Compositions and Methods for Joint Health
20210346447 · 2021-11-11
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
A61K36/22
HUMAN NECESSITIES
International classification
A61K36/22
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
Abstract
Compositions for and methods of maintaining bone structure, cartilage structure or both, minimizing bone reabsorption, preventing cartilage degradation, increasing bone density, promoting healthy joints by protecting cartilage integrity, diminishing the action of enzymes that affect bone health, cartilage health, or both, improving joint movement or function, alleviating joint pain, alleviating joint discomfort, alleviating joint pain and discomfort, alleviating joint stiffness, improving joint range of motion or flexibility, promote mobility, or the like, wherein the composition comprises a therapeutically effective amount of a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content.
Claims
1. A composition for reducing joint stiffness and discomfort and/or for improving cartilage rebuild or cartilage renewal function in a mammal in need thereof comprising a therapeutically effective amount of a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content.
2. The composition according to claim 1, wherein the therapeutically effective amount of the botanical extract in the composition is at least an amount of about 500.0 mg/kg or greater, based on human equivalent dosing.
3. The composition according to claim 2, wherein the therapeutically effective amount of the botanical extract in the composition is an amount of about 500.0 mg/kg to about 2000.0 mg/kg, based on human equivalent dosing.
4. The composition according to claim 3, wherein the therapeutically effective amount of the botanical extract in the composition is an amount of about 1000.0 mg/kg to about 2000.0 mg/kg, based on human equivalent dosing.
5. The composition according to claim 1, wherein the botanical extract is standardized to a total catechin content of at least about 15.00% by weight, based on total weight of the extract.
6. The composition according to claim 1, wherein the composition ameliorates cyclooxygenase and 5-lipoxygenase mediated inflammation in the mammal having joint stiffness and discomfort.
7. The composition according to claim 1, wherein the botanical extract is further enriched for total polyphenol content
8. The composition according to claim 1 further comprising a pharmaceutically acceptable carrier.
9. The composition according to claim 1, wherein the composition is a dietary supplement.
10. A method for reducing joint stiffness and discomfort in a mammal in need thereof comprising administering a therapeutically effective amount of a composition comprising a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content.
11. A composition comprising a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content for use in the treatment of reducing joint stiffness and discomfort in a mammal.
12. The method according to claim 10, wherein the botanical extract is standardized to a total catechin content of at least about 15.00% by weight, based on total weight of the extract.
13. The method according to claim 10, wherein the botanical extract is further enriched for total polyphenol content.
14. The method according to claim 10, wherein the method ameliorates cyclooxygenase and 5-lipoxygenase mediated inflammation in the mammal having joint stiffness and discomfort.
15. A method for improving cartilage rebuild or renewal function in a mammal in need thereof comprising administering a therapeutically effective amount of a composition comprising a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content.
16. A composition comprising a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content for use in improving cartilage rebuild or renewal function in a mammal.
17. The method according to claim 15, wherein the botanical extract is standardized to a total catechin content of at least about 15.00% by weight, based on total weight of the extract.
18. The method according to claim 15, wherein the botanical extract is further enriched for total polyphenol content.
19. The method according to claim 15 wherein the method ameliorates cyclooxygenase and 5-lipoxygenase mediated inflammation in the mammal in need of cartilage rebuild or renewal function.
20. A composition comprising a botanical extract of the testa of Anacardium occidentale L, wherein the botanical extract is enriched for total catechin content for use in the treatment of reducing joint stiffness and discomfort in a mammal and/or in improving cartilage rebuild or renewal function in a mammal.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0071] The present invention is based on the surprising discovery that the testa of the cashew (Anacardium occidentale Linn) is substantially high in certain flavonoids. In particularly, it has been discovered that the extract of cashew testa comprises catechin and epicatechin as major components, as well as procyanidins. Data noted herein demonstrates that cashew testa extract may have anti-inflammatory applications.
[0072] Other aspects of the present invention relate to methods of using compositions of this disclosure, such as for maintaining bone structure, cartilage structure or both, minimizing bone reabsorption, preventing cartilage degradation, increasing bone density, promoting healthy joints by protecting cartilage integrity, diminishing the action of enzymes that affect bone health, cartilage health, or both, improving joint movement or function, alleviating joint pain, alleviating joint discomfort, alleviating joint pain and discomfort, alleviating joint stiffness, improving joint range of motion or flexibility, promote mobility, or the like.
[0073] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of this disclosure. However, one skilled in the art will understand that the invention may be practiced without these details.
[0074] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the terms “about” and “consisting essentially of” mean ≅20% of the indicated range, value, or structure, unless otherwise indicated. The terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising,” as well as synonymous terms like “include” and “have” and variants thereof, are to be construed in an open, inclusive sense; that is, as “including, but not limited to”.
[0075] 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, the 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. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0076] For the present application, the term “composition” refers to a product that treats, improves, promotes, increases, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition associated with a natural state, biological process or disease or disorder. For example, a composition improves the inhibition of oxidation and/or reduces inflammation, and the like in a subject. The term composition includes, but is not limited to, pharmaceutical (i.e., drug), over-the counter (OTC), cosmetic, food, food ingredient or dietary supplement compositions that include an effective amount of an extract, at least one component thereof, or a mixture thereof. Exemplary compositions include cream, cosmetic lotion, pack or powder, or as an emulsion, lotion, liniment foam, tablets, plasters, granules, or ointment. Compositions can also include beverages, for example, beverages infused with an effective amount of an extract, or a tea satchel containing an effective amount of an extract. Non-limiting examples of food compositions containing an effective amount of an extract include baked goods, protein powders, meat products, dairy products, and confectionary.
[0077] Further, a “pharmaceutical composition” or “nutraceutical composition” as used herein refers to a formulation of a botanical extract of this disclosure and a medium generally accepted in the art for the delivery of the biologically active extract to mammals, e.g., humans. For example, a pharmaceutical composition of the present disclosure may be formulated or used as a stand-alone composition, or as a component in a prescription drug, an over-the-counter (OTC) medicine, a botanical drug, an herbal medicine, a homeopathic agent, functional foods, or any other form of health care product reviewed and approved by a government agency. Exemplary nutraceutical compositions of the present disclosure may be formulated or used as a stand-alone composition, or as a nutritional or bioactive component in food, a novel food, a functional food, a beverage, a bar, a food flavor, a food additive, a medical food, a dietary supplement, or an herbal product. A medium generally accepted in the art includes all pharmaceutically or nutraceutically acceptable carriers, diluents, or excipients therefor.
[0078] As used herein, the term “extract” or “botanical extract” refers to a solid, viscid, or liquid substance or preparation that includes one or more active ingredients of a substance of at least the plant genus Anacardium (e.g., Anacardium humile, Anacardium othonianum, Anacardium giganteum, Anacardium nanum, Anacardium negrense, and/or Anacardium occidentale), preferably Anacardium occidentale L. Preferably, the active ingredient is derived from the extract of the testa of the cashew. The extract is prepared using a solvent such as water, lower alcohols of 1 to 4 carbon atoms (e.g., methanol, ethanol, butanol, etc.), ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N,N-dimethylformamide (‘DMF’), dimethyl sulfoxide (‘DMSO’), 1,3-butylene glycol, propylene glycol, and combinations thereof, but also a fraction of the crude extract in such a solvent. So long as it assures the extraction and preservation of the active ingredient(s), any extraction method may be employed.
