression. In addition to its role in maintaining metabolic homeostasis, PPARs also regulate the expression of genes involved in lipid metabolism, adipogenesis and inflammation.

There are at least 3 PPAR subtypes (α, β and γ) with diverse tissue expression, suggesting that each of these subtypes may have specific functions. Among them, PPAR- γ is known to have two isoforms, PPAR- γ 1 and PPAR- γ 2. PPAR- γ 1 is abundantly expressed in adipose tissue, large intestine and hematopoietic cells, and to a lesser extent in kidney, liver, muscle, pancreas and small intestine. In contrast, PPAR- γ 2 is restricted to white and brown adipose tissue.

Activation of PPAR- γ is one of the key steps in the differentiation of preadipocyte precursor cells into adipocytes with a final role in the regulation of glucose metabolism. For example, thiazolidinediones (also known as "TZDs" or glitazones, such as troglitazone, rosiglitazone and pioglitazone), which are potent exogenous agonists of PPAR- γ, are known to improve insulin responsiveness by this route, increasing glucose uptake and lipid storage by adipocytes, making them a good intervention option for diabetes.

Botanicals play an important role in the management of most of these diseases, and plants are a potential source of natural regulators of metabolic disorders. Thus, there is increasing interest in the study of plants containing regulators and plant components that promote health as potential therapeutic agents. The medicinal plants provide a safe, cost-effective, ecological alternative to chemical modifiers which may be toxic after prolonged exposure.

Cranberries (Vaccinium macrocarpon) are introduced by native Americans to European residents who use the berries to treat kidney stones and urinary tract health problems. Since then, cranberries have been used by a large portion of the north american population to treat a variety of different diseases, including urinary tract infections, gastric disease, scurvy, vomiting, and weight loss. Many cranberry fruit extracts exist on the market, and cranberry juice alone or in combination with other juices is a common and popular beverage. Furthermore, the health benefits of cranberry fruit-based products are highly appreciated by the public.

Numerous scientific studies have demonstrated the contribution of edible berries to three goals of functional foods: (a) maintaining health; (b) reducing the risk of obesity; (c) reducing the risk of diet-related chronic diseases such as cardiovascular disease, type 2 diabetes and metabolic syndrome. In addition to the fruit, the leaves of the berry plant are also used for traditional therapies. The american citizen and others often use leaf extracts against several diseases such as cold, urinary tract inflammation, diabetes and eye dysfunction.

However, the composition of berry plant leaves and their beneficial properties are poorly understood. It is known that the major bioactive compounds in berry leaves are close to those found in their fruits (i.e. phenolic acids and esters, flavonols, anthocyanins and procyanidins). It is also known that the concentration of these compounds varies between different families in the genus Vaccinium (Vaccinium).

As part of a healthy lifestyle and a balanced healthy diet, supplementation is considered an important means of regulating a variety of different metabolic disorders. As mentioned above, there is a need for effective, non-toxic natural compounds with such modulating activity. The present invention provides one such solution.

Disclosure of Invention

Disclosed herein is a composition comprising a plant extract of cranberry (Vaccinium macrocarpon) leaves, wherein the plant extract exhibits modulation of one or more metabolic disorders. The plant extract may be present in an amount of about 1.0 μ g/mL or more. Preferably, the plant extract is present in an amount of about 1.0 μ g/mL to about 2000.0 μ g/mL.

In one aspect, the compositions exhibit MMP-9 inhibition. In such cases, the plant extract is present in the composition in an amount from about 1.0 μ g/mL to about 2000.0 μ g/mL.

In another aspect, the composition exhibits PPAR-gamma agonist activity. In such cases, the plant extract is present in the composition in an amount from about 50.0 μ g/mL to about 2000.0 μ g/mL.

Also disclosed herein is a dietary supplement having modulating properties for one or more metabolic disorders. The supplement comprises a therapeutically effective amount of a plant extract of cranberry (Vaccinium macrocarpon) leaves. The plant extract of said leaves exhibits MMP-9 inhibitory and/or PPAR-gamma agonist activity. The plant extract of cranberry (Vaccinium macrocarpon) leaves is present in the supplement in an amount of about 1.0 μ g/mL or more.

The present invention also provides a method of modulating one or more metabolic disorders in a subject by administering a composition comprising a plant extract of cranberry (Vaccinium macrocarpon) leaves at a concentration of about 1.0 μ g/mL to about 2000.0 μ g/mL.

Drawings

Figure 1 provides the chemical structures (non exhaustive) of the various procyanidins and flavonoids identified in cranberry fruit extract (E1).

