阅读说明：本技术 植物抗氧化剂 (Plant antioxidant ) 是由 贾廷德·拉纳 凯莉·米切尔 于 2019-07-12 设计创作，主要内容包括：一种表现出抗氧化活性的植物提取物,其中所述植物提取物至少是来自于腰果属(Anacardium)的提取物。(A plant extract exhibiting antioxidant activity, wherein the plant extract is at least an extract from cashew (Anacardium).)

1. A composition comprising a plant extract of the seed coat of cashew nut (Anacardium occidentale L.), wherein said plant extract inhibits γ -H2AX activity.

2. The composition of claim 1, wherein the plant extract is present in an amount of about 4.0 μ g/mL or more.

3. The composition of claim 2, wherein the plant extract is present in an amount of about 4.0 μ g/mL to about 2000.0 μ g/mL.

4. A dietary supplement having antioxidant properties comprising a therapeutically effective amount of cashew nut shell extract, wherein said plant extract inhibits gamma-H2 AX activity.

5. The dietary supplement of claim 4, wherein the cashew nut shell extract is present in an amount of about 4.0 μ g/mL or more.

6. A plant extract comprising catechins, wherein the extract has been standardized to a catechin content of about 15.0 wt/wt% or more based on the total weight of the extract, wherein the plant extract exhibits antioxidant activity, and wherein the plant extract comprises at least an extract from the genus Anacardium (Anacardium).

7. The plant extract of claim 6, wherein the extract from cashew is at least an extract from cashew nut.

8. The plant extract of claim 7, wherein the extract from cashew nuts is derived from at least the testa of cashew nut seeds.

9. A method of inhibiting oxidation-induced DNA damage in a subject, the method comprising administering a composition comprising a plant extract of cashew nut shells in a concentration of about 4.0 μ g/mL to about 2000.0 μ g/mL.

Technical Field

The present invention relates generally to inhibitors of oxidation-induced DNA damage, and more particularly to plant inhibitors of oxidation-induced DNA damage and the use of such plant inhibitors as antioxidants.

Background

Oxygen is a highly reactive atom that can become part of a potentially damaging molecule called a "radical". Free radicals, commonly referred to as reactive oxygen species ("ROS"), contain one or more unpaired electrons in their outermost orbitals. Common examples of reactive oxygen species include hydroperoxyl Radicals (ROO), superoxide anion (O)₂Active hydroxyl (OH), and hydrogen peroxide (H)₂O₂) A free radical. These free radicals are produced spontaneously during metabolism in living organisms. Since free radicals are very unstable, they react with other molecules in their vicinity (e.g., proteins, lipids, or DNA), gaining stability by extracting electrons from those molecules, causing cell damage and initiating chain reactions for free radical generation.

An imbalance between free radical production and intracellular antioxidants can cause oxidative stress. Oxidative stress occurs when an oxygen atom splits into a monatomic with unpaired electrons, called a free radical. Since the electron preference occurs in pairs, these free radicals clean the body looking for other electrons with which they pair, causing damage to cells, proteins and DNA during this period. The term oxidative stress is used to describe the condition of oxidative damage caused when the key balance between free radical generation and antioxidant defense is unfavorable.

Oxidative stress, which occurs as a result of an imbalance between free radical production and antioxidant defense, is associated with damage to a wide range of molecular substances, including lipids, proteins and nucleic acids. Excessive oxidative stress can lead to the oxidation of lipids and proteins, which is accompanied by changes in their structure and function. Short-term oxidative stress may occur in tissues damaged by trauma, infection, thermal injury, hyperoxia, toxins and excessive movement. These damaged tissues produce increased free radical generating enzymes (e.g., xanthine oxidase, lipoxygenase, cyclooxygenase), activation of phagocytes, release of free iron, copper ions or destruction of the electron transport chains of oxidative phosphorylation, and excess ROS production. Oxidative stress is associated with the etiology of several degenerative diseases, such as stroke, parkinson's disease, alzheimer's disease, rheumatoid arthritis, diabetes, peptic ulcers, gene mutations and cancer, cardiac and blood disorders, and inflammatory diseases. Oxidative stress is now considered to contribute significantly to all inflammatory diseases (arthritis, vasculitis, glomerulonephritis, lupus erythematosus, adult respiratory disease syndromes), ischemic diseases (heart disease, stroke, intestinal ischemia), hemochromatosis, acquired immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers, hypertension and preeclampsia, neurological disorders (alzheimer's disease, parkinson's disease, muscular dystrophy), alcoholism, diseases associated with smoking and many other diseases.

