JP2014177454A - Food and drink and pharmaceutical preparation containing nejime biwa tea extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lipase inhibitor, an α-glucosidase inhibitor, or an anti-inflammatory agent which contains extract or purified product of Eriobotrya japonica leaves or Eriobotrya japonica tea as an active ingredient.SOLUTION: A method for production of a lipase inhibitor, an α-glucosidase inhibitor, or an anti-inflammatory agent comprises (a) a process of subjecting Nejime Biwa Tea to hot water extraction to obtain an extract which is a concentrate or dry powders, (b) a process of subjecting the hot water extract of process (a) to column chromatography to obtain a fraction by extraction with a mixed solution which contains ethanol solution of 30%-70% or water and acetone in a ratio of 1:1.

Description

  The present invention relates to a food or drink or pharmaceutical having, for example, a lipase inhibitory action, an α-glucosidase inhibitory action and / or an anti-inflammatory action, and a method for producing the same.

  In the patent document 1, the applicant of the present application disclosed that loquat leaves have a high blood glucose level lowering action, a hypertension inhibiting action, a cancer cell proliferation inhibiting action, a cancer cell apoptosis inducing action, a reactive oxygen species producing action, an antihyperlipidemic action, etc. It is clarified that it has an effect, and loquat leaves can be used for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension, cancer, etc., antihyperlipidemic action, antihypertensive action, cancer cell growth inhibition Food / beverage products or pharmaceuticals having an action, cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemia lowering action and / or antioxidant action, and a method for producing the same.

  In addition, in the patent document 2, the applicant of the present application discloses a method and production equipment for producing a large amount of flavored loquat tea using loquat leaves. Concerning the produced Nemebiwa tea, consumers say that the blood sugar level of diabetes has decreased and the blood pressure of hypertension has become normal (Non-patent Document 1).

  Conventionally, it has been disclosed that loquat has the following activity or utilization method.

  Patent Document 3 discloses that loquat or an extract thereof has a matrix metalloproteinase (MMP-1) inhibitory activity.

  Patent Document 4 discloses that the leaves of loquat trees are baked into charcoal to make a powdered food material.

  Patent Document 5 describes a Chinese medicine characterized by cutting raw loquat leaves, steaming with steam, scouring while sending hot air, drying, shaping, and then drying again to increase aroma and umami. A method for producing tea is disclosed.

  Patent Document 6 discloses a method for producing a health tea containing a larger amount of γ-aminobutyric acid in which leaf green health tea and loquat tea leaves are mixed.

  Patent Document 7 discloses that loquat extracts excluding fruit parts have α-glucosidase inhibitory activity.

  Patent Document 8 discloses a composition for enhancing nutritional tonic health in which peony and loquat leaves are mixed.

  Patent Document 9 discloses a healthy tea obtained by mixing roasted yellow persimmon leaves with loquat tea.

  Patent Document 10 discloses a health tea composed of a mixture of sweet tea and loquat.

  Patent Document 11 discloses that a composition containing an organic extract belonging to the genus Rosaceae inhibits cyclooxygenase-2 (COX-2) activity in an organism.

Further, the present applicant, in Patent Document 12, with a tea compound, COX-2 inhibitors and / or prostaglandin E2 (PGE2 or PGE ₂₎ discloses a food or beverage or pharmaceutical having an activity of biosynthesis inhibition. The applicant of the present application in Patent Document 12, “40 kinds of tea compounds separated from green tea, oolong tea, and black tea were tested, and the enzyme COX-2 that causes chronic inflammation and PGE2 that is a product thereof were studied as targets. As a result, it was found that 18 types of tea compounds suppress the expression of COX-2 and / or PGE2 production. ”As a result, the tea compound was used to inhibit COX-2 and / or PGE2 biosynthesis. Provided food or drink or medicine.

Japanese Patent No. 4974116 Japanese Patent No. 3435351 JP 2005-008539 A JP 2004-154108 A JP 2004-105036 A Japanese Patent Laid-Open No. 2002-065227 JP 2001-163795 Japanese Patent Laid-Open No. 2001-163792 Japanese Unexamined Patent Publication No. 7-274832 Japanese Patent Laid-Open No. 5-056772 Special Table 2004-529079 JP 2012-56952 A

Totsukawa Farm, Agricultural Production Corporation, “Nemebiwa Tea Brochure”, 2004

  As described above, although the existence of various biological activities and active compounds contained in Nemebiwa tea has been clarified, Nemebiwa tea has a lipase inhibitory action, an α-glucosidase inhibitory action, and a COX-2 inhibitory action. And / or it has not been known to have effects such as PGE2 biosynthesis inhibition action (so-called anti-inflammatory action). If these effects can be confirmed, Nejimebiwa tea can be used for the prevention or treatment of metabolic syndrome such as diabetes, or as a COX-2 inhibitor and / or PGE2 biosynthesis inhibitor for the prevention or treatment of various cancer diseases. It is considered a thing.

  Therefore, in view of the above-mentioned circumstances, the present invention has a lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE2 biosynthesis inhibitory action (so-called anti-inflammatory action) using Nemebiwa tea. It aims at providing the food-drinks or pharmaceutical which have, and its manufacturing method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have used Nemebiwa tea for hot water extraction, and the obtained extract has a lipase inhibitory action and an α-glucosidase inhibitory action, In addition, in RAW264 cells (mouse-derived macrophages) in which inflammation was induced using lipopolysaccharide (LPS), the nitric oxide production in the cells was decreased, the expression of COX-2 and iNOS was suppressed, and PGE2 It was found to suppress production. Furthermore, the extract derived from Nemebiwa tea suppresses the production of cytokines (IL-5, IL-6, IL-12, GM-CSF, MCP-1, and TNF-α) that should be called markers of inflammation. I found out. In addition, the extract derived from Nemebiwa tea suppresses phosphorylation of p38, ERK, and JNK in the MAPK cell signal transduction system among the inflammatory signal transduction systems involved in the production of inflammatory factors as described above, and NF-κB It was found that phosphorylation of NF-κB (p65), IKK, and TAK1 in the cell signaling system was suppressed. Furthermore, it was confirmed that the crude fraction obtained from the extract of Nemebiwa tea had a strong antioxidant action, and the present invention was completed.

  The present invention includes the following.

  (1) A food or beverage product containing an extract or purified product of loquat leaf or loquat tea as an active ingredient and having at least one action selected from the group consisting of lipase inhibitory action, α-glucosidase inhibitory action and anti-inflammatory action Or medicine.

  (2) The food or drink or medicine according to (1), wherein the loquat tea is Nememebiwa tea.

  (3) The food or drink has an indication that it has one or more actions selected from the group consisting of a lipase inhibitory action, an α-glucosidase inhibitory action and an anti-inflammatory action, (1) The described food or beverage or medicine.

  (4) The food or drink or pharmaceutical according to (1), wherein the anti-inflammatory action is a prostaglandin E2 (PGE2) biosynthesis inhibiting action or a cyclooxygenase-2 (COX-2) inhibiting action.

  (5) selected from the group consisting of a lipase inhibitory action, an α-glucosidase inhibitory action and an anti-inflammatory action, comprising a step of subjecting loquat leaves or loquat tea to hot water extraction or solvent extraction to obtain an extract A method for producing a food or drink or pharmaceutical product having one or more actions.

  (6) The method according to (5), further comprising a step of subjecting the extract to a purification means to obtain a purified product.

  (7) The method according to (6), wherein the purification means is column chromatography.

  (8) The method according to (5), wherein the loquat tea is Nememebiwa tea.

  (9) The production method according to (5), wherein the anti-inflammatory action is a PGE2 biosynthesis inhibitory action or a COX-2 inhibitory action.

  Moreover, this invention includes the following.

  (1 ') (a) A step of subjecting Nemebiwa tea to hot water extraction to obtain an extract, wherein the extract is a concentrate or a dry powder; and (b) step (a) Subjecting the hot water extract to column chromatography and obtaining a fraction by extraction with a 30% to 70% aqueous ethanol solution or a mixture containing water and acetone in a ratio of 1: 1. A method for producing a lipase inhibitor, an α-glucosidase inhibitor or an anti-inflammatory agent.

  (2 ′) The method according to (1 ′), wherein the 30% to 70% ethanol aqueous solution is a 30%, 50% or 70% ethanol aqueous solution.