Figure 2 provides the chemical structures (non exhaustive) of the various procyanidins and flavonoids identified in cranberry leaf extract (E2).

FIG. 3 is an LC/MS TIC chromatogram of cranberry fruit extract (E1).

FIG. 4 is an LC/MS TIC chromatogram of cranberry leaf extract (E2).

FIG. 5 is an LC/PDA (wavelength of 280 and 350nm) chromatogram of cranberry fruit extract (E1).

FIG. 6 is an LC/PDA (wavelength of 280 and 350nm) chromatogram of cranberry leaf extract (E2).

FIG. 7 is a LC/MS TIC chromatogram comparison between cranberry fruit extract (E1) and cranberry leaf extract (E2).

Fig. 8 provides the chemical structures of the 5 anthocyanins identified in cranberry fruit extract (E1), which were present in the extract at 1.90mg/g total anthocyanins.

Fig. 9 is a graphical representation of a calibration curve for anthocyanins in cranberry fruit extract (E1).

Fig. 10 is a graph showing MMP-9 inhibition of cranberry leaf extract at 10 different concentrations.

Figure 11 is a graph showing PPAR-gamma ligand binding of cranberry leaf extract at 10 different concentrations.

Detailed Description

Disclosed herein is a plant extract of a fruit and/or leaf of a plant comprising a plurality of procyanidins and bioflavonoids, wherein the fruit extract has been standardized to an anthocyanin content of about 1.90mg/g, based on the total weight of anthocyanin-3-galactoside, anthocyanin-3-arabinoside, methyl anthocyanin-3-galactoside, methyl anthocyanin-3-arabinoside and malvidin-3-galactoside in the fruit extract, and wherein the plant extract comprises at least an extract from the genus Vaccinium.

The present invention is also based on the surprising discovery that the leaves of the cranberry plant (cranberry) are significantly higher in certain flavonoids than the cranberry fruit. In particular, the extract from the leaves has a flavonoid content that is at least 20 times higher than the flavonoid content of the fruit of the cranberry plant. In another embodiment, the extract from the leaves comprises at least 23 times and 700 times higher amounts of procyanidin trimers and procyanidin tetramers than the amounts of procyanidin trimers and procyanidin tetramers, respectively, of the fruits of the cranberry plant. Thus, in one embodiment, the plant extract is derived from at least the leaves of cranberry (Vaccinium macrocarpon). Furthermore, the plant extract at least from the leaves of cranberry (Vaccinium macrocarpon) may be useful in modulating one or more metabolic disorders.

When the plant extract is at least a leaf of the plant, the plant extract may be present in the composition in an amount of about 1.0 μ g/mL or more. For example, the leaf extract can be present in the composition in an amount of about 1.0 μ g/mL to about 1000.0 μ g/mL.

For purposes of this application, the term "composition" refers to a product that treats, ameliorates, promotes, enhances, manages, controls, maintains, optimizes, modifies, alleviates, inhibits or prevents a particular condition associated with a natural state, biological process or disease or disorder. For example, the composition improves inhibition of tumor metastasis and/or reduces inflammation and the like in a subject. The term composition includes, but is not limited to, a pharmaceutical (i.e., drug), Over The Counter (OTC), cosmetic, food ingredient or dietary supplement composition comprising an effective amount of the extract, at least one component thereof, or a mixture thereof. Exemplary compositions include creams, cosmetic lotions, masks or powders, or as emulsions, lotions, liniment foams, tablets, plasters, granules or ointments. The composition may also include a beverage, such as a beverage to which an effective amount of the extract is added or a tea bag containing an effective amount of the extract. Non-limiting examples of food compositions containing an effective amount of the extract include baked goods, protein powders, meat products, dairy products, and confectioneries.

As used herein, the term "extract" or "plant extract" refers to a solid, semi-fluid or liquid substance or formulation of one or more active ingredients comprising at least material of the plant genus Vaccinium, such as cranberry (Vaccinium macrocarpon) and/or cranberry (Vaccinium oxycoccus). Preferably, the active ingredient is derived from an extract of the leaves of the plant. The extract can be prepared using solvents such as water, short chain alcohols of 1 to 4 carbon atoms (e.g., methanol, ethanol, butanol, etc.), ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, methylene chloride, N-dimethylformamide ("DMF"), dimethyl sulfoxide ("DMSO"), 1, 3-butanediol, propylene glycol, and combinations thereof, but can also be prepared using fractions of the crude extract in such solvents. Any extraction method may be used as long as it ensures extraction and protection of the active ingredient.