[0079] As used herein, “enriched for” refers to a plant extract or other preparation having at least a two-fold up increase in the amount of or activity of one or more active compounds as compared to the amount or activity of the one or more active compounds found in the weight of the raw plant material or other source before extraction or other preparation. In certain embodiments, the weight of the plant material or other source before extraction or other preparation may be dry weight, wet weight, or a combination thereof.
[0080] As used herein, the term “effective amount” or “therapeutically effective amount” of a pure compound, composition, extract, extract mixture, component of the extract, and/or active agent or ingredient, or a combination thereof refers to an amount effective at dosages and for periods of time sufficient to achieve a desired result. More specifically, “effective amount” or “therapeutically effective amount” refers to that amount of an extract or composition containing the extract of this disclosure that, when administered to a mammal, such as a human, is sufficient to effect treatment, including any one or more of: (1) treating or preventing loss of bone and cartilage in a mammal; (2) promoting bone and cartilage health; (3) suppressing loss of bone and cartilage in a mammal; (4) increasing bone density in a mammal; (5) treating or preventing osteoporosis in a mammal; (6) modifying inflammation of bone and cartilage in a mammal; (7) protecting bone and cartilage integrity; and (8) reducing joint stiffness and discomfort. The amount of a compound or composition of this disclosure that constitutes a “therapeutically effective amount” will vary depending on the amount of the major active ingredient, the condition being treated and its severity, the manner of administration, the duration of treatment, or the body weight and age of a subject to be treated but can be determined by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
[0081] The term “pharmaceutically acceptable” means those drugs, medicaments, extracts or inert ingredients, which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, and the like, commensurate with a reasonable benefit/risk ratio.
[0082] The terms “administer”, “administered”, “administers”, and “administering” are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intra-arterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In preferred embodiments, oral routes of administering a composition are suitable.
[0083] As used herein, the term “subject” or “individual” includes mammals 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) or the like. In some embodiments, the subject is a non-human mammal, and in some embodiments, the subject is human.
[0084] As used herein, the term “carrier” refers to a composition that aids in maintaining one or more plant extracts in a soluble and homogeneous state in a form suitable for administration, which is nontoxic, and which does not interact with other components in a deleterious manner.
[0085] “Supplements” as used herein refers to a product that improves, promotes, supports, increases, regulates, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition, structure or function associated with a natural state or biological process (i.e., are not used to diagnose, treat, mitigate, cure, or prevent disease). In certain embodiments, a supplement is a dietary supplement. For example, with regard to bone and cartilage health-related conditions, dietary supplements may be used to maintain bone and cartilage integrity, minimize bone reabsorption, minimize cartilage degradation, promote healthy bone and cartilage by protecting bone and cartilage integrity, diminish the action of enzymes that affect bone and cartilage health, improve osteoporosis condition, support bone rebuild, alleviate pain, alleviate discomfort, alleviate stiffness, improve range of motion, improve flexibility, promote mobility, or the like. In certain embodiments, dietary supplements are a special category of diet, food, or both, and are not a drug.
[0086] Unless indicated otherwise, all proportions and percentages recited throughout this disclosure are by weight.
[0087] In certain embodiments, compounds and compositions (e.g., pharmaceutical, nutraceutical) of the present disclosure may be administered in an amount sufficient to promote bone health; improve bone health; maintain bone health; treat or manage bone disorders; support bone health; support a normal and comfortable range of motion and/or flexibility; improve range of motion and/or flexibility; reduce the action of harmful enzymes that break down bones; alter the action of enzymes that affect bone absorption; improve movement with normal bone function; improve physical mobility; manage and/or maintain physical mobility; alleviate pain and/or stiffness due to bone loss; improve physical function; promote or enhance flexibility and comfortable movement; promote healthy bone function and comfort; relieve bone discomfort; relieve bone discomfort caused by exercise, work, overexertion or any combination thereof; promote healthy bones by protecting cartilage integrity; maintain joint cartilage; support joint cartilage; treat, prevent, or manage cartilage degradation; minimize cartilage degradation; promote joint health or comfort by maintaining synovial fluid for joint lubrication; support joint stability and joint flexibility; revitalize joints and promote mobility; promote flexible joints and strong cartilage; maintain steady blood flow to joints to support enhanced flexibility and/or strength; promote joint comfort and a wide range of motion after exercise, work, overexertion, or any combination thereof; or any other associated indication described herein, and generally with acceptable toxicity to a patient.
[0088] The present invention provides a botanical extract that exhibits anti-inflammatory activity and thus promotes joint health. More particularly, the present invention is directed towards a botanical extract of the cashew testa from the genus Anacardium. As shown herein, such botanical extracts have been found to reduce joint stiffness and discomfort and improve joint function. Further, botanical extracts according to the invention provide cartilage protection based on reduction of μCTX-II and protection of joint structure integrity. Botanical extracts according to the invention provide improved cartilage rebuild or renewal. Finally, botanical extracts according to the invention appear to be more effective than glucosamine/chondroitin supplements in improving symptoms of OA, suppressing catabolic pathways, protecting joint structure integrity, and improving cartilage rebuild or renewal function.
[0089] As previously stated, useful joint health botanical extracts according to the present invention include botanical extracts from the genus Anacardium. More particularly, the extract is a botanical extract chosen from one or more of the species Anacardium humile, Anacardium othonianun, Anacardium giganteum, Anacardium nanum, Anacardium negrense, and/or Anacardium occidentale. Preferably, the botanical extract is from the species Anacardium occidentale L. In one embodiment, the botanical extract is from the testa of the species Anacardium occidentale L.
[0090] Joint health compositions according to the present invention may include one or more compounds that may function as active ingredients. The compound may be a component of the botanical extract. For example, the compound can be a phytochemical present in the plant from which the plant extract is obtained. The compound may be at least partially responsible for exhibiting anti-inflammatory activity. The compound can be any compound capable of promoting joint health. In one embodiment, the compound is chosen from the phytochemicals catechins, epicatechins, and/or procyanidins (e.g., A, B, trimer, tetramer).
[0091] Generally, one or more parts of a plant can be used to produce a plant extract including, but not limited to, the root, the stem, the leaf, the flower, the fruit, the seed, and the testa of the seed. In the present invention, at least the testa of the seed is used—alone or with other plant parts—to produce the plant extract. The testa from the Anacardium plant can be commercially obtained from various sources. The extract of the cashew testa can be obtained using any suitable extraction technique.
[0092] In this regard, one or more parts of the plant, particularly the testa of the plant, can be collected and milled. Thereafter, the milled material can be extracted using a suitable solvent. The solvent can be removed in a concentration step. For example, the extracted material can be screened or filtered to create a supernatant and a cake. The cake can be pressed to remove a substantial portion of the liquid, which can be added to the supernatant. The cake can then be dehydrated and used as a fiber source. The supernatant can 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 can be dried (e.g., by spray drying) to provide a dried plant extract. This dried plant extract can be assayed and/or standardized as described herein. Preferably, the dried plant extract is derived from Anacardium occidentale, particularly the testa of the plant Anacardium occidentale L.