Antioxidants stabilize or inactivate free radicals before they attack cells. The use of externally sourced antioxidants can help to cope with oxidative stress. These include synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole; however, recently, these synthetic antioxidants have been reported to be harmful to human health. Thus, the search for effective, non-toxic natural compounds with antioxidant activity has been intensified in recent years.

Antioxidants are reducing agents, examples of which include antioxidants of nutrient origin such as ascorbic acid (vitamin C), tocopherols and tocotrienols (vitamin E), carotenoids and polyphenols, antioxidases such as superoxide dismutase, glutathione peroxidase and glutathione reductase, metal binding proteins such as ferritin, lactoferrin, albumin and ceruloplasmin, and trace metals such as zinc and molybdenum. These antioxidants can scavenge active oxygen and inhibit chain reactions by donating electrons to free radicals. The antioxidant defense system supported by dietary antioxidants protects the body from free radicals. However, during oxidative stress, antioxidants are insufficient to maintain homeostasis. In such cases, the antioxidants may be provided as supplements, the consumption of which may significantly reduce the risk of free radical related diseases.

Botanicals play an important role in the management of most of these diseases, and plants are a potential source of natural antioxidants. Studies have shown that the consumption of polyphenols present in tea, fruits and vegetables is associated with a low risk of these diseases. Accordingly, there is growing interest in the development of plants containing antioxidants and plant components that promote health as potential therapeutic agents. The medicinal plants provide a safe, cost-effective, ecological alternative to chemical antioxidants that may be toxic upon prolonged exposure.

Cashew nut (Anacardium occidentale Linn) originally originated in the Amazon area and was later transplanted to India, east Africa and other countries for planting. Such trees produce very exotic fruits or fruits in the form of swelled fruit stalks. On the outside of the end of this stalk, cashew nuts grew into their own gray kidney-shaped crust. This shell has a soft, leathery outer skin and a thin, hard inner skin, called the shell or seed coat, surrounding the kernels. Between the two skins is a honeycomb structure containing cashew nut shell liquid. This liquid contains anacardic acid, cardanol and cardol, among other ingredients. Anacardic acid is a salicylic acid, while cardanol and cardanol are substituted phenols.

The use of various parts of the fruit has been studied. In addition to being an edible food, juice from cashew nuts is used in beverages, while fruit extracts show benefits in weight management. Cashew nutshell liquids have been extracted for a variety of different industrial and agricultural applications, including friction linings, coatings, laminating resins, rubber compounding resins, cashew cements, polyurethane-based polymers, surfactants, epoxy resins, casting chemicals, chemical intermediates, pesticides, and fungicides. Cashew nut shells have been used in tanning materials.

As part of a healthy lifestyle and a balanced healthy diet, the supplementation of antioxidants is considered to be an important means of improving free radical protection. As mentioned above, there is a need for effective, non-toxic natural compounds with antioxidant activity. The present invention provides one such solution.

Disclosure of Invention

Disclosed herein is a plant extract comprising catechins, wherein the extract has been standardized to a catechin content of about 15.0 w/w% or more based on the total weight of the extract, wherein the plant extract exhibits antioxidant activity, and wherein the plant extract comprises at least an extract from the genus Anacardium (Anacardium). In one aspect, the plant extract may be obtained from a plant selected from the group consisting of Anacardium humile, Anacardium othioninum, Anacardium giganteum, Anacardium nanum, Anacardium regrense, and/or cashew nut (Anacardium occidentale). Preferably, the plant extract is from at least cashew nut (Anacardium occidentale L). In one embodiment, the plant extract is from at least the seed coat of the fruit of cashew nut (Anacardium occidentale L.).

In another aspect, the present invention provides a composition comprising a plant extract of the seed coat of cashew nut (cashew occidentale L.), wherein the plant extract exhibits antioxidant activity. The plant extract may be present in the composition in an amount of about 4.0 μ g/mL or more. For example, the plant extract may be present in the composition in an amount of about 4.0 μ g/mL to about 2000.0 μ g/mL.