As used herein, the term "effective amount" or "therapeutically effective amount" of a pure compound, composition, extract, mixture of extracts, extract components, and/or active agent or ingredient, or combination thereof, refers to an amount sufficient to achieve a desired result, in doses and for a length of time. For example, the "effective amount" or "therapeutically effective amount" refers to an amount of a pure compound, composition, extract, plant extract, extract mixture, plant extract mixture, extract component, and/or active agent or ingredient, or combination thereof, of the present invention that, when administered to a subject (e.g., a mammal such as a human), is sufficient to effect a treatment, e.g., improve inhibition of oxidation and/or reduce inflammation, etc., in the subject. The amount of a composition, extract, plant extract, mixture of extracts, mixture of plant extracts, extract components, and/or active agent or ingredient of the present disclosure that constitutes an "effective amount" or a "therapeutically effective amount" will vary with the active agent or compound, the condition to be treated and its severity, the mode of administration, the duration of administration, or the age of the subject to be treated, but can be routinely determined by one of ordinary skill in the art based on his or her own knowledge and the present disclosure.

The term "pharmaceutically acceptable" means those drugs, medicaments, extracts or inert ingredients which are suitable for use in contact with humans and lower animals without undue toxicity, incompatibility, instability, irritation, and the like, commensurate with a reasonable benefit/risk ratio.

The term "administering" is defined as providing the composition to the subject by a route known in the art, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal administration. In a preferred embodiment, the oral route of administration of the composition is suitable.

As used herein, the term "subject" or "individual" includes a mammal to which a composition may be administered. Non-limiting examples of mammals include humans, non-human primates, canines, felines, equines, bovines, rodents (including transgenic and non-transgenic mice), and the like. In certain embodiments, the subject is a non-human mammal, and in certain embodiments, the subject is a human.

As used herein, the term "carrier" refers to a composition that helps maintain one or more plant extracts in a soluble and homogeneous state in a form suitable for administration, that is non-toxic and does not interact with other components in a deleterious manner.

As used herein, the term "modulate" or "modulator" generally refers to a substance that indirectly affects (or modulates) one or more metabolic disorders.

As used herein, the term "metabolic disorder" refers to an abnormal chemical reaction that alters a normal metabolic process. Non-limiting examples of metabolic disorders include glucose metabolism disorders, DNA repair deficiency disorders, lipid metabolism disorders, malabsorption disorders, and calcium metabolism disorders. Symptoms of such disorders are often present in a range of conditions known as metabolic syndrome, including hypertension (elevated blood pressure), abdominal obesity (excess body fat in the waist), and dyslipidemia (abnormal cholesterol or triglyceride levels), which occur together, increasing the risk of a person suffering from heart disease, stroke, and diabetes.

All ratios and percentages recited throughout this disclosure are by weight unless otherwise indicated.

The present invention provides a plant-based extract capable of modulating one or more metabolic disorders. More specifically, the present invention is directed to plant extracts from the leaves of cranberry plants of the genus Vaccinium. Such plant extracts have been found to be capable of inhibiting MMP-9 and act as agonists of PPAR-gamma, thereby limiting undesirable enzymatic activity in the event of MMP-9 inhibition and/or promoting ligand binding when acting as agonists of PPAR-gamma. PPAR-gamma affects the insulin sensitivity of peripheral tissues by controlling the expression of many factors secreted by adipose tissues, such as adiponectin, leptin, resistin, and tumor necrosis factor-alpha (TNF-alpha). PPAR- γ also directly upregulates the type 4 glucose transporter (Glut4) and thus regulates glucose homeostasis. By limiting MMP-9 and/or promoting PPAR-gamma activity, one or more metabolic disorders, such as inflammation, tumor metastasis, and/or insulin sensitivity, may be reduced. In addition, one or more symptoms of metabolic syndrome, including hypertension, obesity, and/or dyslipidemia, may be reduced by limiting MMP-9 and/or promoting PPAR- γ activity.