  (3 ′) (a) a step of subjecting Nemebiwa tea to hot water extraction to obtain an extract, wherein the extract is a concentrate or a dry powder; and (b) step (a) Subjecting the hot water extract to column chromatography, obtaining a fraction by extraction with 50% to 70% aqueous ethanol, and (c) subjecting the fraction of step (b) to column chromatography, A lipase inhibitor comprising a step of obtaining a fraction by extraction with a mixture containing water and methanol at a ratio of 80%: 20%, 70%: 30% or 50%: 50%, α- A method for producing a glucosidase inhibitor or an anti-inflammatory agent.

  (4 ′) The method according to (3 ′), wherein the 50% -70% ethanol aqueous solution is a 50% or 70% ethanol aqueous solution.

  (5 ′) A lipase inhibitor, α-glucosidase inhibitor or anti-inflammatory agent containing the fraction obtained by the method according to any one of (1 ′) to (4 ′) as an active ingredient.

  (6 ′) The anti-inflammatory agent according to (5 ′), which is a PGE2 biosynthesis inhibitor.

  (7 ′) The anti-inflammatory agent according to (5 ′), which is a COX-2 inhibitor.

  According to the present invention, it has a lipase inhibitory action, an α-glucosidase inhibitory action, a COX-2 inhibitory action and / or a PGE2 biosynthesis inhibitory action (so-called anti-inflammatory action), and is an extract or purified product of loquat leaf or loquat tea. The food-drinks or pharmaceutical containing this is provided. The food and drink according to the present invention (especially health supplements or foods for specified health use) or pharmaceuticals have lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE2 biosynthesis inhibitory action (so-called anti-inflammatory action) Therefore, it can be used as an anti-inflammatory agent having a lipase inhibitor, an α-glucosidase inhibitor, a COX-2 inhibitory action and / or a PGE2 biosynthesis inhibitory action.

FIG. 1 shows the results of examining the antioxidant effect of Nemebiwa tea extract (M) and fractions “AD” in Example 1. The vertical axis shows the antioxidant capacity in percentage (%), the horizontal axis shows the extract of Nemebiwa tea (M), fractions “A to D”, and each powder has a concentration of 50 μg / ml. Adjusted and tested. FIG. 2 shows a thin-layer chromatograph (TLC) of a crude fraction / fraction “C1 to C9” of Nememebiwa tea in Example 2. The TLC was directly irradiated with ultraviolet light (UV) having a wavelength of 252 nm for detection. As the developing solvent, four types of solvents of benzene-ethyl formate-formic acid-water were mixed at a ratio of 4: 31: 5: 1. The numbers 1 to 9 correspond to the crude fraction and fraction “C1 to C9” of Nememebiwa tea. FIG. 3 shows TLC of crude fractions and fractions “C1 to C9” of Nememebiwa tea in Example 2. A 10% sulfuric acid aqueous solution is sprayed directly on the TLC, and a substance that develops color from brown to light blue is detected. As the developing solvent, four types of solvents of benzene-ethyl formate-formic acid-water were mixed at a ratio of 4: 31: 5: 1. The numbers 1 to 9 correspond to the crude fraction and fraction “C1 to C9” of Nememebiwa tea. FIG. 4 shows the results of examining the antioxidant effect of the crude fraction / fraction “C1 to C9” of Nemebiwa tea in Example 2. As a control, the fraction “C” of Nejimebiwa tea used in Example 1 was used. The vertical axis shows the antioxidant capacity in percentage (%), and the horizontal axis shows the fraction “C” of Nemebiwa tea and the crude fraction / fraction “C1 to C9”. Tested at a concentration of 10 μg / ml. FIG. 5 shows the results of examining the lipase inhibitory action of Nememeiwa tea extract (M) and fractions “AD” in Example 3. The vertical axis represents the effective concentration of 50%, and the horizontal axis represents the extract of Nemebiwa tea (M) and fractions “A to D”. FIG. 6 shows the results of examining the lipase inhibitory action of crude fractions and fractions “C1 to C9” of Nemebiwa tea in Example 4. As a control, a fraction “C” of Nememebiwa tea was used. The vertical axis shows the effective concentration of 50%, and the horizontal axis shows the fraction “C” of Nemebiwa tea and the crude fractions and fractions “C1 to C9”. FIG. 7 shows the results of examining the α-glucosidase inhibitory action of the extract of Nemebiwa tea (M) and fractions “AD” in Example 5. The vertical axis represents the effective concentration of 50%, and the horizontal axis represents the extract of Nemebiwa tea (M) and fractions “A to D”. FIG. 8 shows the results of examining the α-glucosidase inhibitory action of crude fractions and fractions “C1 to C4” of Nemebiwa tea in Example 6. As a control, a fraction “C” of Nememebiwa tea was used. The vertical axis shows the effective concentration of 50%, and the horizontal axis shows the fraction “C” of Nemebiwa tea and the crude fractions and fractions “C1 to C4”. FIG. 9 shows the results of examining the inhibitory action on nitric oxide production of Nemebiwa tea extract (M) and fractions “AD” in Example 7. The vertical axis shows the amount of nitric oxide induced by LPS as 100%, expressed as a percentage of this, and the horizontal axis shows LPS that induced inflammation with LPS, Nemebiwa tea extract (M) And the fractions “AD” (concentration of 50 μg / ml each). FIG. 10 shows the results of examining the inhibitory action on nitric oxide production of Nememebiwa tea fraction “C” and crude fraction / fraction “C2” in Example 8. The vertical axis indicates the amount of nitric oxide induced and generated by LPS as 100%, and is expressed as a percentage value. The horizontal axis indicates LPS that induced inflammation with LPS, and the fraction "C" of Nemebiwa tea. And the result of investigating the concentration of the crude fraction / fraction “C2” at 12.5 to 100 μg / ml or more is shown. FIG. 11 shows the electrophoresis obtained by examining the expression inhibitory action of COX-2 and iNOS of the extract of Nemebiwa tea (M) and fractions “AD” in Example 10. FIG. 12 shows an electrophoresis diagram obtained by examining the COX-2 and iNOS expression inhibitory action of the fraction “C” of Nemebiwa tea in Example 10. FIG. 13 shows an electrophoresis diagram obtained by examining the COX-2 and iNOS expression-inhibiting action of the fraction “C2” of Nemebiwa tea in Example 11. FIG. 14 is a graph showing that IL-5 production is decreased by increasing the concentration of crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 15 is a graph showing that IL-6 production is reduced by increasing the concentration of the crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 16 is a graph showing that IL-12 production is reduced by increasing the concentration of crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 17 is a diagram showing that the production of GM-CSF is decreased by increasing the concentration of the crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 18 is a diagram showing that the production of MCP-1 is decreased by increasing the concentration of the crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 19 is a graph showing that the production of TNF-α is decreased by increasing the concentration of the crude fraction “C2” of Nemebiwa tea to 100, 200, 200 μg / ml or more in Example 12. FIG. 20 is a diagram comparing the degree of MAPK cell signal transmission inhibitory action of crude fraction “C2” of Nememebiwa tea with LPS in Example 13. Specifically, an electrophoretic diagram of the expression genes of p38, ERK, and JNK and the expression levels of these phosphorylated proteins is shown. FIG. 21 is a diagram comparing the degree of inhibitory action of the crude fraction “C” of Nememebiwa tea in Example 13 with that of LPS induced by LPS. Specifically, an electrophoretic diagram of the expression genes of p38, ERK, and JNK and the expression levels of these phosphorylated proteins is shown. FIG. 22 is a graph comparing the degree of inhibitory action of NF-κB cell signal transmission system of crude fraction “C2” of Nememebiwa tea in Example 14 with LPS. Specifically, a diagram of electrophoresis of the concentration of IκB, IKK, and TAK1 expression genes and the expression level of those phosphorylated proteins is shown. FIG. 23 is a diagram comparing the degree of inhibitory action of the crude fraction “C” of Nemebiwa tea in Example 14 with the LPS induced by LPS. Specifically, a diagram of electrophoresis of the concentration of IκB, IKK, and TAK1 expression genes and the expression level of those phosphorylated proteins is shown. 24 is a graph comparing prostaglandin E2 production amounts of crude fractions “A” to “D” of Nememebiwa tea in Example 15. FIG. 25 is a graph comparing prostaglandin E2 production amounts of crude fractions “C” and “C2” of Nemebiwa tea in Example 16. FIG. FIG. 26 shows a photograph in which the extract of Nemebiwa tea improved foot edema in ICR mice in Example 17. Ctl is a control, LPS is a group that did not give Nemebiwa tea, and Biwa + LPS is a photo of ICR mice that received LPS treatment and gave Nemebiwa tea. FIG. 27 shows the content of relative phenolic components contained in the powder of Nemebiwa tea extract “M” and crude fractions “A to D” in Example 18. FIG. 28 shows the content of relative phenolic components contained in the powder of crude fraction / fraction “C1 to C9” of Nemebiwa tea in Example 18. FIG. 29 shows the results of analyzing the functional components of the extract of Nemebiwa tea and loquat leaf in Example 19 using high performance liquid chromatography (HPLC).