In one aspect, the composition comprising a plant extract of cashew nut shells inhibits γ -H2AX activity. In one embodiment, the plant extract is present in the composition in an amount from about 4.0 μ g/mL to about 2000.0 μ g/mL. The composition may include, for example, a food and/or beverage composition to which the extract is added.

Also provided herein are dietary supplements having antioxidant properties, wherein the supplement comprises a therapeutically effective amount of cashew nut shell extract. For example, the cashew nut shell extract may be present in the dietary supplement in an amount of about 4.0 μ g/mL to about 2000.0 μ g/mL.

The present invention also provides a method of inhibiting oxidation-induced DNA damage in a subject by administering a composition comprising a plant extract of the seed coat of cashew nuts (Anacardium occidentale L.) at a concentration of about 4.0 μ g/mL to about 2000.0 μ g/mL.

Drawings

Fig. 1 is an HPLC chromatogram of cashew nut shell extract at 275nm with retention time from 0 min (start) to 20 min.

FIG. 2 is LC/MS and LC/PDA (wavelength 280 and 350nm) chromatograms of cashew nut shell extract.

Fig. 3 is a graph showing the efficacy of cashew nut shell extract in inhibiting DNA damage compared to catechin and soliprin standards.

Fig. 4 is a graph showing cell viability of cell cultures when dosed with various doses of cashew nut shell extracts compared to catechin and soliprin standards.

Detailed Description

The present invention is based on the surprising discovery that the seed coat of cashew nuts (Anacardium) contains significantly higher levels of certain flavonoids. In particular, it has been found that the extract of cashew nut shells contains catechins and epicatechins as major components as well as proanthocyanidins. The data presented herein demonstrate that cashew nut shell extract is beneficial in protecting DNA from oxidative stress induced damage.

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 oxidation 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 material or formulation comprising one or more active ingredients of at least cashew plants (e.g., Anacardium milk, Anacardium othionium, Anacardium giganteum, Anacardium annum, Anacardium regrense, and/or cashew nuts (Anacardium occidentale)), preferably cashew nuts (Anacardium occidentale L). Preferably, the active ingredient is derived from an extract of cashew nut shells. 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.

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

The present invention provides a plant-based inhibitor capable of inhibiting DNA damage caused by oxidative stress. More specifically, the present invention is directed to a plant extract from cashew nut shells of the genus cashew. Such plant extracts have been found to be able to inhibit oxidative stress-induced DNA damage by neutralizing free radicals, thereby terminating the chain reaction produced by said free radicals. By terminating the chain reaction, damage caused by the free radicals through their reaction with important macromolecules such as DNA, proteins, lipids or cell membranes is prevented or inhibited.

Useful plant extracts capable of inhibiting DNA damage caused by oxidative stress according to the present invention include plant extracts from the genus cashew. More specifically, the plant-based inhibitor is a plant extract of one or more species selected from the group consisting of Anacardium humile, Anacardium othionium, Anacardium giganteum, Anacardium nanum, Anacardium regrense, and/or cashew nuts (Anacardium occidentale). Preferably, the plant extract is from the cashew (Anacardium occidentale) species. In one embodiment, the plant extract is from the seed coat of the cashew (Anacardium occidentale) species.

The composition capable of inhibiting DNA damage caused by oxidative stress according to the present invention may include one or more compounds capable of functioning as active ingredients. The compound may be a component of the 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 DNA damage caused by oxidative stress. The compound may be any compound capable of inhibiting DNA damage caused by oxidative stress. In one embodiment, the compound is selected from the phytochemicals catechins, epicatechins, 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 seed coat of the seed is used alone or together with other plant parts to produce the plant extract. Seed coats from cashew plants are commercially available from a variety of different sources. The extract of cashew nut shells can be obtained using any suitable extraction technique.

In this connection, one or more parts of the plant, in particular the seed coat of the plant, can 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 seed coat of the cashew nut (cashew apple), in particular the cashew nut (cashew apple).

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 used in the form of a mixture. 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 carbon dioxide 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. In one embodiment, the plant extract is standardized to a catechin content of about 15.0 wt% or more based on the total weight of the extract.