Useful plant extracts according to the invention that are capable of inhibiting MMP-9 and/or act as agonists of PPAR-gamma include plant extracts from the genus Vaccinium. More specifically, the plant extract may be obtained from a plant selected from the group consisting of Vaccinium archamphylos, Vaccinium macrocarpon, Vaccinium bracteatum (Vaccinium macrocarpon), Vaccinium bracteatum (Vaccinium oxycodon), Vaccinium macrocarpon (Vaccinium microcarpum), Vaccinium macrocarpon, Vaccinium myrtilon (Vaccinium macrocarpon), Vaccinium arborescens (Vaccinium arborescens), Vaccinium viticola, Vaccinium coenarium, Vaccinium caesium, Vaccinium caespitosum, Vaccinium macrocarpon, Vaccinium myrtillus, Vaccinium vitium purpureum, Vaccinium vitium myrtillus, Vaccidium dichroium, Vaccinium vitium myrtillus, Vaccinium myrtillus, Vaccidium purpureum, Vaccidium perecium cornium, Vaccinium vitium, Vaccidium purpureum, Vaccidium grandis, Vaccidium vitium, Vaccidum cornium, Vaccidum film or Vaccidum cornium, Vaccidum. Preferably, the plant extract is derived from at least cranberry (Vaccinium macrocarpon), red cranberry (Vaccinium oxycoccus), small red cranberry (Vaccinium microcarpum) and/or small red cranberry (Vaccinium microcarpum). More preferably, the plant extract is derived from at least cranberries (Vaccinium macrocarpon), even more preferably, the plant extract is derived from leaves of cranberries (Vaccinium macrocarpon).

The composition capable of inhibiting MMP-9 and/or acting as an agonist of PPAR-gamma according to the present invention may comprise one or more compounds capable of acting as active ingredients and being a component of said plant extract. For example, the compound may be a phytochemical present in the plant from which the plant extract was obtained. The compounds may be at least partially responsible for inhibiting MMP-9 and/or acting as agonists of PPAR-gamma. The compound may be any compound capable of inhibiting MMP-9 and/or acting as an agonist of PPAR-gamma. In one embodiment, the compound is selected from the phytochemicals isoquercetin, quercetin-3-glycoside, kaempferol glycoside and/or procyanidins (e.g., A, B, trimers, tetramers).

Generally, one or more parts of a plant can be used to produce a plant extract, including but not limited to roots, stems, leaves, flowers, fruits, seeds, and seed coats of seeds. In the present invention, at least the leaves of the plant are used, alone or together with other plant parts, in particular fruits, to produce the plant extract. Fruits and leaves from plants of the genus Vaccinium are commercially available from a variety of different sources. The extracts of the fruits and leaves may be obtained using any suitable extraction technique.

In this regard, one or more parts of the plant, particularly the leaves of the Vaccinium plant, may be collected and comminuted. The comminuted material can then be extracted using a suitable solvent. The solvent may be removed in a concentration step. For example, the extracted material can be screened or filtered to produce a supernatant and a cake. The filter cake can be pressed to remove a significant portion of the liquid, which can be added to the supernatant. The filter cake can then be dewatered and used as a fiber source. The supernatant may be distilled to remove the solvent or a portion thereof to form a plant extract liquid concentrate. The removed solvent can be recycled. The concentrate may be dried (e.g., by spray drying) to provide a dried plant extract. Such dried plant extracts may be assayed and/or standardized as described herein. Preferably, the dried plant extract is derived from the leaves of a cranberry (Vaccinium macrocarpon) plant, in particular a cranberry (Vaccinium macrocarpon) plant.

Suitable solvents for the extraction process include water, alcohols or mixtures thereof. Exemplary alcoholic solvents include, but are not limited to, C₁-C₇Alcohols (e.g., methanol, ethanol, propanol, isopropanol, and butanol), water-alcohols or mixtures of alcohols and water (e.g., aqueous ethanol), polyols (e.g., propylene glycol and butylene glycol), and fatty alcohols. Any of these alcoholic solvents may be in the form of a mixtureThe preparation is used. In one embodiment, the plant extract is extracted using ethanol, water, or a combination thereof (e.g., a mixture of about 70% ethanol and about 30% water). In another embodiment, the plant extract is extracted using water only.

In one embodiment, the plant extract may be obtained using organic solvent extraction techniques. In another embodiment, the plant extract may be obtained using a solvent sequential separation technique. Whole water-ethanol extraction techniques can also be used to obtain the plant extract. Typically, this is referred to as a one-time extraction.

Whole ethanol extraction may also be used. This technique uses ethanol as a solvent. This extraction technique can produce plant extracts that have fat-soluble and/or lipophilic compounds in addition to water-soluble compounds.