  Hereinafter, the present invention will be described in detail.

The food / beverage product or pharmaceutical product according to the present invention contains an extract or purified product of loquat leaf or loquat tea as an active ingredient, and has a lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE ₂ production. It has one or more actions selected from the group consisting of synthesis inhibitory action (so-called anti-inflammatory action). Metabolic syndrome such as diabetes can be prevented or treated by ingesting or administering the food or drink or pharmaceutical product according to the present invention to an animal such as a human. In addition, by ingesting or administering the food or drink or pharmaceutical product according to the present invention to an animal such as a human, production of COX-2 and PGE2 can be suppressed and inflammation can be suppressed.

  Here, the lipase inhibitory action means reducing the decomposition and absorption of ingested neutral fat. In addition, the α-glucosidase inhibitory action means that decomposition and absorption of ingested saccharides to monosaccharides are suppressed.

COX-2 inhibitory action and is meant to suppress the synthesis of PGE _2. Moreover, iNOS inhibitory action means suppressing synthesis of nitric oxide (NO). Furthermore, the inhibitory action of PGE ₂ and NO production means suppression of inflammation.

  Inhibiting the production of cytokines (IL-5, IL-6, IL-12, GM-CSF, MCP-1, TNF-α, etc.) means suppressing inflammation.

  Inducing inflammation with LPS means inducing inflammation.

  In the present invention, loquat leaves or loquat tea is used. As loquat leaves, fresh leaves or dried leaves can be used as they are. As the loquat tea, any loquat tea may be used, for example, Nemebiwa tea (trade name). Nemebiwa tea is scorched by high-temperature heating with tourmaline stone, and can be prepared by the method described in Patent Document 2 described above. Nemebiwa tea (Neguro tea) is commercially available from Totsugawa Farm, Kagoshima Agricultural Production Corporation.

  The extract used for the food or drink or pharmaceutical product according to the present invention can be obtained by subjecting loquat leaves or loquat tea to hot water extraction or solvent extraction. For example, loquat leaves or loquat tea is subjected to extraction using hot water, and this is repeated once or several times (for example, 3 times) to obtain loquat leaves or loquat tea extracts. Alternatively, for example, loquat leaves or loquat tea can be obtained by subjecting loquat leaves or loquat tea to extraction using water, an organic solvent such as lower alcohol or acetone. In the present invention, the loquat leaf or loquat tea extract means an extract or various solvent extracts obtained by the above extraction method, a diluted solution thereof, a concentrated solution thereof or a dry powder thereof.

  In the present invention, the above-described loquat leaf or loquat tea extract is subjected to purification means such as filtration, centrifugation, or purification treatment, whereby a purified product from which impurities are removed can be used. Examples of the purification means include column chromatography, normal phase or reverse phase chromatography, ion exchange chromatography, and gel filtration, and column chromatography is particularly preferable.

For example, water extraction solution or hot water extract (concentrate or dry powder) of loquat leaf or loquat tea directly, open column chromatography of gel for reverse phase (for example, MCI gel CHP-20P (Mitsubishi Chemical) etc.) Attached. Use water as liquid A and ethanol as liquid B in the mobile phase, and change the ratio of liquid A and liquid B to 100: 0, 30:70 (70% ethanol aqueous solution), and 70:30 (30% ethanol aqueous solution). Perform chromatography. Alternatively, chromatography is performed using acetone as the liquid C and the ratio of the liquid A and the liquid C being 50:50. A crude fraction (e.g., "Fraction B" in the Examples below) that elutes at a ratio of A to B of 70:30 (30% aqueous ethanol), and the ratio of A to B is 30 : A crude fraction eluted at 70 (70% ethanol aqueous solution) (for example, “Fraction C” in the Examples below) and a crude fraction eluted at a ratio of A liquid to C liquid of 50:50 ( For example, “Fraction D” in the Examples below shows a strong lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE ₂ biosynthesis inhibitory action (so-called anti-inflammatory action). . Therefore, these crude fractions can be suitably used for foods and beverages or pharmaceuticals according to the present invention as purified products of loquat leaves or loquat tea. Thus, for example, using a 30% to 70% aqueous ethanol solution (for example, 30%, 50% or 70% aqueous ethanol solution), a mixed solution containing water and acetone at a ratio of 1: 1, and the like, the eluted fraction The product can be preferably used as a purified product of loquat leaf or loquat tea in a food or drink or a pharmaceutical product according to the present invention.

Alternatively, for example, as described above, loquat leaves or purified loquat tea (fraction) eluted with 50 to 70% ethanol aqueous solution (for example, 50% or 70% ethanol aqueous solution) in column chromatography is removed with a vacuum evaporator. After leaving, the residue is again subjected to open column chromatography on a reverse phase gel (eg, ODS column). Chromatography is performed with a linear gradient from liquid A to liquid B, using water as liquid A and methanol as liquid B in the mobile phase. The crude fraction eluted by this method shows a strong lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE ₂ biosynthesis inhibitory action (so-called anti-inflammatory action). Thus, for example, fractions eluted using water and methanol in a ratio of 80%: 20%, 70%: 30% or 50%: 50% (for example, the following examples) "Fraction C1", "Fraction C2", and "Fraction C4") can be suitably used in the food and drink or the pharmaceutical product according to the present invention as a purified product of loquat leaf or loquat tea.

  By using the loquat leaf or loquat tea extract or purified product described above as an active ingredient, the food or drink or pharmaceutical product according to the present invention can be produced. The food or drink or pharmaceutical product according to the present invention has at least one action selected from the group consisting of a lipase inhibitory action, an α-glucosidase inhibitory action and an anti-inflammatory action as the action of the loquat leaf or loquat tea extract or purified product contained therein. Can be attached.

  The effective amount of the extract or purified product of the present invention is added or enclosed in any form such as tablets, capsules, granules, drinks, PET bottles, etc. Goods can be obtained. The food and drink according to the present invention is a food or drink having a lipase inhibitory action, an α-glucosidase inhibitory action, a COX-2 inhibitory action and / or a PGE2 biosynthesis inhibitory action (so-called anti-inflammatory action), particularly a health supplement or specified health It can be used as a food product. Preferably, it is a health supplement or specific health food in the form of tablets, capsules, granules, drinks, PET bottles and the like.

  Examples of the food and drink include, but are not limited to, confectionery, retort food, juices, teas, and dairy products. In addition, sweeteners, seasonings, emulsifiers, suspending agents, preservatives, etc. may be added to foods and drinks as necessary, or vitamins, nutrients, immune enhancers (for example, propolis or Mushroom extract etc.) may be added.

  The amount of loquat leaf or loquat tea extract or purified product added to the food or drink product according to the present invention is an amount within a range corresponding to 0.1 to 200 mg per kg of adult body weight to be ingested or 50 mg to 1 g per product, for example. It is not limited to this range.

  On the other hand, the pharmaceutical product according to the present invention includes an effective amount of an extract or purified product of loquat leaf or loquat tea. The pharmaceutical agent according to the present invention can be used as a lipase inhibitor, α-glucosidase inhibitor, COX-2 inhibitor and / or PGE2 biosynthesis inhibitor (so-called anti-inflammatory agent or anti-inflammatory agent).

  In addition to the loquat leaf or loquat tea extract or purified product, the pharmaceutical product according to the present invention further includes a pharmaceutically acceptable carrier (excipient or diluent), a binder, a bulking agent, a lubricant, and a disintegrant. , Wetting agents, emulsifying agents, buffering agents, suspending agents, preservatives, coloring agents, flavoring agents, sweetening agents and the like may be added as appropriate. Carriers and additives that are commonly used for formulation can be used in the production of the pharmaceutical product according to the present invention. For example, examples of the binder include starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose and the like. Examples of the bulking agent include lactose and microcrystalline cellulose. Examples of lubricants include talc, silica, magnesium stearate and the like. Examples of the disintegrant include starch and sodium starch glycolate. Examples of the wetting agent include sodium lauryl sulfate. Examples of emulsifiers include cellulose derivatives and sorbitol. Examples of preservatives include methyl-p-hydroxybenzoate and sorbic acid. However, the additives that can be used in the present invention are not limited to the examples of these additives.