The amount of plant extract present in the composition for inhibiting oxidative stress-induced DNA damage may depend on several factors, including the desired level of inhibition of oxidative stress-induced DNA damage, the level of inhibition of oxidative stress-induced DNA damage by 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 50.00 wt%, based on the total weight of the composition.

The composition for inhibiting oxidative stress-induced DNA damage may include one or more acceptable carriers. The carrier may help incorporate the plant extract into a composition having a form suitable for administration to a subject that inhibits oxidative stress-induced DNA damage. 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 is ProSolv, available from Penwest Pharmaceutical coSilicon 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 quercitrin. In addition, 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 composition for inhibiting oxidative stress-induced DNA damage may comprise 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 composition for inhibiting oxidative stress-induced DNA damage may be manufactured 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 4 μ g/mL or more, preferably from about 4.0 μ g/mL to about 2000.0 μ g/mL, more preferably from about 15.0 μ g/mL to about 1000.0 μ g/m, even more preferably from about 30.0 μ g/mL to about 500.0 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 with antioxidant properties containing a therapeutically effective amount of cashew nut shell 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 4.0 μ g/mL to about 2000.0 μ g/mL of the plant extract.

Examples

Example-materials and chemistry Profile

Example 1 preparation of cashew nut shell extract using 70% ethanol solvent

Dried cashew nut (Anacardium occidentale) seed coat powder (60g) was loaded into 3 100ml stainless steel test tubes and using Thermo Scientific^TM Dionex^TMASE 350 accelerated solvent extraction or two extractions with 70% ethanol solvent in DI water at a temperature of 80 ℃ and a pressure of 1500 psi. Filtering and collecting the extract solution. The combined ethanol extract solution was evaporated under vacuum with a rotary evaporator to give crude cashew nut shell extract.

The extraction results are provided in table 1 below.

TABLE 1 extraction of cashew nut skins

Example 2 quantitation of catechins from cashew nut shell extract

Use of a C18 reverse phase column (HPLC/PDA) with Hitachi high performance liquid chromatography with photodiode array detector5μm C18(2)LC column 250x 4.6mm, can be selected fromTorrance, California, US) to determine the free catechins present in the cashew nut shell extract. For mobile phase A, the solvent was 0.10% phosphoric acid ("H") in water₃PO₄") and for mobile phase B, solvent B was acetonitrile (" ACN ") which was used for elution at a flow rate of 1.0ml/min and UV absorption at 275nm and a column temperature of 35 ℃. The catechin reference standard used was from Sigma-Aldrich Co. The reference standard was dissolved in methanol ("MeOH"): 0.1% H₃PO₄(1:1 ratio), wherein the catechin (C1251) concentration was 0.5mg/ml, and the epicatechin (E1753) concentration was 0.1 mg/ml. Test samples were prepared in a volumetric flask at a concentration of 2mg/ml in 0.1% H with 50% MeOH₃PO₄In (c), sonicated until dissolved (about 10 minutes), then cooled to room temperature, mixed well, and filtered through a 0.45 μm nylon syringe filter. HPLC analysis was performed by injecting 20 μ l of sample into the HPLC. Table 2 below provides a gradient table of HPLC analytical methods.

TABLE 2 gradiometer for HPLC analysis methods

Time (min)	Mobile phase A	Mobile phase B
			0.0	85.0	15.0
7.0	85.0	15.0
			12.0	10.0	90.0
16.5	10.0	90.0
			16.6	85.0	15.0
24.0	85.0	15.0

Quantitation of HPLC catechins in cashew nut shell extract provided that catechin content was 9.40% and epicatechin content was 6.12% and total catechin content was 15.52% based on total weight of the extract. Thus, the cashew nut shell extract can be standardized to a total catechin content of about 15.00 wt% or more based on the total weight of the extract. An HPLC chromatogram of cashew nut shell extract at 275nm wavelength is provided in fig. 1.

Example 3 chemical assay of cashew nut coat extract

Using ultra-high pressure liquid chromatography ("HPLC") and Mass Spectrometry (MS) ((M))UPLC class I andGS-XT-QTof system, both of which are available from Water Corporation, Milford, available from Massachusetts USA), the presence of flavonoids in the cashew nut shell extract was determined. The column used isUPLC HSS T32.1x100mm, 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 3 below.