Another example of an extraction technique that can be used to obtain the plant extract is supercritical fluid extraction ("SFE"). During such extraction, the material to be extracted may not be exposed to any organic solvent. Instead, carbon dioxide under supercritical conditions (>31.3 ℃ and >73.8 bar) with or without a modifier can be used as the extraction solvent. One skilled in the art will recognize that temperature and pressure conditions may be varied to obtain optimal yields of extract. This technique, similar to the all-hexane and ethyl acetate extraction techniques, can produce extracts of fat-soluble and/or lipophilic compounds.

The plant extract produced in the process may include a wide variety of phytochemicals present in the extracted material. The phytochemical ingredient may be fat-soluble or water-soluble. After collecting the extract solution, the solvent may be evaporated to obtain the extract.

The plant extract may be standardized to a specified amount of a particular compound. For example, the plant extract may be standardized to the active or phytochemical components present in a defined amount of the extract.

The amount of plant extract present in the MMP-9 inhibitor and/or PPAR-gamma agonist composition may depend on several factors, including the desired level of MMP-9 inhibition and/or PPAR-gamma activity increase, the level of MMP-9 inhibition and/or PPAR-gamma activity increase for a particular plant extract or component thereof, and other factors. Preferably, the plant extract is present in an amount of about 0.005 wt% or more, for example about 0.005 wt% to about 99.00 wt%, based on the total weight of the composition.

The MMP-9 inhibitor and/or PPAR-gamma agonist compositions may include one or more acceptable carriers. The carrier may assist in incorporating the plant extract into a MMP-9 inhibitor and/or PPAR-gamma agonist composition having a form suitable for administration to a subject. A wide variety of acceptable carriers are known in the art, and the carrier can be any suitable carrier. The carrier is preferably suitable for administration to animals, including humans, and is capable of acting as a carrier without significantly affecting the desired activity of the plant extract and/or any active ingredient. The carrier may be selected on the basis of the desired route of administration and dosage form of the composition.

Suitable dosage forms include liquid and solid forms. In one embodiment, the composition is in the form of a gel, syrup, slurry or suspension. In another embodiment, the composition is in a liquid dosage form, such as an oral liquid (drink shot) or a liquid concentrate. In another embodiment, the composition is in a solid dosage form, such as a tablet, pill, capsule, dragee, or powder. When in liquid or solid dosage form, the composition may take the form of a food delivery form suitable for incorporation into a food for delivery. Examples of carriers suitable for use in solid forms (particularly tablet and capsule forms) include, but are not limited to, organic and inorganic inert carrier materials such as gelatin, starch, magnesium stearate, talc, gums, silicon dioxide, stearic acid, cellulose and the like. The carrier may be substantially inert.

As an example, silicified microcrystalline cellulose may be used as a carrier or binder. Silicified microcrystalline cellulose is a physical mixture of microcrystalline cellulose and colloidal silicon dioxide. One such suitable form of silicified microcrystalline cellulose isProSolv available from Penwest Pharmaceutical Co., Patterson, N.J90. Silicon dioxide may also be added to the composition as a processing aid in addition to that provided by the silicified microcrystalline cellulose. For example, silicon dioxide may be included as a glidant to improve powder flow during tableting in the manufacture of solid dosage units such as tablets.

In another embodiment, the carrier is at least a functional carrier such as buckwheat or spelt. By adding a functional carrier to the composition, additional benefits may be provided, such as a lower glycemic index compared to, for example, the standard carriers mentioned above. Furthermore, functional carriers may be non-allergenic (e.g. buckwheat), and by adding them to the production process, the plant extracts of the invention may benefit from the flavonoids of these functional carriers, such as rutin and quercetin. The high fiber content of these functional carriers can also facilitate and regulate intestinal passage. Finally, the additional mineral benefit of selenium present in spelt wheat may contribute to metabolism.

The MMP-9 inhibitor and/or PPAR-gamma agonist compositions may contain other inert ingredients such as lubricants and/or glidants. The lubricant aids in handling the tablet during manufacturing, such as during ejection from a die. Glidants improve powder flow during tableting. Stearic acid is an example of an acceptable lubricant/glidant.

The MMP-9 inhibitor and/or PPAR-gamma agonist compositions can be formulated into solid dosage forms such as tablets and capsules. This form provides a product that can be easily transported by an individual to a dining location, such as a restaurant, and taken before, during, or after eating food. The composition may be formulated as dosage units containing appropriate amounts of the plant extract and/or active ingredient, allowing the individual to determine the appropriate number of units to take according to appropriate parameters such as body weight, food amount or carbohydrate (e.g. sugar) content.