  The pharmaceutical product according to the present invention is formulated for oral administration or parenteral administration (intravenous, intraarterial, intraperitoneal, intrarectal, subcutaneous, intramuscular, sublingual, intranasal, intravaginal, etc.). obtain. Although it does not specifically limit as a form of a formulation, For example, a solution agent, a tablet, a powder agent, a granule, a capsule, a suppository, a spray, a controlled release agent, a suspension agent, a drink agent etc. are mentioned.

  The dose of loquat leaf or loquat tea extract or purified product contained in the pharmaceutical product according to the present invention can vary depending on factors such as the age, weight, sex, condition, severity, etc. of the patient. The daily dose of loquat leaf or loquat tea extract or purified product administered to a patient is, for example, in the range of 0.1 to 200 mg, preferably 1 to 100 mg per kg of the patient's body weight, but is not limited to this range. If necessary, the dose may be divided and administered in several divided doses, for example, 2 to 3 times. In addition, the medicament according to the present invention may be used in combination with other lipase inhibitors, α-glucosidase inhibitors, COX-2 inhibitors and / or PGE2 biosynthesis inhibitors (so-called anti-inflammatory agents) having the same or different therapeutic uses. It can also be administered to a patient.

  The food / beverage products or pharmaceutical products according to the present invention can be subjected to pharmacological evaluation as follows, for example.

  As the pharmacological evaluation of the anti-inflammatory action of the food or drink or medicine according to the present invention, for example, the model animal is allowed to ingest the food or drink or medicine according to the present invention, and the degree of occurrence of LPS-induced foot edema during or after breeding A method of comparison by measuring the circumference of the foot is mentioned. When the edema that occurs in the paw is significantly reduced in the animal that has taken the food or drink or medicine according to the present invention, compared with the animal that has not taken the food or drink or medicine according to the present invention, the present invention It can be judged that the food / beverage products or pharmaceutical products have an anti-inflammatory effect. In addition, examples of the pharmacological evaluation of the anti-inflammatory action of the food or drink or pharmaceutical product according to the present invention include a method using mouse macrophage-like cells (RAW264). That is, by culturing RAW264 cells in the absence of the food or drink or pharmaceutical product according to the present invention, compared to the inflammation onset group (control group) in which inflammation was induced by LPS, in the presence of the food or food product or pharmaceutical product according to the present invention In an inflammation suppression group in which RAW264 cells are cultured and inflammation is induced by LPS, when the expression of inflammation is suppressed, it can be determined that the food or drink or the pharmaceutical product according to the present invention has a good anti-inflammatory effect . As an evaluation method for knowing the degree of inflammation suppression in this case, quantification of COX-2 and iNOS proteins in RAW264 cells, quantification of PGE2, cytokines (IL-5, IL-6, IL-12, GM- Quantification of CSF, MCP-1, TNF-α, etc., and measurement of the expression of MAPK and NF-κB cell signaling systems and the amount of phosphorylated protein produced by these genes Can know by.

  As a pharmacological evaluation of the lipase inhibitory action of foods and drinks or pharmaceuticals according to the present invention, for example, the food or foods or pharmaceuticals according to the present invention are ingested for the prevention of high blood fat, blood glucose in the blood after a certain period (1-2 months) The method of comparing by measuring a value and neutral fat is mentioned. Compared with high-fat food animals that have not ingested (administered) the food or drink or pharmaceutical product according to the present invention, in the animals that have ingested (administered) the food or food product or pharmaceutical product of the present invention, neutral fat is significantly reduced. In that case, it can be determined that the food or drink or pharmaceutical product according to the present invention has a lipase inhibitory action. Alternatively, according to the method described in Nakai, M., et al., J. Agric. Food Chem., 53 (11), 4593-4598, 2005, that is, in vitro, the food or drink or pharmaceutical product according to the present invention In the present invention, the fluorescence emission of 4-methylumbelliferone (4-MU) produced by degradation of the substrate 4-methylumbelliferyl oleate (4-MUO) by lipase in the presence is measured, and the lipase activity inhibition rate is calculated from the measured value. The lipase inhibitory action of the food / beverage products or pharmaceutical products can be evaluated.

  As the pharmacological evaluation of the α-glucosidase inhibitory action of the food or drink or pharmaceutical product according to the present invention, for example, the food or food product or pharmaceutical product according to the present invention is ingested for the prevention of hyperglycemia, and comparison is made by measuring the blood glucose level after breeding. The method of doing is mentioned. Compared with animals that have not ingested (administered) the food or drink according to the present invention, in animals that have ingested (administered) the food or drink according to the present invention, when hyperglycemia is significantly reduced, It can be determined that the food or drink or the pharmaceutical product according to the present invention has an α-glucosidase inhibitory action. Alternatively, according to the method described in Miwa I., et al., Clin. Chem. Acta, 37, 219-224, 1972, ie, using maltose (maltose) or saccharose as a substrate, the present invention is applied in vitro. The degree of α-glucosidase inhibitory action in the presence of a food or drink according to the present invention can be calculated using the amount of glucose produced as an index, and the α-glucosidase inhibitory action of the food or drink according to the present invention can be evaluated.

In roasted loquat leaves and non-roasted loquat leaves, lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE ₂ biosynthesis inhibitory action (so-called anti-inflammatory action) are not observed at all. On the other hand, in the present invention, by using an extract or purified product of loquat leaf or loquat tea, lipase inhibitory action, α-glucosidase inhibitory action, COX-2 inhibitory action and / or PGE2 biosynthesis inhibitory action (so-called anti-inflammatory) It is possible to produce foods and beverages or pharmaceuticals having an action. These foods and drinks or pharmaceuticals are useful for the prevention or treatment of diseases such as diabetes and cancer.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

  Note that Nemebiwa tea used in the following examples is commercially available from Totsukawa Farm, Kagoshima Agricultural Production Corporation.

  In addition, the results in the figure accompanying each example are shown as an average value or an average value ± standard deviation of each group, and an asterisk mark (*) is 1% or less for statistically significant 5% or less and a significant difference. Those with significant difference are marked with a double asterisk (**).

[Example 1] Antioxidative effect of extract of Nemebiwa tea (1) Preparation of extract and fractions "AD" of Nemebiwa tea Extraction method of fraction from commercially available Nemebiwa tea Was performed using the method described in the following patent document (Japanese Patent No. 4974116).

  Commercial Nemebiwa tea (about 1 kg) was repeatedly extracted with hot water. Specifically, commercially available Nemebiwa tea leaves were boiled in about 400 ml of hot water for about 100 g every 15 minutes and extracted in 10 portions. The obtained hot water extract was cooled and sequentially subjected to open column chromatography of MCI gel CHP-20P. The amount of gel is preferably about 1 kg as a dry weight. The glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass, acrylic material, or the like. The solvent is eluted using water (2L), 30% aqueous ethanol (2L), 70% aqueous ethanol (2L) and water-acetone (1: 1 ratio) (2L) to obtain four fractions. It was. The dried crude fraction eluted with water (2 L) is referred to as fraction “A”. The dried crude fraction eluted with 30% aqueous ethanol (2 L) is referred to as fraction “B”. The dry crude fraction (hereinafter referred to as fraction “C”) eluted with 70% aqueous ethanol (2 L) was 13.18 g. The dry crude fraction eluted with water-acetone (1: 1 ratio) (2 L) is referred to as fraction “D”.

  As a method of obtaining the fraction “C”, after cooling the hot water extract extracted by boiling for 15 minutes with the hot water described above, if a precipitate is formed, the supernatant is treated with MCI gel CHP. It was obtained by -20P open column chromatography. In addition, in order to obtain fraction C by subjecting to MCI gel CHP-20P open column chromatography, the fraction can be separated by elution with 50% aqueous ethanol (2L) instead of elution with 70% aqueous ethanol (2L). Picture C was obtained.

  This “Fraction A, B, C, D” can be stored at −20 ° C. in a freezer or the like.

  In the following examples, in experiments conducted using fractions “A to D” (that is, other than fraction “C”, which is the starting material for the preparation of fractions “C1 to C9” in Example 2), Fraction “C” obtained by elution with 50% ethanol was used.

(2) Experiment on Antioxidant Action of Extract and Fraction “A to D” of Nemebiwa Tea Antioxidant experiment was conducted using the method described in the following literature (Rabah, IO, et al , J. Agric. Food Chem., 52, 7152-7157, 2004).

  The sample was prepared by adjusting the four crude fractions of fractions “A to D” obtained in (1) above at a concentration of 50 μg / ml. Was adjusted to a concentration of 50 μg / ml. Add these samples to a flat-bottomed plate (96-well), add 190 μL of DPPH (1,1-diphenyl-2-picrylhydrazyl, adjusted to the concentration of micromolar), mix for 10 seconds, and then shield from light. Left for a minute. After this reaction, the reaction solution in each well was measured with a microplate reader using the absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using the Trolox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from the Trolox radical scavenging ability standard curve.