[0093] Suitable solvents for the extraction process include water, alcohol, or mixtures thereof. Exemplary alcoholic solvents include, but are not limited to, C.sub.1-C.sub.7 alcohols (e.g., methanol, ethanol, propanol, isopropanol, and butanol), hydro-alcohols or mixtures of alcohol and water (e.g., hydro-ethanol), polyhydric alcohols (e.g., propylene glycol and butylene glycol), and fatty alcohols. Any of these alcoholic solvents can 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 only water.
[0094] In one embodiment, the plant extract can be obtained using an organic solvent extraction technique. In another embodiment, solvent sequential fractionation can be used to obtain the plant extract. Total hydro-ethanolic extraction techniques can also be used to obtain the plant extract. Generally, this is referred to as a lump-sum extraction.
[0095] Total ethanol extraction can also be used. This technique uses ethanol as the solvent. This extraction technique can generate a plant extract having fat soluble and/or lipophilic compounds in addition to water soluble compounds.
[0096] Another example of an extraction technique that can be used to obtain the plant extract is supercritical fluid carbon dioxide extraction (‘SFE’). In this extraction procedure, the material to be extracted may not be exposed to any organic solvents. Rather, carbon dioxide can be used as the extraction solvent—with or without a modifier—in super-critical conditions (>31.3° C. and >73.8 bar). Those skilled in the art will appreciate that temperature and pressure conditions can be varied to obtain the best yield of extract. This technique can generate an extract of fat soluble and/or lipophilic compounds, similar to a total hexane and ethyl acetate extraction technique.
[0097] The plant extract generated in the process can include a broad variety of phytochemicals present in the extracted material. The phytochemicals can be fat soluble or water soluble. Following collection of the extract solution, the solvent can be evaporated, resulting in the extract. The plant extract can be standardized to a specified amount of a particular compound. For example, the plant extract can be standardized to a specified amount of an active ingredient or phytochemical. In one embodiment, the plant extract is standardized to a catechin content of about 15.0 wt % or greater, based on total weight of the extract.
[0098] The amount of plant extract present in the joint health composition can depend upon several factors, including the desired level of inflammation inhibition, the inflammation inhibiting level of a particular plant extract or component thereof, and other factors. Preferably, the plant extract is present in an amount of from about 0.005 wt % or greater, for example, from about 0.005 wt % to about 50.00 wt %, based on total weight of the composition.
[0099] The joint health composition can include one or more acceptable carriers. The carrier can aid in enabling incorporation of the plant extract into an anti-inflammatory composition having a suitable form for administration to a subject. A wide number of acceptable carriers are known in the art, and the carrier can be any suitable carrier. The carrier is preferable suitable for administration to animals, including humans, and can be able to act as a carrier without substantially affecting the desired activity of the plant extract and/or any active ingredient. The carrier can be chosen based upon the desired administration route and dosage form of the composition.
[0100] Suitable dosage forms include liquid and solid forms. In one embodiment, the composition is in the form of a gel, a syrup, a slurry, or a suspension. In another embodiment, the composition is in a liquid dosage form such as a drink shot or a liquid concentrate. In a further embodiment, the composition is present in a solid dosage form, such as a tablet, a pill, a capsule, a dragée, or a powder. When in liquid or solid dosage form, the composition can be in a food delivery form suitable for incorporation into food for delivery. Examples of suitable carriers 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 can be substantially inert.
[0101] As an example, silicified microcrystalline cellulose can 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 SMCC® 90, available from Penwest Pharmaceutical Co., Patterson, N.J. Silicon dioxide, in addition to that provided by the silicified microcrystalline cellulose, may be added to the composition as a processing aid. For example, silicon dioxide can be included as a glidant to improve the flow of powder during compression in the manufacturing of solid dosage units, such as tablet.
[0102] In another embodiment, the carrier is at least a functional carrier such as buckwheat or spelt. By the addition of functional carriers into the composition, additional benefits may be provided such as lower glycemic index compared to standard carriers such as those mentioned above. Further, functional carriers can be allergen free (e.g., buckwheat), and by adding them into the production process, the botanical extracts of the invention may benefit from the flavonoids of these functional carriers, such as rutin and quercetin. Further, the high fiber content of these functional carriers may also facilitate and regulate intestinal transit. Finally, the added mineral benefit of selenium found in spelt may aid in metabolism.
[0103] The anti-inflammatory composition can include other inert ingredients, such as lubricants and/or glidants. Lubricants aid in the handling of tablets during manufacturing, such as during ejection from dies. Glidants improve powder flow during tablet compression. Stearic acid is an example of an acceptable lubricant/glidant.
[0104] The anti-inflammatory composition can be made in solid dosage form, such as tablets and capsules. This form provides a product that can be easily transported by an individual to a place of eating, such as a restaurant, and taken prior to, during, or after consumption of a foodstuff. The composition can be formulated into dosage units containing suitable amounts of the plant extract and/or active ingredient that permit an individual to determine an appropriate number of units to take based upon appropriate parameters, such as body weight, foodstuff size, or carbohydrate (e.g., sugar) content.
[0105] In further embodiments, a composition according to the present disclosure comprises an Anacardium extract enriched for flavans containing catechin, epicatechin, or a combination thereof. In certain embodiments, major active ingredients in an extract of Anacardium comprise flavan containing catechin, epicatechin, or a combination thereof, wherein the extract is enriched for these active ingredients from the testa.
[0106] In one embodiment, the botanical extract is present in the composition in a therapeutically effective amount, such as an amount of about 500.0 mg/kg or greater, preferably from about 500.0 mg/kg to about 2000.0 mg/kg, more preferably from about 1000.0 mg/kg to about 2000.0 mg/kg. The composition can be administered, for example, in a dosage of from about 500.00 mg/kg to about 2000.0 mg/kg per day of the plant extract for human equivalent dosing. The composition can be administered as a single dose, or in multiple doses. In one example, the compound is administered in up to three doses per day. For example, the compound may be administered prior to a meal, during a meal, or after a meal. In one embodiment, the composition is a dietary supplement having anti-inflammatory properties containing cashew testa extract in a therapeutically effective amount.
[0107] The dosage can be chosen to provide a level of inhibitory effect in a single unit that may be effective for some individuals and/or some foodstuffs, while also allowing for relatively simple dosage increases to provide other levels of inhibitory effects that can be effective for other individuals and/or other foodstuffs.
[0108] The inhibiting composition can be in a form adapted for oral ingestion. This form can be configured as a single dosage form intended to provide a specified dose of the plant extract. For example, the single dosage form can be a powder, a pill, a tablet, a capsule, or a drink shot. The single dosage form can include, for example, from about 500.0 mg/kg to about 2000.0 mg/kg of the plant extract for human equivalent dosing.
EXAMPLES
Examples—Materials and Chemical Profiling
Example 1—Total Catechin (Flavanols) and Polyphenol Quantification of Raw (Pre-Extraction) Cashew Testa Material
[0109] Quantification of flavanols were performed by HPLC with the results presented in the following Table 1—
TABLE-US-00001 Total Catechin Catechin equivalents Catechin 43.4 mg/g Epicatechin 40.1 mg/g
In weight percentage, total catechin content of the cashew testa raw material was 7.000%, based on total weight of the raw material.
[0110] Total polyphenols (anthocyanins, flavanols, hydroxycinnamic acids, and soluble proanthocyanidins) can be quantified by the method of Folin-Ciocalteu. Gallic acid is generally recognized as the reference standard of choice, and thus total polyphenol results are reported as gallic acid equivalents.