In one embodiment, the plant extract is present in the composition in a therapeutically effective amount, for example, an amount of about 1.0 μ g/mL or more, preferably from about 1.0 μ g/mL to about 1000.0 μ g/mL, more preferably from about 15.0 μ g/mL to about 750.0 μ g/mL. The compositions may be administered as a single dose or in multiple doses. In one example, the compound is administered up to three doses per day. For example, the compound may be administered before, during or after a meal. In one embodiment, the composition is a dietary supplement having MMP-9 inhibitor and/or PPAR-gamma agonist properties comprising a therapeutically effective amount of cranberry leaf extract.

The dosage may be selected to provide a level of inhibition that may be effective in certain individuals and/or certain food products in a single unit, while also allowing for relatively simple dose escalation to provide other levels of inhibition that may be effective in other individuals and/or other food products.

The inhibitory composition may take a form suitable for oral ingestion. This form may be configured as a single dosage form intended to provide a prescribed dose of the plant extract. For example, the single dosage form may be a powder, a pill, a tablet, a capsule, or an oral liquid. The single dosage form may comprise, for example, from about 1.0 μ g/mL to about 2000.0 μ g/mL of the plant extract.

Examples

Example-materials and chemistry Profile

Example 1 preparation of 70% ethanol extracts from cranberry fruits and cranberry leaves

Dried cranberry fruit powder (Vaccinium macrocarpon) (60g) was loaded into 3 100ml stainless steel tubes and used with Thermo Scientific^TM Dionex^TMASE 350 accelerated solvent extractor extraction twice with 70% ethanol solvent in DI water at a temperature of 80 ℃ and a pressure of 1500 psi. The extract solution was automatically filtered and collected. The combined ethanol extract solutions were evaporated under vacuum using a rotary evaporator to give crude 70% ethanol fruit extract ("E1").

Mixing dried and ground cranberry leaf powder (cranberry: (a))Vaccinium macrocarpon) (140g) were loaded into 7 100ml stainless steel tubes and Thermo Scientific was used^TM Dionex^TMASE 350 accelerated solvent extractor extraction twice with 70% ethanol solvent in DI water at a temperature of 80 ℃ and a pressure of 1500 psi. The extract solution was automatically filtered and collected. The combined ethanol extract solutions were evaporated under vacuum using a rotary evaporator to give crude 70% ethanol leaf extract ("E2").

The extraction results are provided in table 1 below.

TABLE 1 cranberry fruit and cranberry leaf extraction

Example 2 chemical profiling of cranberry fruit and cranberry leaf extracts

Using ultra-high pressure liquid chromatography ("HPLC") and Mass Spectrometry (MS) ((M))UPLC class I andGS-XT-QTof system, both available from Water Corporation, Milford, Massachusetts USA), identified the flavonoid compounds present in cranberry fruit extract E1 and cranberry leaf extract E2. The column used isUPLC HSS T32.1x100 mm, 1.8 μm, column temperature 40 ℃ and sample temperature 15 ℃. For the mobile phase, solvent a was water (containing 0.1% formic acid) containing 10% acetonitrile ("ACN") and solvent B was ACN. The collection range is 100-1500 daltons ("Da") and the collection mode is electrospray ionization ("ESI") (-). HPLC conditions are provided in table 2 below.

TABLE 2 HPLC conditions for analysis of E1 and E2 extracts

Extract of plant	Run time (min)	Sample volume (μ L)	Concentration of
				E1	20.00	1.00	5mg/mL
E2	20.00	2.00	1mg/mL

Identification of peaks is based solely on accurate mass. Due to database limitations, multiple isomers may have been identified as the same compound. For example, eight (8) procyanidin B1-B8 compounds with the same molecular weight of 578.528 were not distinguished in this analysis.

On an accurate mass basis, relatively low levels of procyanidins and flavonoid glycosides such as quercetin, isoquercetin and myricetin 3-arabinofuranoside are detected and identified in E1. The chemical structure of the compound detected in E1 (non exhaustive) is shown in figure 1. The following table lists compounds identified in E1 on the basis of accurate mass.

TABLE 3 Compounds identified in E1

Abundant bioflavonoids were identified in E2, including astragaloside, isoquercitrin, kaempferol, glycosides, etc. The chemical structure of the compound detected in E2 (non exhaustive) is shown in figure 2. The following table lists compounds identified in E2 on the basis of accurate mass.