  The experimental results of the antioxidant action are shown in FIG. In FIG. 1, sample symbols A to D correspond to the respective crude fractions “A to D”. Further, the symbol “M” indicates the hot water extract “M” of Nemebiwa tea.

  As shown in FIG. 1, it can be seen that the crude fraction “C” has a strong antioxidant effect. It can also be seen that the antioxidant effect of the crude fraction “C” is about 1.5 times stronger than the hot water extract “M” of Nemebiwa tea.

[Example 2] Effect of antioxidant action of crude fraction of Nemebiwa tea (1) Preparation of crude fraction and fraction "C1 to C9" of Nemebiwa tea Crude from commercially available Nejimebiwa tea The method for producing the fraction was performed using the method described in the following patent document (Japanese Patent No. 4974116).

The dry crude fraction / fraction “C” (13.18 g) obtained in “Example 1” was subjected to ODS column chromatography. The fraction “C” is the fraction “C” eluted in Example 1 with a 70% aqueous ethanol solution. The amount of gel is preferably about 1 kg as a dry weight. The glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass, acrylic material, or the like. The conditions of ODS open column chromatography were as follows: Chromatolex ODS (Chromatorex ODS, Fuji Silysia Chemical LTD.) Was used for the stationary phase, and distilled water (H ₂ O), B as liquid A was used for the moving bed. Methanol (MeOH) was used as a liquid, and various chromatographies were performed by increasing the content of B liquid from A liquid. Specifically, as a solvent, 80% H ₂ O-20% MeOH (1 L), 70% H ₂ O-30% MeOH (1 L), 60% H ₂ O-40% MeOH (1 L), 50% H ₂ O-50% MeOH (1 L), 40% H ₂ O-60% MeOH (1 L), 30% H ₂ O-70% MeOH (1 L), 20% H ₂ O-80% MeOH (1 L), 10 Elution was performed using 1% H ₂ O-90% MeOH (1 L) and 100% MeOH (1 L), and each 1 L elution fraction was fractionated, and a total of 9 fractions designated “C1” to “C9”, respectively. A picture was obtained. As a result, the yield of the nine crude fractions referred to as crude fractions `` C1 to C9 '' is 540 mg of crude fraction `` C1 '', 570 mg of crude fraction `` C2 '', 720 mg of crude fraction `` C3 '', 630 mg of crude fraction “C4”, 420 mg of crude fraction “C5”, 490 mg of crude fraction “C6”, 1.71 g of crude fraction “C7”, 1.78 g of crude fraction “C8”, crude fraction “C9” was 3.33 g.

  The crude fraction “C1 to C9” can be stored at −20 ° C. in a freezer or the like.

Of these nine crude fractions, TLC (thin layer chromatography) of the crude fractions “C1 to C9” is shown in FIG. 2 and FIG. 2 and 3, numbers 1 to 9 indicate the results of the crude fractions “C1 to C9”, respectively. As the developing solvent used for TLC, four solvents of benzene-ethyl formate-formic acid-water were mixed at a ratio of 4: 31: 5: 1. As the coloring reagent, a compound in which sulfuric acid (H ₂ SO ₄ ) was dissolved in about 5 to 10% water was used, and the compound was color-detected by spraying directly on TLC. As shown in FIG. 2, it can be seen that what appears to be shaded by UV irradiation at 252 nm is a phenolic substance. Moreover, as shown in FIG. 3, it turns out that the substance which develops color from brown to light blue by the heating after spraying of 10% sulfuric acid aqueous solution is contained.

(2) Antioxidant action experiment of crude fraction and fraction “C1 to C9” of Nemebiwa tea Antioxidant experiment was carried out using the method described in the following literature (Rabah, IO, et al., J. Agric. Food Chem., 52, 7152-7157, 2004).

  The sample was prepared by adjusting 9 crude fractions of fractions “C1 to C9” obtained in (1) above at a concentration of 10 μg / ml. Was adjusted at a concentration of 10 μg / ml.

  The antioxidant action experiment was performed in the same manner as in Example 1.

  The experimental results of the antioxidant action are shown in FIG. In FIG. 4, sample numbers 1 to 9 correspond to the respective crude fractions “C1 to C9”. "C" indicates Nememebiwa tea extract / fraction "C".

  As shown in FIG. 4, it can be seen that the crude fractions “C1”, “C2” and “C4” have a strong antioxidant effect. It can also be seen that the crude fractions “C1”, “C2” and “C4” have an antioxidant effect that is about 1.4 times stronger than the extract / fraction “C” of Nemebiwa tea.

[Example 3] Effect of lipase inhibitory action of extract and fraction "A to D" of Nemebiwa tea The experiment of lipase inhibitory action was performed using the method described in the following literature (Nakai, M. , et al., J. Agric. Food Chem., 53 (11), 4593-4598, 2005).

For the sample, four crude fractions of Nemebiwa tea fractions “A to D” obtained in “Example 1” were used, and the degree of lipase inhibitory activity was adjusted to a concentration of IC ₅₀ (mg / ml). Calculated. As a control, hot water extract “M” of Nemebiwacha was used.

  In the lipase activity measurement, the fluorescence emission of 4-methylumbelliferone (4-MU) produced by the decomposition of the substrate 4-methylumbelliferyl oleate (4-MUO) by lipase is measured, and the lipase activity inhibition rate is calculated from the value. The experiment was performed as follows. To 96 wells (black), 50 μl of Biwa tea sample and 20 μl of 4-MUO solution were added and mixed for 1 minute, and then 25 μl of lipase solution was added and mixed for 30 seconds. After incubating at 37 ° C. for 30 minutes, 100 μl of 0.1M sodium citrate was added to stop the reaction, and fluorescence was measured at 355 nm (excitation wavelength) and 460 nm (emission wavelength) using a fluorescence multilabel counter. The lipase activity inhibition rate is calculated from the value.

  The experimental results of the lipase inhibitory action are shown in FIG. In FIG. 5, sample symbols A to D correspond to the respective crude fractions “A to D”. Further, the symbol “M” indicates the hot water extract “M” of Nemebiwa tea.

  As shown in FIG. 5, it can be seen that the crude fraction “C” has a strong lipase inhibitory action. It can also be seen that the lipase inhibitory action of the crude fraction “C” is about 2.5 times stronger than the hot water extract “M” of Nemebiwa tea.

[Example 4] Effect of lipase inhibitory action of crude fraction and fraction "C1 to C9" of Nemebiwa tea The experiment of lipase inhibitory action was carried out using the method described in the following literature (Nakai, M., et al., J. Agric. Food Chem., 53 (11), 4593-4598, 2005).

For the sample, nine crude fractions of Nemebiwa tea crude fractions and fractions “C1 to C9” obtained in “Example 2” were used to determine the degree of lipase inhibitory action and the concentration of IC ₅₀ ( mg / ml). As a control, extract and fraction “C” of Nemebiwa tea was used.

  The lipase inhibitory action was performed in the same manner as in Example 3.

  The experimental results of the lipase inhibitory action are shown in FIG. In FIG. 6, sample numbers 1 to 9 correspond to the respective crude fractions “C1 to C9”. "C" indicates Nememebiwa tea extract / fraction "C".

  As shown in FIG. 6, it can be seen that the crude fraction “C2” has a strong lipase inhibitory action. The lipase inhibitory action of the crude fraction “C2” was equivalent to the extract / fraction “C” of Nemebiwa tea.

[Example 5] Effect of α-glucosidase inhibitory action of extract and fraction “A to D” of Nemebiwa tea The experiment of α-glucosidase inhibitory action was performed using the method described in the following literature ( Miwa I., et al., Clin. Chem. Acta, 37, 219-224, 1972).

As a sample, four crude fractions of Nemebiwa tea fractions “A to D” obtained in “Example 1” were used to determine the degree of α-glucosidase inhibitory action and the concentration of IC ₅₀ (mg / ml). ). As a control, hot water extract “M” of Nemebiwacha was used. Maltose (maltose) and saccharose were used as substrates.

  In the experiment, glucose CII test Wako (Wako Pure Chemical Industries) was used to measure the glucose calibration curve and the α-glucosidase inhibitory action of Biwa tea. 10 μl each of substrate, crude enzyme solution and Biwa tea sample were added to the microtube and reacted at 37 ° C. for 60 minutes. Then, heat the reaction at 100 ° C for 10 minutes to stop the reaction, add 1.1 ml of color reagent and 7.33 μl of sample to the microtube, mix well, react at 37 ° C for 5 minutes, and measure with a spectrophotometer at 505 nm did. The inhibition rate of α-glucosidase activity was calculated from the decrease in the amount of glucose produced by subtracting a blank from the Biwa tea sample.