[0111] A stock solution of gallic acid (1 mg/mL) was serially diluted and used to generate standard curves for the estimation of total polyphenols. The sample cashew testa and gallic acid standards were added to a 96 well plate alone with diluted folin reagent (7% in water) and allowed to incubate at room temperature for 10 minutes, followed by addition of 200 g/L Na.sub.2CO.sub.3. After shaking, the 96 well plate was incubated at 40° C. for 20 minutes, and then analyzed at 755 nm by spectrophotometry.
[0112] The quantification of total polyphenols was performed by UV-Vis spectroscopy at 755 nm wavelength. Quantification of total polyphenols by the method of Folin-Ciocalteu resulted in total polyphenols of 1420 mg/g, expressed as gallic acid equivalents (mg/g). In weight percentage, total polyphenol content of the cashew testa raw material was approximately 25.000%, based on total weight of the raw material.
Example 2—Preparation of 70% Ethanol Extracts from Cashew Testa
[0113] Dried cashew testa powder (Anacardium occidentale L.) (60 g) was loaded into three 100 ml stainless steel tubes and extracted twice using a solvent of 70% ethanol in DI water with a Thermo Scientific™ Dionex™ ASE 350 Accelerated Solvent Extractor at a temperature of 80° C. and pressure of 1500 psi. The extract solution was filtered and collected. The combined ethanol extract solution was evaporated with a rotary evaporator under vacuum to give a crude cashew testa extract.
[0114] The extraction results are provided in the following Table 2—
TABLE-US-00002 TABLE 2 Extraction of cashew testa Plant Powder Extract Weight Extraction Yield Plant Part (g) (g) (wt %) Testa 60 23.78 39.63%
Example 3—Catechin Quantification of Cashew Testa Extract
[0115] Free catechins present in the cashew testa extract were determined using a C18 reversed-phase column (Luna® 5 μm C18(2) 100 Å LC Column 250×4.6 mm, available from Phenomenex®, Torrance, Calif., US) together with a Hitachi high performance liquid chromatograph with photodiode array detector (‘HPLC/PDA’). For mobile phase A, the solvent was 0.10% phosphoric acid (‘H.sub.3PO.sub.4’) in water, and for mobile phase B, the solvent B was acetonitrile (‘ACN’), which was used for elution at a flow rated of 1.0 ml/min with UV absorbance at 275 nm and a column temperature of 35° C. Catechin reference standards used were from Sigma-Aldrich Co. The reference standards were dissolved in methanol (‘MeOH’):0.1% H.sub.3PO.sub.4 (1:1 ratio) with catechin (C1251) at a concentration of 0.5 mg/ml and epicatechin (E1753) at 0.1 mg/ml. Testing samples were prepared at 2 mg/ml in 50% MeOH in 0.1% H.sub.3PO.sub.4 in a volumetric flask and sonicated until dissolved (approximately 10 minutes), and then cooled to room temperature, mixed well, and filtered through a 0.45 μm nylon syringe filter. HPLC analysis was performed by injecting a 20 μl sample into the HPLC. Table 2 below provides the gradient table of HPLC analytical method—
TABLE-US-00003 TABLE 2 Gradient Table of HPLC Analytical Method 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
HPLC Catechin quantification results in cashew testa extract provided a catechin content of 9.40% and an epicatechin content of 6.12%, for a total catechin content of 15.52% by weight, based on total weight of the extract. Accordingly, the cashew testa extract can be standardized to a total catechin content of about 15.00% or greater by weight, based on total weight of the extract. The HPLC chromatogram for cashew testa extract at 275 nm wavelength is provided in
[0116] Total polyphenols in the cashew testa extract was about 55.00% by weight, based on total weight of the extract. Accordingly, the cashew testa extract according to the present invention is enriched for total polyphenols.
Example 4—Chemistry Profiling of Cashew Testa Extract
[0117] Flavonoid compounds present in the cashew testa extract were determined using ultra high-pressure liquid chromatography (‘HPLC’) and mass spectrometry (ACQUITY® UPLC I-Class and XEVO® GS-XT-QTof system, both available from Water Corporation, Milford, Mass. USA). Column used was an ACQUITY® UPLC HSS T3 2.1×100 mm, 1.8 μm, with a column temperature of 40° C. and a sample temperature of 15° C. For the mobile phase, Solvent A was 10% acetonitrile (‘ACN’) in water (0.1% Formic Acid), and Solvent B was ACN. The acquisition range was 100-1500 Daltons (‘Da’), and the acquisition mode was electrospray ionization (‘ESI’) (−). Table 3 below provides the HPLC conditions—
TABLE-US-00004 TABLE 3 HPLC conditions for analyzing cashew testa extract Run Time (min) Injection Volume (μL) Concentration 20.00 2.00 1 mg/mL
[0118] Peak identification was based on accurate mass only. Flavan-3-ols digalloyl catechin, catechin and epicatechin were identified as the major components for cashew testa extract, having the following general structures—
##STR00001##
Procyanidin flavonoids were detected in the extract as well, including A- and B-type procyanidins, procyanidin tetramer, and procyanidin trimer, with B-type procyanidins being the major component of the procyanidins.
##STR00002##
Procyanidin B2, or (−)-Epicatechin-(4β→8)-(−)-epicatechin
[0119] Compounds identified, in addition to those just mentioned, included vaccihein A, 6″-p-coumaroylprunin, and dunalianoside B, among others. LC/MS and LC/PDA chromatograms of cashew testa extract obtained from the analysis are illustrated in
Examples 5-7—In Vitro Bioassays
[0120] Extracts of cashew testa were prepared with food-grade ethanol, and then filtered and dried as described above. Research grade reagents were used for the rest of the assay preparations. Extracts were dissolved in dimethyl sulfoxide (‘DMSO’) to a final concentration of 50 mg/mL, and then diluted in appropriate buffer for each bioassay to working concentrations.
Example 5—COX-1 and COX-2 Inhibition
[0121] Cashew testa extract was tested for COX-1 inhibition using the cyclooxygenase-1 (‘COX-1’) Inhibitor Screening Kit (catalog #K548) from BioVision (Milpitas, Calif., US). This screening kit measures the production of the organic peroxide prostaglandin G2, a product generated by the COX enzyme, over a time course. Extracts were dissolved to working concentrations in DMSO with COX Assay Buffer to a final concentration of 5% DMSO. SC-560 COX-1 inhibitor was used as a positive control. COX-1 enzyme was reconstituted in sterile water and stored at −80° C. COX cofactor and arachidonic acid solutions were diluted just prior to use. COX probe, COX cofactor, and COX-1 enzyme solution were added to the test samples and controls before the arachidonic acid solution was quickly added to start the reaction. Fluorescence was measured every minute for 10 minutes at the following wavelengths: excitation −535 nm, emission 590 nm. The slope of the linear portion of the curve (
[0122] Cashew testa extract was tested for COX-2 inhibition using the cyclooxygenase-2 (‘COX-2’) Inhibitor Screening Kit (catalog #K547) from BioVision (Milpitas, Calif., US). This screening kit measures the production of the organic peroxide prostaglandin G2, a product generated by the COX enzyme, over a time course. Extracts were dissolved to working concentrations in DMSO with COX Assay Buffer to a final concentration of 10% DMSO. Celecoxib nonsteroidal anti-inflammatory drug (‘NSAID’) was used as a positive control. COX-2 enzyme was reconstituted in sterile water and stored at −80° C. COX cofactor and arachidonic acid solutions were diluted just prior to use. COX probe, COX cofactor, and COX-1 enzyme solution were added to the test samples and controls before the arachidonic acid solution was quickly added to start the reaction. Fluorescence was measured every minute for 10 minutes at the following wavelengths: excitation −535 nm, emission 590 nm. The slope of the linear portion of the curve (
Example 6—5-LOX Inhibition
[0123] Cashew testa extract was tested for 5-LOX inhibition using the Lipoxygenase Inhibitor Screening Assay Kit (available from Cayman Chemical, Ann Arbor, Mich., US) and potato 5-Lipoxygenase enzyme (available from Cayman Chemical). This kit measures hydroperoxides produced in the lipoxygenation reaction.