TABLE 4 Compounds identified in E2

A number of procyanidins were found to be significantly higher in E2 than in E1. Based on the detector counts at 575.11 and 577.13 for the mass to charge ratio ("m/z"), it was found that including both a and B types of procyanidin dimers in E2 was approximately fifty (50) times higher than in E1. The observed presence of procyanidin trimers at m/z at 863.18 was approximately twenty-three (23) times higher in E2 than E1, while procyanidin tetramers at m/z at 1152.24 were more than seven hundred (700) times higher in E2 than E1,

based on LCMS analysis, comprises isoquercetin, quercetin-3-arabinofuranoside, kaempferol glycoside, etcSimilar bioflavonoids were also identified in E2 in much higher abundance. Observed m/z at 463.093, identified molecular formula C₂₁H₂₂O₁₀The peak in E2 was twenty (20) times higher for the retention time ("RT") of 6.38min and thirty-six (36) times higher for the RT of 6.78min, compared to the corresponding peak detected in E1. Based on LCMS analysis, the total detector count of flavonoids in E2 was more than twenty (20) times higher than the flavonoids in E1.

LCMS TIC, PDA 280nm and PDA 350nm chromatograms for E1 and E2 are provided in FIGS. 3 and 4, respectively. Comparison of the LCMS TIC chromatograms between E2 and E1 shown in fig. 5 clearly shows higher levels of procyanidins and bioflavonoids in E2, while higher organic acid levels are seen in E1 (fig. 3).

Example 3 anthocyanin quantification

Methods for the quantification of anthocyanins are adapted from the published methods for HPLC analysis (J.AGRIC.FOOD CHEM., "isolation, identification, quantification and method validation of anthocyanins in plant supplement raw materials by HPLC and HPLC-MS" ("Separation, identification, quantification, and method validation of anti-fungal in cosmetic raw materials by HPLC and HPLC-MS)", Vol.49(8), pp.3515-3521 (2001)). The HPLC system used was a Hitachi D7000 HPLC system with a Phenomenex Luna 10. mu. m C18 column with column size 4.6X250 mm. The solvent used in the mobile phase is H₂0.5% phosphoric acid in O (solvent A) and H₂O/ACN/acetic acid/H₃PO₄(50%: 48.5%: 1.0%: 0.5%) (solvent B). The UV wavelength was 480 nm.

Reference standard anthocyanin-3-glucoside was purchased from ChromaDex (Chicago, Illinois US). Anthocyanin-3 glucoside was prepared in a 5mL volumetric flask at a concentration of 1mg/mL in a methanol solution containing 2% (v/v) HCl. The stock solutions were further diluted 1/5, 1/10, 1/20 and 1/100 fold with methanol containing 2% (v/v) HCl to provide five concentrations of anthocyanin-3-glucoside solutions of 1.00, 0.20, 0.10, 0.05 and 0.01mg/mL, respectively. Calibration curves were generated using the five solutions. Triplicate samples were injected at 10. mu.L each. The calibration curve is determined on the basis of the integrated peak areas. The value of the correlation coefficient (R2) for anthocyanin-3-glucoside was determined to be 0.9985.

Samples for analysis were prepared as follows. 12.5, 25.0, 50.0 and 100.0mg of E1 were weighed out. To each sample was added 1mL of methanol containing 2% (v/v) HCl, then each sample was mixed by sonication for fifteen (15) minutes and vortexed at 10,000rpm for five (5) minutes. 20 μ L of supernatant from each solution was injected to the HPLC in triplicate. The quantitative analysis of five (5) anthocyanin compounds at different concentrations shows linearity, and the correlation coefficient R²0.9953 to 0.9982 (FIG. 7). For samples at concentrations of 25mg/mL and 50mg/mL, the amount of each anthocyanin was calculated separately for 0.05mg/mL of anthocyanin-3-glucoside on the basis of the integrated peak areas.

The five anthocyanosides in E1 were quantified at a total content of 1.903mg/g E1 dry weight. Aoac int., "Determination of anthocyanins in Cranberry fruits and Cranberry fruit products by High Performance Liquid Chromatography with uv Detection, Single Laboratory Validation" (Determination of anthocyanins in Cranberry fruits and Cranberry fruit products by High-Performance Liquid Chromatography with ultra Detection), vol.94 (2); comparison of the data disclosed in pp.459-466(2011), which include anthocyanin-3-galactoside ("C3 Gla"), anthocyanin-3-arabinoside ("C3-Ara"), methylcyanin-3-galactoside ("P3-Gla"), methylcyanin-3-arabinoside ("P3-Ara"), and malvidin-3-galactoside ("Mal 3-Gla"). These compounds are shown in figure 6. No anthocyanins were detected in E2.