  The experimental results of the α-glucosidase inhibitory action are shown in FIG. In FIG. 7, sample symbols A to D correspond to the respective crude fractions “A to D”. Further, the symbol “M” indicates the hot water extract “M” of Nemebiwa tea.

  As shown in FIG. 7, it can be seen that the crude fraction “C” has a very strong α-glucosidase inhibitory action.

[Example 6] Effect of α-glucosidase inhibitory action of crude fraction and fraction “C1 to C4” of Nemebiwa tea An experiment of α-glucosidase inhibitory action was performed using the method described in the following literature. (Miwa I., et al., Clin. Chem. Acta, 37, 219-224, 1972).

For the sample, the crude fraction of Nemebiwa tea obtained in “Example 2” and the four crude fractions of fractions “C1 to C4” were used to determine the degree of α-glucosidase inhibitory activity of IC ₅₀ Calculated as concentration (mg / ml). As a control, extract and fraction “C” of Nemebiwa tea was used. Maltose (maltose) and saccharose were used as substrates.

  The experiment of α-glucosidase inhibitory action was performed in the same manner as in Example 5.

  The experimental results of the α-glucosidase inhibitory action are shown in FIG. In FIG. 8, sample numbers 1 to 4 correspond to the respective crude fractions “C1 to C4”. "C" indicates Nememebiwa tea extract / fraction "C".

  As shown in FIG. 8, it can be seen that the crude fraction “C2” has a relatively strong α-glucosidase inhibitory action.

[Example 7] Effect of inhibitory action on nitric oxide production of extract and fraction "A to D" of Nemebiwa tea The experiment described in the following literature used the experiment of the inhibitory action on nitric oxide production. (Archer, S. FASEB J., 7, 349-360, 1993).

  Samples were obtained by using LPS to induce the inhibition of nitric oxide production using the four crude fractions of Nemebiwa tea fractions “AD” obtained in “Example 1”. As 100%, the degree was compared as a percentage. As a control, hot water extract “M” of Nemebiwacha was used.

Nitric oxide was measured by the Griess method. The Griess method is a method for detecting a diazonium chloride compound caused by NO ₂ generated by oxidation of NO using azo coupling of naphthylethylenediamine. More specifically, 100 μl each of the medium supernatant of macrophage cells (RAW264.7) co-cultured with loquat tea was dispensed into a 96-well plate, 100 μl of Griess reagent was added, and the reaction was allowed to proceed at room temperature for 10 minutes. Thereafter, measurement was performed at 550 nm with a myoplate reader, and the amount of NO produced according to the intensity of absorbance was converted.

  The experimental results of the inhibitory effect on nitric oxide production are shown in FIG. In FIG. 9, sample symbols A to D correspond to the respective crude fractions “A to D”. Further, the symbol “M” indicates the hot water extract “M” of Nemebiwa tea.

  As shown in FIG. 9, it can be seen that the crude fraction “C” has an effect of suppressing LPS-induced nitric oxide production by about 70%.

[Example 8] Effect of inhibitory action of nitric oxide production on crude fraction and fraction "C2" of Nemebiwa tea For the experiment of inhibitory action on nitric oxide production, the method described in the following literature was used. (Archer, S. FASEB J., 7, 349-360, 1993).

  The sample was a crude fraction of Nemebiwa tea obtained in "Example 2" and a crude fraction of fraction "C2", and the degree of inhibition of nitric oxide production was induced with LPS. As a percentage, the degree was compared as a percentage. As a control, extract and fraction “C” of Nemebiwa tea was used.

  Experiments on the inhibitory effect on nitric oxide production were carried out in the same manner as in Example 7.

  The experimental results of the inhibitory effect on nitric oxide production are shown in FIG. In FIG. 10, the crude fraction and fraction “C2” of Biwa tea are shown as white columns, and the extract and fraction “C” are shown as black columns.

  As shown in FIG. 10, it can be seen that the extract / fraction “C” and the crude fraction “C2” have the effect of suppressing nitric oxide production induced by LPS in a concentration-dependent manner to the same extent as the control. .

[Example 9] Method for investigating anti-inflammatory effect The experimental method for investigating the anti-inflammatory effect was carried out using the method described in the following patent document (JP 2012-56952 A).

[Experiment 1] Cell culture method (1) Cell types
Mouse macrophage-like cells (Cell No. RCB0535, hereinafter referred to as “RAW264”) obtained from RIKEN CELL BANK were used.

(2) Preparation of medium
9.5 g of D-MEM (Dulbecco's Modified Eagle Medium “Nissui”) was dissolved in 1 L of water (H ₂ O) and sterilized at high temperature and pressure in an autoclave. After cooling, 15 ml of a 10% NaHCO ₃ solution was added, and 10 mL of an antibiotic mixed reagent (PSG [penicillin, streptomycin, glutamin]: Gibco 100 mL Lot No. 1185891) was added. FBS (Fetal Bovine Serum: Equitech-Bio 500 mL Lot No. SFB30-1463) solution was added to the prepared solution at a rate of 10% to prepare a medium.

(3) Cell Passage RAW264 cells previously stored in a freezer (-80 ° C.) were thawed, 10 mL of the adjusted medium (hereinafter referred to as “medium”) was added and mixed with the cells. Lightly mixed, centrifuged (1000 rpm, 5 min), the supernatant was discarded, and this operation was repeated twice. The supernatant was removed, 1 mL of trypsin (Trypsin-EDTA: Gibco 100 mL) was added, 9 mL of medium was added and stirred, and then centrifuged (1000 rpm, 5 min), the supernatant medium was removed, and 10 mL of medium was added. The medium containing the cells was placed in a 10 cm petri dish. The concentration of RAW264 cells in each dish was adjusted to about 1 to ³ × 10 ⁵ cells. The cells were cultured in an incubator (hereinafter referred to as a “CO ₂ incubator”) in which 5% carbon dioxide (CO ₂ ) was mixed in an air stream at 37 ° C.

[Experiment 2] Induction method of COX-2 in cells (1) Culture of control cells In a petri dish (6 mL size), the concentration of RAW264 cells was adjusted to 1 × 10 ⁶ cells. This was cultured in advance at 37 ° C. for 21 hours in a CO ₂ incubator. After the culture, the cells were placed on a petri dish (3 mL size). At this time, no FBS solution was added to the medium. Subsequently, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 hours. After culturing, lipopolysaccharide adjusted to 5 ng / μL (hereinafter referred to as “LPS”) was added to a 24 μL medium and cultured in a CO ₂ incubator at 37 ° C. for 12 hours. By this method, in addition to COX-2, the induction of COX-1 and iNOS was also investigated.

(2) Preparation of test compound Biwa tea extract and crude fraction / fraction were dissolved at a concentration of 12.5 to 100 μM. Water (H ₂ O) and dimethyl sulfoxide (hereinafter referred to as “DMSO”) were used as the solvent. Hereinafter, the sample adjusted to this concentration is referred to as “test compound”.

(3) Culture of test cells In a petri dish (6 mL size), the concentration of RAW264 cells was adjusted to 1 × 10 ⁶ cells. This was cultured in advance at 37 ° C. for 21 hours in a CO ₂ incubator. After culturing, the cells were placed in a petri dish (3 mL size). At this time, no FBS solution was added to the medium. Subsequently, the cells were cultured in a CO ₂ incubator at 37 ° C. for 2.5 hours. After culturing, the prepared test compound was added and cultured in a CO ₂ incubator at 37 ° C. for 30 minutes. To this, a lipopolysaccharide adjusted to 5 ng / μL (hereinafter referred to as “LPS”) was added to a 24 μL medium and cultured in a CO ₂ incubator at 37 ° C. for 12 hours. By this method, in addition to COX-2, the induction of COX-1 and iNOS was also investigated.

[Experiment 3] Recovery and detection method of COX-2 protein in cells (1) Recovery of COX-2 protein The cells cultured in Experiment 2 (1) and (3) were recovered. The collected cells were washed with PBS (KCl: 0.02%, KH ₂ PO ₄ : 0.02%, NaHPO ₄ · 12H ₂ O: 0.29%, NaCl: 0.8% aqueous solution), and then SDS reagent (1M Tris-HCl 20 mL of 6.8 mL of pH6.8, 0.4 g of SDS (2% w / v), 2 mL of Glycerol (10%), 1 mL of 1M DTT (50 mM), 0.02 g of bromophenol blue (0.1%) was added. The protein in the cells was recovered by boiling at 100 ° C. for 5 minutes.