[0124] The extracts were dissolved in methanol to final working concentrations. 5-LOX enzyme, Chromagen, and Linoleic Acid solutions were prepared immediately before use. Nordihydroguaiaretic acid (‘NDGA’) was used as a positive control. 5-LOX enzyme was added to the test samples and controls and incubated for five minutes at room temperature to allow for enzyme/inhibitor interaction. Linoleic acid substrate was added to the plate to initiate the reaction, and the plate was then shaken at room temperature for 10 minutes. Chromagen was added to visualize the hydroperoxides formed during the reaction and the plate was shaken at room temperature for another five minutes. The absorbance was then read at 492 nm. Percent inhibition of the extract concentration was calculated in comparison to the uninhibited control wells.
[0125] Cashew testa extract was tested for its 5-LOX inhibition activity at 10 different concentrations (0.7, 1.5, 3.0, 6.0, 11.9, 15.6, 31.2, 62.5, 125.0 and 250.0 μg/mL). NDGA was used as a positive control at 100 μM with a 100% 5-LOX enzyme inhibition. Referring to
Example 7—HMGB1 Inhibition
[0126] HMGB1 Experimental Procedure—
[0127] Ce/l Culture. Murine macrophage-like cells (available as RAW 264.7 (ATCC® TIB-71™) from American Type Culture Collection (ATCC), Manassas, Va., US) were cultured in Dulbecco's Modified Eagle's Medium (‘DMEM’) ((DMEM) (ATCC® 30-2002™), from American Type Culture Collection (ATCC), Manassas, Va., US) supplemented with 10% fetal bovine serum (from Atlanta Biologicals, Lawrenceville, Ga., US). The cells were maintained under normoxic conditions (5% CO.sub.2/21% 02), allowed to grow to 70-80% confluency, and subcultured every two (2) days.
[0128] Extract/Drug Preparation. Cashew testa extract was stored in powder form at −20° C. Prior to treating cells with extract, a stock solution volume of the extract was adjusted to a final concentration of 50 mg/mL in dimethyl sulfoxide (‘DMSO’) (from AMRESCO, Inc., Solon, Ohio, US) and stored at −20° C. Extract was diluted to a final concentration of 0.25 mg/mL in serum-free Opti-MEM™ I medium (from Gibco-BRL, Gaithersburg, Md., US) and filtered sterilized by 0.2 μm PES syringe filter (from VWR, Radnor, Pa., US). Sodium salicylate (from AMRESCO, Inc., Solon, Ohio, US) was prepared at 2-20 μM as a positive control, which can attenuate hyperoxia-induced HMGB1 release from macrophages.
[0129] Hyperoxia Exposure. The exposure of murine macrophage RAW 264.7 cells to hyperoxia was achieved in sealed, humidified Plexiglas chambers (from Billups-Rothenberg, Del Mar, Calif., US) flushed with 95% O.sub.2/5% CO.sub.2 at 37° C. for 24 hours.
[0130] HMGB1 ELISA. To determine the levels of extracellular HMGB1, RAW 264.7 cells were cultured in serum-free Opti-MEM™ I medium (from Gibco-BRL, Gaithersburg, Md., US) in 6-well plates and were kept at either 21% 02 (room air) or exposed to 95% 02 with or without the cashew testa extract for 24 hours. After hyperoxic exposure, the levels of HMGB1 in the culture media were measured by ELISA (enzyme-linked immunosorbent assay). Cell culture media was collected and pelleted at 500 g for 5 minutes at 4° C. Equal volumes of cell culture supernatant were then approximately 6-x's concentrated using Amicon Ultra-4 centrifugal units (from EMD Millipore, Burlington, Mass., US). Just after concentration, equal volumes of cell culture supernatant concentrate were loaded onto a 96-well plate for determination of HMGB1 by ELISA according to manufacturer's instructions (from Chondrex, Inc., Redmond, Wash., US). Plate absorbances were determined by reading the optical density (‘OD’) value at 450 nm (with 630 nm used as a reference) on a Thermo Multiscan Ex microplate reader (from Thermo Scientific, Waltham, Mass., US). HMGB1 levels were determined in sample cell culture supernatant by comparison to a standard curve and further corrected by applying concentration factors.
[0131] Statistical Analysis. Data was presented as the mean±standard error of the mean (SEM) of one to three independent experiments. Data was analyzed by use of one-way analysis of variance (ANOVA) using Fisher's Least Significant Difference (‘LSD’) post-hoc analysis and GraphPad Prism version 6 software (from GraftPad Software, La Jolla, Calif., US). A P-Value of <0.05 was considered statistically significant.
[0132] HMGB1 Experimental Results—
[0133] Referring to
[0134] The above data illustrates that the botanical extract of the testa of Anacardium occidentale L. has one or more compounds that exhibit anti-inflammatory activity. More particularly, the cashew testa extract may have reasonable activities in ameliorating the activity or release of COX-1, COX-2, 5-LOX, and/or HMGB1.
Example 8—Efficacy of Anacardium occidentale L. Extract in Collagen-Induced Rat Paw Arthritis Induction
[0135] The collagen-induced arthritis (CIA) model in rats is the most commonly studied autoimmune model of rheumatoid arthritis (RA), with several pathological features resembling the immune mediated polyarthritis in human RA. Its shortest duration between immunization and disease manifestations makes the model more feasible for therapeutic efficacy evaluations. During its pathophysiology, following inoculation of heterogenic type II collagen (CII) from bovine nasal septum, rats will mount both humoral and cellular responses for the antigen. This sensitization subsequently will lead the host to fail to recognize self and to attack its own type II collagen where exclusively present in the joint cartilages. Upon induction, rats will experience inflammatory pain and swelling, cartilage degradation, synovial hyperplasia, panus formation, mononuclear cell infiltration, deformity, and immobility. Therefore, this model is ideal to evaluate the effectiveness of cashew testa extract administered orally at low, mid and high doses in reducing signs and symptoms associated with arthritis.