Amounts of five anthocyanins calculated in tables 5-E1

mg/g	R1–25mg/mL	R1–50mg/mL
			C3-Gla	0.506	0.503
C3-Ara	0.276	0.275
			P3-Gla	0.591	0.587
P3-Ara	0.200	0.195
			Mal-3-Gla	0.330	0.331

Example-bioassay methods

Extracts of cranberry fruits (E1) and cranberry leaves (E2) were prepared using food grade ethanol, then filtered and dried as described above. For the remainder of the assay preparation, research grade reagents were used. The extract was dissolved in dimethyl sulfoxide ("DMSO") to a final concentration of 50mg/mL, and then diluted to the working concentration in a buffer suitable for each bioassay.

Example 4-MMP-9 inhibition

The assay uses an MMP-9 inhibitor screening assay kit (colorimetric) from abcam (Cambridge, United Kingdom; product number ab 139448). E1 and E2 were diluted in assay buffer for testing MMP-9 inhibition in a dose curve and added to the wells of a 96-well half-volume microplate. As a positive control, 1.3 μ M of the broad-spectrum MMP inhibitor NNGH was used. The MMP-9 enzyme was diluted 1:60 in assay buffer and added to the test wells and positive and negative controls to a final concentration of 0.9 units per well. The plate was incubated at 37 ℃ for 30 minutes to allow the inhibitor to bind to the enzyme. The MMP-9 substrate was diluted 1:25 in assay buffer and added to the wells at a final concentration of 100 μ M. The plates were then read continuously for absorbance at 405nm, taking readings every minute for a total of 20 minutes. The slope over the linear range (first 10 minutes) was calculated for each well and the percent inhibition of the test compound and positive control was determined using the negative (untreated) control wells as a 100% score.

Referring to fig. 10, various degrees of MMP-9 inhibition were observed depending on the concentration of cranberry leaf extract. No inhibition of cranberry fruit extract was observed. MMP-9 inhibition, and IC of cranberry leaf extract, is observed at about 1 μ g/mL or more, more specifically about 1 μ g/mL to at least about 1000 μ g/mL, even more specifically about 15 μ g/mL to about 750 μ g/mL₅₀579. mu.g/mL.

Example 5 PPAR-gamma activation

PPAR-gamma ligand screening/characterization assay kit (product number: K437-100) from BioVision was used to test the ability of cranberry fruit (E1) and cranberry leaf (E2) extracts to bind to and activate PPAR-gamma. This assay kit relies on the test sample to displace the fluorescent probe bound to the PPAR-gamma protein. When the test sample displaced the fluorescent probe and bound to PPAR-gamma, there was an observable decrease in fluorescence intensity. PPAR-gamma assay probes were diluted 1:100 in DMSO. A master mix of PPAR-gamma protein, PPAR-gamma assay probe, PPAR-gamma assay buffer and DMSO (final concentration 10%) was prepared and added to test samples in 384 well black plates for a total of 25 μ Ι _ per well. The plates were incubated for 5 minutes at room temperature and then read on a fluorescence plate reader at the following wavelengths: excitation-405 nm, emission-460 nm. Samples were also read in the absence of PPAR-gamma assay probes or PPAR-gamma protein and these blanks were subtracted from the experimental values to correct for interference. Percent inhibition was calculated as the difference in fluorescence intensity between the untreated control (which had 100% binding of the fluorescent probe to the PPAR-gamma protein) and the test sample divided by the value of the untreated control and expressed as a percentage.

Referring to FIG. 11, various degrees of intensity of PPAR- γ ligand binding activity were observed for E2. 10 different concentrations of E2(3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000, and 2000. mu.g/mL) were tested. E2 activation was observed at about 50.0 μ g/mL to at least about 2000 μ g/mL, more specifically about 100 μ g/mL to about 1000 μ g/mL, even more specifically about 125 μ g/mL to about 500 μ g/mL. IC was observed for E2₅₀At 384. mu.g/mL. No observable binding activity was noted for E1.

The above data indicate that a plant extract of cranberry (Vaccinium macrocarpon) leaves has one or more compounds that may contribute somewhat in addressing the imbalance between normal physiological conditions and uncontrolled enzyme expression/activity during tissue remodeling or repair, i.e., the extract exhibits modulation of one or more metabolic disorders.

The above description discloses several methods and materials of the present invention. The present invention is susceptible to modifications in materials and methods, and to variations in manufacturing methods and apparatus. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed herein, but that the invention will cover all modifications and alternatives falling within the true scope and spirit of the invention as embodied in the following claims.