(2) Detection of COX-2 protein The protein solution recovered in (1) above is 10 μL for examining the expressed COX-2 protein, 30 μL for examining the expressed COX-1 protein, and expressed iNOS. In order to examine the protein, 37 μL was adsorbed to each electrophoresis well. Electrophoresis was performed at a constant current. After activating the membrane and washing it well, it was immersed in a transfer buffer, and the gel plate and filter paper were mounted together and transferred at a constant current. The recovered membrane was subjected to Western blot, the antibody reaction was repeated, and the protein was detected with a chemiluminescence detector. The electrophoretic image was preserve | saved using Lumi Vision Analyzer 140 about the detected protein.

[Example 10] Effect of inhibitory action on expression of COX-2 and iNOS of extract and fraction "AD" of Nemebiwa tea
Experiments on the COX-2 and iNOS expression-suppressing action were performed using the methods described in the following patent documents (Japanese Patent Laid-Open No. 2012-56952).

  The sample was obtained by using the four crude fractions of Nemebiwa tea fractions “A to D” obtained in “Example 1”, and the degree of COX-2 and iNOS expression suppression action by LPS and COX- The expression of 2 and iNOS expression were compared with the induced electrophoretic figures. As a control, hot water extract “M” of Nemebiwacha was used. In addition, the concentration-dependent suppression of COX-2 and iNOS expression was investigated for the fraction "C" of Nemebiwa tea.

  FIG. 11 shows the experimental results of COX-2 and iNOS expression inhibitory action. In FIG. 11, sample symbols A to D correspond to the respective crude fractions “A to D”. Further, the symbol “M” indicates the hot water extract “M” of Nemebiwa tea. Each experiment was conducted at a concentration of 200 μg / ml. LPS induced inflammation at 40 ng / ml.

  As shown in FIG. 11, it can be seen that the crude fractions “C” and “D” have an effect of suppressing the expression of COX-2 and iNOS induced by LPS.

  FIG. 12 shows the experimental results of the concentration-dependent COX-2 and iNOS expression-suppressing action of the crude fraction “C” of Biwa tea. It can be seen that increasing the concentration of the crude fraction “C” of Biwa tea to 50, 100, and 200 μg / ml increases the suppression of COX-2 and iNOS expression.

[Example 11] Effect of inhibitory action of COX-2 and iNOS expression of crude fraction and fraction "C2" of Nememebiwa tea
Experiments on the COX-2 and iNOS expression-suppressing action were performed using the methods described in the following patent documents (Japanese Patent Laid-Open No. 2012-56952).

  Using the crude fraction of Nemebiwa tea obtained in “Example 2” and the crude fraction of fraction “C2”, the degree of COX-2 and iNOS expression suppression effect was induced by LPS. The percentage was compared as a percentage. Specifically, the concentration-dependent suppression of COX-2 and iNOS expression was investigated for the crude fraction and fraction “C2” of Nemebiwa tea.

  FIG. 13 shows the concentration-dependent experimental results of COX-2 and iNOS expression inhibitory action. In FIG. 13, the sample symbol “C2” corresponds to the crude fraction “C2”, and experiments were performed at concentrations of 50, 100, and 200 μg / ml. LPS induced inflammation at 40 ng / ml.

  It can be seen that increasing the concentration of crude fraction “C2” of Biwa tea to 50, 100, 200 μg / ml increases the suppression of COX-2 and iNOS expression (FIG. 13).

[Example 12] Effect of suppressing the cytokine production of the crude fraction of nejimebiwa tea / fraction "C2" The cytokine production inhibitory experiment was carried out using a mouse-cytokine assay kit manufactured by Bio-Rad. I went by instrument.

  Samples were obtained by using LPS to induce the suppression of cytokine production using the crude fraction of Nemebiwa tea obtained in “Example 2” and the crude fraction of fraction “C2”. Comparison was made as the concentration of production. Specifically, the effect of inhibiting the production of cytokines in a concentration-dependent manner was investigated for the crude fraction / crop “C2” of Nemebiwa tea.

Cytokine production inhibitory action experiments were performed with a suspension array system using a mouse cytokine assay kit from Bio-Rad. Specifically, mouse macrophage-like cells RAW264.7 (ATCC, TIB-71, USA) were seeded in a 12-well plate at a concentration of 1.2 × 10 ⁵ cells / 1 ml / well and pre-cultured for 21.5 hours. After the preculture, the medium was changed to a DMEM medium not added with FBS (fetal calf serum) and starved for 2.5 hours. Thereafter, the loquat tea fraction was added, and LPS (final concentration 40 ng / ml) was added 0.5 hours later. After culturing for 12 hours, 1 ml of the supernatant was recovered. Measurement of 23 types of mouse cytokines was performed using a measurement kit (Bio-Plex Pro Mouse Cytokine 23-Plex Panel, M60-009RDPD: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFNγ, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα) were used. In order to bind cytokine and each antibody, 50 μl of primary antibody bead solution and each Biwa tea culture supernatant (or Standard solution) are dispensed into black 96 well plates, shielded with aluminum, and shaken for 60 minutes (1100 rpm for 1 min, 300 rpm 59 min). After washing with MAG × 3 solution, 25 μl of biotinylated antibody was dispensed, mixed with light shielded with aluminum, and shaken for 30 minutes (1100 rpm 1 min, 300 rpm 29 min). After washing with MAG × 3 solution, 25 μl of streptavidin-phycoerythrin (PE) solution was dispensed, mixed with light shielding with aluminum for 10 minutes (1100 rpm 1 min, 300 rpm 9 min). After washing with MAG × 3 solution, 125 μl of Assay Buffer was added, mixed for 1 minute with vibration (1100 rpm for 1 min), and measured with a suspension array system. The absolute value of fluorescence intensity and the concentration value converted by Standard (pm / ml) were also determined from the data.

(1) About IL-5
FIG. 14 shows the concentration-dependent experimental results of IL-5 production inhibitory action. In FIG. 14, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that IL-5 production is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 14).

(2) About IL-6
The concentration-dependent experimental results of IL-6 production inhibitory action are shown in FIG. In FIG. 15, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that IL-6 production is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 15).

(3) About IL-12
FIG. 16 shows the concentration-dependent experimental results of IL-12 production inhibitory action. In FIG. 16, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that IL-12 production is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 16).

(4) About GM-CSF
FIG. 17 shows the concentration-dependent experimental results of the GM-CSF production inhibitory action. In FIG. 17, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that the production of GM-CSF is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 17).

(5) About MCP-1
FIG. 18 shows the concentration-dependent experimental results of the MCP-1 production inhibitory action. In FIG. 18, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that the production of MCP-1 is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 18).

(6) About TNF-α
FIG. 19 shows the concentration-dependent experimental results of the TNF-α production inhibitory action. In FIG. 19, the sample symbol “C2” corresponds to the crude fraction “C2”, and the sample symbol “C” corresponds to the crude fraction “C”, which is 100, 200, 200 μg / ml or more, respectively. Experimented with concentration. LPS induced inflammation at 40 ng / ml.

  It can be seen that the production of TNF-α is suppressed by increasing the concentration of the crude fraction “C2” of Biwa tea to 100, 200, 200 μg / ml or more (FIG. 19).

[Example 13] Effect of inhibitory action on crude MAPK cell signal transduction system of crude fraction and fraction "C2" of Nemebiwa tea
Experiments on the MAPK cell signal transduction system inhibitory action were performed using the method described in the following literature (Hou. DX et al., Carcinogenesis, 25: 29-36, 2004).

  The sample was obtained by using LPS to induce the degree of inhibitory action of MAPK cell signal transmission system using the crude fraction of Nemebiwa tea obtained in “Example 2” and the crude fraction of fraction “C2”. We compared the concentration of p38, ERK, and JNK expression genes with the concentration of their phosphorylated protein expression levels. Specifically, the effect of concentration-dependent suppression of the MAPK cell signal transduction system was investigated for the crude fraction and fraction “C2” of Nemebiwa tea.

  Experiments on the MAPK cell signal transduction system inhibitory action were performed using cell culture, cell protein recovery and detection methods according to [Experiment 1], [Experiment 2] and [Experiment 3] of Example 9. The detection antibodies used were ERK, JNK, p38 and their phosphorylated antibodies.

  FIG. 20 shows the results of electrophoresis concentration-dependent changes in the expression levels of p38, ERK, and JNK and the expression levels of these phosphorylated proteins. In FIG. 20, the sample symbol “C2” corresponds to the crude fraction “C2”, the sample symbol “C” corresponds to the crude fraction “C”, and the concentrations are 50, 100, and 200 μg / ml, respectively. Experimented with. LPS induced inflammation at 40 ng / ml. For comparison, an experiment of crude fraction “C” was also added. The results are shown in FIG.