[0136] Cartilage is the main component of articular structure and consists of chondrocytes that are embedded in a dense and highly organized extracellular matrix (‘ECM’). ECM is synthesized by the chondrocytes and is composed of a collagenous network that primarily contains type II collagen, along with glycosaminoglycans (‘GAGs’) and associated proteoglycans. While the exact pathological sequences are unknown, all structural components of the joint are involved in the pathogenesis of arthritis. Along with aggrecan breakdown, degradation of collagen is a central feature of arthritis. Pro-inflammatory cytokines such as tumor necrosis factor (‘TNF’)-α and interleukin (‘IL’)-1β are known to play important roles in cartilage matrix degradation in the articular cartilage through a cascade of events that lead to stimulation of aggrecanase and matrix metalloproteinase (such as MMP13) production. While TNF-α is known as the driving force for the inflammatory process, IL-1β is believed to orchestrate recruiting other proinflammatory cytokines and chemokines. Together they can amplify, sustain, and perpetuate the disease process. Degraded cartilage is one of the major clinical manifestations in both rheumatoid arthritis (‘RA’) and osteoarthritis (‘OA’). In particular, Urinary C-terminal telopeptide of type II collagen (‘μCTX-II’) has been by far the most studied and frequently referred to biomarker of cartilage degradation that can be used for the purpose of diagnosis, determination severity of disease or prediction disease progression, prognosis and monitoring efficacy of treatment. As a result, suppression of any of these mediators could have therapeutic advantage in OA/RA. It is also noted that at the early stages of arthritis, there is an effort by the chondrocyte to rebuild and replenish the degrading extra cellular matrix such as collagen and aggrecan. This anabolic property can be assessed by measuring the serum level of PIIANP representing collagen synthesis.
[0137] Collagen induced arthritis (CIA) in rats was developed and utilized to evaluate efficacy of orally administered cashew skin extract for three weeks post disease induction. The study included seven groups of rats (n=9 rats/group). The rats were purpose bred male Sprague-Dawley rats (7-8 weeks old. Charles River Laboratories Inc., Wilmington, Mass.). Animals were acclimated for two weeks to achieve the required body weight before being assigned randomly to their respective group. Rats (3/cage) were housed in a polypropylene cage and individually identified by numbers on their tail. Individual cages were identified with a cage card indicating project number, test article, dose level, group, and animal number. Harlan Soft cob bedding (Envigo Tekland 7087, Envigo, Indianapolis, Ind.) was used and changed at least twice/week. Animals were provided with fresh water and rodent chow diet (Teklad 2018, Envigo, Indianapolis, Ind.) ad libitum and housed in a temperature-controlled room (22.2° C.) on a 12-h light-dark cycle throughout the study.
[0138] The study rats were randomized and administered one of seven study articles as noted in Table 4—
TABLE-US-00005 TABLE 4 N (no. Dose Group of rats) (mg/kg) G1 Control + vehicle (0.5% CMC *) 9 0.0 G2 CIA + vehicle (0.5% CMC) 9 0.0 G3 CIA + MTX 9 0.5 G4 CIA + low dose CNT 9 50.0 G5 CIA + mid dose CNT 9 100.0 G6 CIA + high dose CNT 9 200.0 G7 Glucosamine + Chondroitin 9 150.0 G + 120.0 C * CMC = carboxymethylcellulose; MTX = Methotrexate; CNT = cashew testa extract; G = glucosamine; C = chondroitin. Dosing based on Nair, A. B. et al., J BASIC CLIN PHARM, “A simple practice guide for dose conversion between animals and human”, March 2016, Vol. 7, No. 2, pp. 27-31.
[0139] Anacardium occidentale L. extract used in the CIA study was prepared as described in Example 1 above and quantified for total catechin content according to Example 2, with the extract having a total catechin content of 18.4% by total weight of the extract.
[0140] As noted in Table 4 above, the rats were randomized into seven treatment groups, with nine (9) rats in each group, based on their body weight. On treatment start day, the average body weight of the rats was 189.7±11.7 g. The animals were orally treated according to Table 4 with Methotrexate, cashew skin extract at three dosages, and glucosamine and chondroitin daily for three weeks. Methotrexate is an effective immunosuppressant optimally used to treat autoimmune mediated arthritis such as CIA rats. The normal control rats and CIA rats were treated with the carrier vehicle (0.5% Carboxymethyl cellulose) only.
[0141] For two (2) weeks prior to induction, the rats were gavaged with freshly prepared respective test materials suspended in 0.5% CMC at 10 ml/kg/rat. Samples in solution were vortexed before oral administration to maintain the homogeneity of test materials. Ankle diameter, paw thickness and pain sensitivity measurements were taken before induction of arthritis at priming for baseline.
[0142] For induction, collagen type-II from bovine nasal septum (Elastin Products Company, Owensville, Mich.) and Incomplete Freund's adjuvant (‘IFA’, from Sigma, St. Louis, Mo.) were used. All materials were kept at suitable temperature as recommended by the manufacturer. At the time of preparation, 60 mg of collagen was weighed and added to a pre-chilled 15 ml 0.1M acetic acid in a 60 ml size flask with a magnetic stirrer to yield 4 mg/mI concentration. The mixture was dissolved by gently stirring overnight at 4° C. The next morning, the dissolved collagen was emulsified with equal volume of IFA (15 ml) to achieve a final concentration of 2 mg/ml Collagen. Rats sedated with isoflurane were then primed intradermal with 400 μl of the emulsified collagen at the base of their tail at two sites using a 1 ml syringe fitted in 26 g needle. The dissolved mixture was kept in ice bucket and stirred between groups at the time of injection to preserve uniform consistency.
[0143] Post-induction, all the rats continued receiving their respective treatment materials after priming. The rats received a total of 5 weeks of treatment (i.e., 2 weeks before induction and 3 weeks after induction).
[0144] Rats were evaluated for arthritis index before injection of a booster dose. Inoculated a booster dose of 2 mg/ml type II collagen emulsified with equal volume of IFA at 100 μl/rat/site, following the same preparation as indicated in the pre-induction treatment. Paw thickness, ankle diameter and pain sensitivity measurements were taken before injection of the antigen on day 7.
[0145] During the in-life period, arthritis severity index, paw thickness, ankle diameter and pain sensitivity were monitored. Urine and serum were collected at the end of study for biomarker analysis. Necropsy was carried out on day 22 for all the groups post priming. At necropsy, the ankle joint from each rat was collected for histopathology analysis. Urinary cartilage degradation marker (CTX-II), proinflammatory cytokines (TNF-α, IL-1β, and IL-6), cartilage synthesis marker (PIIANP) and matrix degrading proteases (MMP13) were measured.
[0146] All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals, which were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) (Approval number IA-P02-092619). Experimental design is depicted in
[0147] Clinical findings such as arthritis severity index, paw thickness, ankle diameter and pain sensitivity data have been compiled as follows.