TABLE 3 HPLC conditions for analysis of cashew nut coat extract

Run time (min)	Sample volume (μ L)	Concentration of
			20.00	2.00	1mg/mL

Identification of peaks is based solely on accurate mass. Digalloyl catechin, catechin and epicatechin were identified as the main components of the cashew nut shell extract. Proanthocyanidins, including type a and B proanthocyanidins, proanthocyanidin tetramers, and proanthocyanidin trimers, were also detected in the extract, wherein type B proanthocyanidins are the main components of the proanthocyanidins. In addition to those just mentioned, the identified compounds include digallacyl catechin, vaccihein a, 6 "-p-coumaroyl pruneside, dunalioside B, and the like. LC/MS and LC/PDA chromatograms of cashew nut shell extracts obtained from the analysis are shown in fig. 2.

Example-bioassay methods

Extract of cashew nut shell was 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 DNA Damage assay

When DNA damage, whether endogenous or exogenous, forms a double strand break ("DSB"), it is always followed by phosphorylation of histone H2 AX. H2AX is a variant of the H2A protein family that is a component of the histone octamer in the nucleosome. It is phosphorylated by kinases such as the capillary ataxia mutein of the PI3K pathway ("ATM") and the ATM-Rad3 related protein ("ATR"). Protein gamma-H2 AX is the first step in recruiting and focusing DNA repair proteins. DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents, and subsequently γ -H2AX foci are rapidly formed. These foci represent the DSB in a 1:1 manner and can be used as biomarkers of injury. Antibodies can be raised against γ -H2AX, which can thus be visualized by immunofluorescence through the second antibody. Detection and visualization of γ -H2AX by flow cytometry allows assessment of DNA damage, associated DNA damaging proteins, and DNA repair.

Human skin fibroblasts were seeded at 8000 cells/well in 96-well tissue culture plates. After 24 hours, cells were treated with test compounds for 48 hours, and then the cells were treated with 1mM hydrogen peroxide for 4 hours to induce DNA damage. Hydrogen peroxide induces DNA damage by creating breaks in DNA. The cells were then fixed and stained with an antibody against the biomarker γ -H2AX, which is a phosphorylated histone variant found at the double strand break, γ -H2 AX. At the site of the double strand break, the histone is phosphorylated, indicating that the DNA is damaged and needs repair. Antibodies used to phosphorylate histone variant γ -H2AX can be used to identify sites of DNA damage. The nuclei are stained with 4', 6-diamidino-2-phenylindole ("DAPI"), a fluorescent dye that binds to DNA. Pictures were taken using Image Xpress and analyzed using Meta Xpress to calculate the fluorescence intensity for each condition divided by the total number of cells.

DNA damage in response to hydrogen peroxide treatment was measured by the amount of γ -H2AX present in the cells. Cashew nut shell extracts of 50 and 100 μ g/mL were tested for DNA damage inhibition based on the amount of γ -H2AX detected after treatment with hydrogen peroxide to induce DNA damage. As positive controls, 25. mu.g/mL catechin and soliprin were used. As shown in fig. 3, 100 μ g/mL cashew nut shell extract showed significant protection of DNA from damage in response to hydrogen peroxide treatment. Percent inhibition was calculated relative to wells not treated with extract but exposed to hydrogen peroxide. As seen from this example, pretreatment of fibroblasts with 100mg/mL of cashew nut shell extract significantly reduced the amount of γ -H2AX detected, showing the ability of cashew nut shell extract to protect DNA from oxidative stress induced damage.

The percentage of viable cells in each treatment relative to the untreated control was determined using cell count kit-8 ("CCK-8"). CCK-8 reagent was added to the medium (final concentration 10% of the total volume) and incubated at 37 ℃ and 5% CO₂The next incubation was for one (1) hour, and then the absorbance was read at 460nm on a multimodal plate reader to determine the number of viable cells relative to the untreated control wells. The cashew nut shell extract treatment did not differ statistically significantly from the untreated control (fig. 4), and thus was not toxic to human skin fibroblasts.

The above data indicate that a plant extract of the seed coat of cashew nuts (Anacardium occidentale L.) has one or more compounds that exhibit antioxidant activity. More specifically, the cashew nut shell extract may have reasonable activity in improving γ -H2AX activity.

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.