[0148] Arthritis severity index. Rats continued to show a slow progression of disease for the duration of study. As seen in the data below, rats treated with all the treatment groups showed various degrees of severity inhibition. In particular, rats treated with 100 mg/kg and 200 mg/kg CNT showed statistically significant suppression in arthritis severity from day 10 and continued this significance for the duration of study (
TABLE-US-00006 TABLE 5 P-values for arthritis severity index compared to vehicle-treated CIA+ P-value Group Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Control 0.0042 0.0002 0.0005 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 MTX 0.0042 0.0002 0.0005 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 CNT 50 0.0178 0.0107 02163 0.0700 0.0141 0.0085 0.0035 0.0030 0.0074 0.0025 0.0370 0.1245 CNT 100 0.0042 0.0002 0.0005 0.0000 0.0000 0.0002 0.0038 0.0041 0.0015 0.0001 0.0204 0.0726 CNT 200 0.0042 0.0002 0.0005 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 0.0000 0.0002 0.0004 GC(150 + 0.0042 0.2779 0.8382 0.0733 0.0472 0.0340 0.0775 0.0242 0.0225 0.0087 0.0742 0.1423 120)
[0149] Paw thickness. In agreement with the severity score, rats treated with the 100 mg/kg and 200 mg/kg CNT showed statistically significant reduction in paw swelling starting from day 12 and maintained this significance for the duration of study (
TABLE-US-00007 TABLE 6 P-values for paw thickness compared to vehicle-treated CIA+ P-values Group Day 12 Day 13 Day 15 Day 17 Day 19 Day 21 Control 0.0572 0.0000 0.0000 0.0000 0.0000 0.0000 MTX 0.0388 0.0000 0.0000 0.0000 0.0000 0.0000 CNT 50 0.3734 0.0575 0.1725 0.0862 0.0954 0.0702 CNT 100 0.0438 0.0000 0.0061 0.0395 0.0380 0.0338 CNT 200 0.0468 0.0000 0.0004 0.0278 0.0281 0.0014 GC(150 + 120) 0.6574 0.2100 0.4278 0.1508 0.2207 0.3370
[0150] Ankle diameter. Statistically significant, similar pattern in reductions of ankle diameters were observed for rats treated with 100 mg/kg and 200 mg/kg CNT until day 15 post induction (
TABLE-US-00008 TABLE 7 P-values for ankle diameter compared to vehicle treated CIA+ P-values Group Day 12 Day 13 Day 15 Day 17 Day 19 Day 21 Control 0.0196 0.0000 0.0000 0.0000 0.0000 0.0000 MTX 0.0312 0.0000 0.0000 0.0001 0.0000 0.0003 CNT 50 0.3162 0.0228 0.0124 0.0754 0.0300 0.1462 CNT 100 0.0373 0.0002 0.0115 0.1041 0.0375 0.3843 CNT 200 0.0380 0.0011 0.0034 0.0184 0.0083 0.0116 GC(150 + 120) 0.8567 0.0815 0.4278 0.1508 0.2207 0.3370
[0151] Pain sensitivity. Response to pressure as a measure of pain sensitivity was measured using a Randall-Selitto probe attached to an electronic monitor on priming day, boost, day 12, 13, 15, 17, 19 and 21. Both the left and right hind legs were monitored on those days, and their average was used for data analysis. Changes from the vehicle treated CIA rats have been reported as pain tolerance on those days. The highest pain tolerance was observed for rats in the Methotrexate (14.1-67.1% vs Vehicle treated CIA) group followed by the 200 mg/kg (13.5-43.8% vs Vehicle treated CIA) and the 100 mg/kg (11.8-25.8% vs Vehicle treated CIA) (
TABLE-US-00009 TABLE 8 P-values for pain tolerance compared to vehicle treated CIA+ P-values Group Day 12 Day 13 Day 15 Day 17 Day 19 Day 21 Control 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 MTX 0.0000 0.0000 0.0000 0.0001 0.0000 0.0003 CNT 50 0.0003 0.0228 0.0124 0.0754 0.0300 0.1462 CNT 100 0.0000 0.0002 0.0115 0.1041 0.0375 0.3843 CNT 200 0.0000 0.0011 0.0034 0.0184 0.0083 0.0116 GC(150 + 120) 0.0025 0.0815 0.4278 0.1508 0.2207 0.3370
Blomarkers—
[0152] Urine CTX-II—
[0153] Assay. Rat urine samples were diluted 1:3 and the presence of CTX-II was measured using the Rat CTX-II ELISA kit from Mybiosource as follows. Diluted urine was added to a microplate coated with CTX-II antibody and allowed to bind for 2 hours at 37° C. A biotin-conjugated antibody against CTX-II was then added and allowed to bind to the CTX-II from the rat urine for 1 hour at 37° C. The microplate was washed thoroughly to remove unbound urine and antibody before an enzyme-conjugated avidin antibody was added to bind to the biotin-conjugated antibody for specific detection. The avidin antibody was allowed to bind for 1 hour at 37° C. Washing was repeated, enzyme substrate was added, and the plate was developed for 30 minutes at 37° C. After the addition of stop solution, the absorbance was read at 450 nm, multiplied by dilution factor, and the concentration of CTX-1l calculated based on the absorbance readings of a CTX-II standard curve.
[0154] Normalization.
[0155] Creatine—CTX-II amount was normalized to the amount of Creatinine in the urine using a Creatinine Parameter Assay Kit (R&D Systems) as follows. Urine was diluted 1:20, mixed with alkaline picrate (5 parts 0.13% picric acid:1 part 1 N NaOH) in a microplate, and incubated at room temperature for 30 minutes. Absorbance was read at 492 nm, and Creatinine amount in urine was calculated based on the absorbance readings of a Creatinine standard curve.
[0156] Protein—CTX-II amount was normalized to the amount of total protein in the urine using a Pierce BCA Protein Assay kit (ThermoFisher Scientific) as follows. The urine was diluted 1:20, mixed with bicinchoninic acid (BCA) reagent in a microplate, and incubated at 37° C. for 30 minutes. Absorbance was read at 580 nm, and protein concentration in the urine was calculated based on the absorbance readings of a bovine serum albumin standard curve.
[0157] Results.
[0158] As illustrated in
[0159] Cytokines IL-1β/IL-6/TNF-α—
[0160] Sample collection. At completion of study, blood from cardiac puncture was collected for each animal. Blood was spun at 3000 rpm for 15 min. About 700-800 μl of serum was isolated from each rat. Both samples were kept at −80° C. until use.
[0161] ELISA Assay. The presence of cytokines IL-1β/IL-6/TNF-α was measured using the Rat IL-1β/IL-6/TNF-α Quantikine ELISA kit (R&D Systems, Minneapolis, Minn.) as follows. Undiluted serum was added to a microplate coated with polyclonal IL-1β/IL-6/TNF-α antibody and allowed to bind for 2 hours at room temperature. The microplate was washed thoroughly to remove unbound serum, and then a polyclonal enzyme-conjugated IL-1β/IL-6/TNF-α antibody was added and allowed to bind for 2 hours at room temperature. Washing was repeated, enzyme substrate was added, and the plate was developed for 30 minutes at room temperature. After the addition of stop solution, the absorbance was read at 450 nm and the concentration of IL-1β/IL-6/TNF-α calculated based on the absorbance readings of an IL-1β/IL-6/TNF-α standard curve.
[0162] Result for Serum IL-1β, IL-6, and TNF-α.
[0163] Proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 play critical role alone or in concert in initiation, recruiting, progression and perpetuation of inflammation in the pathogenesis of OA/RA. Agents that reduce the level of these cytokines could mitigate the symptoms associated with OA/RA.
[0164] Referring to
[0165] Similarly, the serum level of TNF-α was reduced as a result of cashew skin extract (
[0166] Cashew testa extract treatment had no effect on rat serum IL-6 levels, with all serum values found below the blank. However, the IL-1β and TNFα data reflects what was observed in the in-life study clinical measurements, such as arthritis index, ankle diameter and paw thickness.
[0167] Type IIA Collagen N-Propetide (PIIANP)
[0168] Levels of PIIANP decrease in patients with OA and RA, suggesting that type IIA collagen synthesis may be altered in these diseases. Accordingly, the measurement of type IIA collagen synthesis based on this biomarker is useful in determining the efficacy of cashew testa extract in patients with joint diseases.
[0169] ELISA Assay. The presence of PIIANP was measured using the Rat Procollagen Type IIA N-Prop (PIIANP) ELISA kit (MyBiosource, San Diego, Calif.) as follows. Undiluted serum was added to a microplate coated with PIIANP antibody as well as an HRP-conjugated PIIANP antibody and allowed to bind for one hour at 37° C. The microplate was thoroughly washed and a Chromagen solution was added and allowed to bind for 15 minutes at 37° C. After the addition of stop solution, the absorbance was read at 450 nm and the concentration of PIIANP calculated based on the absorbance readings of a PIIANP standard curve.
[0170] Results for Serum PIIANP
[0171] While the normal control rats showed a 53.0% increase in the level of serum PIIANP, a statistically significant decrease (34.7% vs. control) in serum PIIANP was observed for the CIA rats treated with vehicle compared to the control group (p=0.0002), indicating the induction of the model (see
[0172] Matrix Metalloproteinase 13 (MMP-13)
[0173] Matrix metalloproteinase 13 is a regulator of inflammation and is an enzyme that plays an important role in type II collagen degradation in articular cartilage in osteoarthritis. It also degrades proteoglycan, type IV and type IX collagen, osteonectin, and perlecan in cartilage.
[0174] ELISA Assay. The presence of MMP-13 in undiluted rat serum was measured using the MMP-13 Rat Matrix Metalloproteinase 13 (MMP-13) ELISA Kit (MyBioSource, San Diego, Calif.) as follows. Undiluted serum was added to a microplate coated with MMP-13 antibody. After 2 hours at 37° C., MMP-13 in serum was bound to the plate and unbound serum was aspirated. A biotin-conjugated antibody specific for MMP-13 was added to the wells and allowed to bind for 1 hour at 37° C. The plate was thoroughly washed, and avidin conjugated Horseradish Peroxidase (HRP) was added to the plate. After 1 hour at 37° C., washing was repeated, and enzyme substrate was added to the plate. After developing for 20 minutes at 37° C., a stop solution was added, and the absorbance was read at 450 nm. The concentration of MMP-13 was calculated based on the absorbance readings of an MMP-13 standard curve.
[0175] Results for Serum MMP-13
[0176] Referring to
[0177] Histopathology
[0178] Procedures and Evaluations. At necropsy, the ankle joint was carefully dissected out, fixed in 10% buffered formalin, and sent to Nationwide Histology (Veradale, Wash., USA) for further histopathology analysis. The fixed specimens were then decalcified with Calci-Clear Rapid for one and a half days and embedded in paraffin. Standardized 5 μm serial sections were obtained from each rat and stained with hematoxylin and eosin (HE) and Safranin 0-fast green to enable evaluation of proteoglycan content. A modified Mankin system (Mankin et al., 1981) was used to score structural and cellular alterations of articular components as indications of disease progression and/or treatment efficacy. The histological analysis was conducted by a certified Pathologist at Nationwide Histology.
[0179] Results. The histopathology data were in alignment with the severity score of arthritis. When compared to the normal control rats, vehicle treated CIA rats showed severe synovitis, marked cartilage degradation, synovial hyperplasia, pannus formation and bone erosion (
[0180] Summary of Efficacy of Anacardium occidentale L. Extract in Collagen-Induced Rat Paw Arthritis Induction—
[0181] Collagen induced arthritis (CIA) in rats was developed and utilized to evaluate efficacy of orally administered cashew testa extract for three weeks post disease induction. The study report includes seven groups of rats, with nine (9) rats per group. The rats in three of the groups were orally treated with cashew testa extract at three different dosages—a low dose of 50 mg/kg, a mid-dose of 100 mg/kg, and a high-dose of 200 mg/kg. The effectiveness of the cashew testa extract groups was compared against a group treated with the immunosuppressive drug Methotrexate dosed at 0.5 mg/kg, and a group treated with Glucosamine and chondroitin (150 G+120 C mg/kg) daily for three weeks. The normal control rats and CIA rats were treated with the carrier vehicle 0.5% Carboxymethyl cellulose only. During the in-life period, arthritis severity index, paw thickness, ankle diameter and pain sensitivity were monitored. Urine and serum were collected at the end of the study for biomarker analysis. At necropsy, the ankle joint from each rat was collected for histopathology analysis. Urinary cartilage degradation marker (CTX-II), proinflammatory cytokines (TNF-α, IL-1β, and IL-6), cartilage synthesis marker (PIIANP) and matrix degrading proteases (MMP13) were measured to determine the efficacy of each treatment.
[0182] Induction of disease model was confirmed by the progressively increased arthritis severity index and, later, by accompanied urinary and serum arthritis related biomarkers as well as histopathology findings. The rats showed various degrees of responses to the treatments. Cashew testa extract showed dose correlated and measurable efficacy with significant impact at higher dosages in mitigating the symptoms of arthritis. When the overall data for arthritis severity, paw thickness, ankle diameter and pain sensitivity were compared, CIA rats treated with mid- and high doses of cashew skin extract showed statistically significant reduction in the prime signs of arthritis. Rats in the GC and 50 mg/kg cashew skin extract group showed minimal efficacy in this study.
[0183] Data from the biomarkers were in accordance with in-life observations. Statistically significant reduction (53.4% inhibition vs. vehicle; P=0.04) in μCTX-II was observed for CIA rats treated with 200 mg/kg cashew testa extract when data were normalized with protein. Similarly, statistically significant reductions in serum IL-1 (200 mg/kg cashew testa extract, 53.1% reduction, P=0.01) and TNF-α (100 mg/kg cashew testa extract) levels were observed for rats treated with cashew testa extract when compared to the vehicle treated group. While the low dose cashew testa extract was infective in suppressing IL-1β and TNF-α, the GC treated rats showed statistically significant decrease in TNF-α. The cashew testa extract (100 mg/kg and 200 mg/kg) and GC treated group achieved the level of significance for the increases of anabolic marker when compared to the vehicle treated group.
[0184] Furthermore, the histopathology data were well aligned with the severity score of arthritis. While the vehicle-treated rats experienced severe synovitis, marked cartilage degeneration, diffused necrosis of bone and cartilage, synovial hyperplasia, pannus formation, bone erosion, and loss of architectural structure, CIA rats treated with cashew testa extract and methotrexate had relatively moderate morphological alternations in matrix integrity, and reduced articular bone damage. Cashew testa extract (200 mg/kg) treated rats showed statistically significant reductions in inflammation and bone erosion from the modified Mankin score analysis of histopathology data. Rats treated with GC or 50 mg/kg of cashew testa extract resulted in minimal microscopic improvement for articular structure damage.
[0185] Accordingly, based on data from the in-life measurements (arthritis severity, paw thickness, ankle diameter and pain sensitivity), urinary CTX-II, serum IL-1β and TNF-α, and histopathology analysis, cashew testa extract administered orally at 100 mg/kg or 200 mg/kg performed significantly superior to the GC treated group. Treatment of rats with GC produced statistically significant changes in the anabolic (PIIANP) markers and TNF-α. The collective data support the potential use of cashes testa extract for support joint structure and function.
[0186] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it covers all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.