  It can be seen that by increasing the concentration of the crude fraction `` C2 '' of Biwa tea to 50, 100, 200 μg / ml, the expression levels of p38, ERK, JNK and their phosphorylated proteins are suppressed ( FIG. 20). On the other hand, by increasing the concentration of the crude fraction “C” of Biwa tea to 50, 100, and 200 μg / ml, the concentration of p38, ERK, and JNK expressed genes and the amount of their phosphorylated proteins were investigated. The degree of inhibition was low compared to that of “C2” (FIG. 21).

[Example 14] Effect of inhibitory action of NF-κB cell signal transduction system of crude fraction and fraction “C2” of Nememebiwa tea
The NF-κB cell signal transduction system inhibitory action was performed using the method described in the following literature (Hou.DX et al., Biochemical Pharmacology 74: 742-751, 2007).

  Using the crude fraction of Nemebiwa tea obtained in “Example 2” and the crude fraction of fraction “C2” as a sample, the degree of NF-κB cell signal transmission inhibitory effect was induced with LPS. The concentrations of the IκB, IKK, and TAK1 expression genes were compared with the electrophoretic concentration of their phosphorylated protein expression levels. Specifically, the effect of concentration-dependent suppression of the NF-κB cell signal transduction system on the crude fraction and fraction “C2” of Nemebiwa tea was investigated.

  Experiments on the NF-κB cell signal transmission system inhibitory action were performed using cell culture, cell protein recovery and detection methods according to [Experiment 1], [Experiment 2] and [Experiment 3] of Example 9. As antibodies for detection, p65, IKK, TAK1, IκB, and phosphorylated antibodies thereof were used.

  FIG. 22 shows the results of the concentration-dependent change in electrophoresis of the expression levels of IκB, IKK and TAK1 and the expression levels of these phosphorylated proteins. In FIG. 22, the sample symbol “C2” corresponds to the crude fraction “C2”, and experiments were conducted at concentrations of 50, 100, and 200 μg / ml, respectively. LPS induced inflammation at 40 ng / ml. For comparison, an experiment of crude fraction “C” was also added. The results are shown in FIG.

  It can be seen that by increasing the concentration of the crude fraction `` C2 '' of Biwa tea to 50, 100, 200 μg / ml, the concentrations of IκB, IKK, TAK1 expressed genes and the expression levels of those phosphorylated proteins are suppressed ( FIG. 22). On the other hand, by increasing the concentration of the crude fraction “C” of Biwa tea to 50, 100, 200 μg / ml, the concentration of IκB, IKK, TAK1 expressed genes and the expression level of those phosphorylated proteins were investigated. , “C2” showed the same suppression (FIG. 23).

[Example 15] Effect of inhibitory action on PGE2 production induced by LPS of crude fraction and fraction "AD" of Nememebiwa tea
Experiments on the inhibitory action of PGE2 production by LPS induction were performed using the method described in the following literature (Uto, T., et al., Biochemical Pharmacology 70, 1772-1784, 2005). RAW264.7 cells are seeded to 1.2 × 10 ⁵ / ml / well (12 well plate), cultured for 21 hours, exchanged with medium without fetal bovine serum (FBS-free DMEM), and further 2.5 Incubate for hours. A loquat tea sample was added, and after 30 minutes of incubation, LPS was added to 40 ng / ml and further cultured for 12 hours. The obtained cell culture was collected and measured using a PGE ₂ measurement kit.

  About the crude fraction and fraction "AD" of Nemebiwa tea, the inhibitory effect test of PGE2 production was conducted. The result is shown in FIG. In FIG. 24, “A to D” sample symbols correspond to the crude fraction “A to D”, and experiments were performed at a concentration of 200 μg / ml. LPS also induced inflammation at a concentration of 40 ng / ml. Extract “M” was also added for comparison. It can be seen that the crude fraction and fractions “A to D” of Nemebiwa tea very strongly suppressed the production of PGE2 induced by LPS (FIG. 24). In the figure, the broken line indicates the cell viability. In the figure, those marked with an asterisk statistically significantly suppressed LPS-induced PGE2 production.

[Example 16] Effect of inhibitory action of PGE2 production by LPS induction of crude fraction and fraction "C2" of Nememebiwa tea The experiment was carried out in the same manner as in Example 15.

  An inhibitory effect test of PGE2 production was conducted on the crude fraction and fraction “C2” of Nemebiwa tea. The result is shown in FIG. In FIG. 25, the sample symbol “C2” corresponds to the crude fraction “C2”, and experiments were conducted at concentrations of 100, 200, and 200 μg / ml or more, respectively. LPS also induced inflammation at a concentration of 2%. The crude fraction “C” was also added for comparison. It can be seen that both the crude fractions and fractions “C2” and “C” of Nemebiwa tea suppressed LPS-induced PGE2 production (FIG. 25). In the figure, the broken line indicates the cell viability.

[Example 17] Anti-inflammatory effect of foot edema induced by LPS in ICR mice with extract of Nemebiwa tea (1) Model experiment on foot edema induced by LPS in ICR mice
The body weight of ICR mice was equalized to 25 ± 2 g, and lighting was ensured during the day with a cycle of 12 hours each day and night, and food and water were randomly fed. Biwa tea extract was injected subcutaneously every day for 4 days in a volume of 200 mg / kg as 3 groups in 1 group. As a control, an isotonic solution containing 0.9% sodium chloride was subcutaneously injected every day for 4 days in the same volume. Control ICR mice were left untouched (Ctl group). 0.1% LPS (weight / volume on the sole of the foot on day 4 for 3 ICR mice from another group subcutaneously injected daily for 4 days at a volume of 200 mg / kg with 0.9% sodium chloride in isotonic solution ) 25 μL was injected subcutaneously (LPS group). Similarly, three ICR mice injected daily with Biwacha extract at a dose of 200 mg / kg for 4 days were injected subcutaneously with 25 μL of 0.1% LPS (weight / volume) on the sole of the foot on the 4th day. (LPS + Biwa group). The thickness of the legs of the three groups was measured using a digital caliper every hour after subcutaneous injection of 25 μL of 0.1% LPS (weight / volume). The foot thickness was measured every hour, and the suppression of foot edema by loquat tea was compared.

(2) Measurement of foot circumference (diameter) due to foot edema in ICR mice Table 1 shows the circumference (diameter) of 3 groups of feet measured after subcutaneous injection of 25% of 0.1% LPS (weight / volume) Indicates. It can be seen that in the group to which Biwacha extract was administered, the thickness of the foot was significantly reduced as compared to the group to which Biwacha extract was not administered, which has the effect of suppressing foot edema induced by LPS.

(3) Actual state of foot edema in ICR mice Observed with a microscope
ICR mice were dissected 6 hours after LPS subcutaneous injection, paw edema was compared, and the degree of injury was observed. The photograph is shown in FIG. It can be seen that the foot edema of the LPS + Biwa group ICR mice is greatly improved compared to the LPS group ICR mice (FIG. 26).

[Example 18] Measurement of polyphenol content in extract "M", crude fraction "AD" and crude fraction "C1-C9" of Nememebiwa tea The amount of phenolic component contained in the powder of the extract “M” / crude fraction “A to D” and the crude fraction / fraction “C1 to C9” was measured. Specifically, each fraction powder was treated with sodium carbonate solution for 3 minutes, then treated with 50% Folin-Ciocalteau reagent for 30 minutes at room temperature, measured at a wavelength of 595 nm, and compared with gallic acid. Was calculated.

  The results are shown in FIGS. In FIG. 27, it can be seen that the crude fraction “C” has a relatively high polyphenol content. In FIG. 28, it can be seen that the crude fraction / fraction “C2” has a relatively high polyphenol content.

[Example 19] Quantification and characterization of functional components of Nemebiwa tea extract The functional components of the extract of Nemebiwa tea and loquat leaves were analyzed using high performance liquid chromatography (HPLC). HPLC separation was performed at 40 ° C. using a CresPak C ₁₈ T-5 column (4.6 mm id × 250 mm).

  The results are shown in FIG.

  In the panel of (A) in FIG. 29, the results of HPLC separation of the extract of Nemebiwa tea and loquat leaf are shown ((a) loquat leaf, (b) hot water extract M of Nemebiwa tea, (c ) Fraction C, and (d) Fraction C2). Moreover, the panel of (B) shows the result of measuring the amount of phenolic components carried out in accordance with the method described in Example 18 (“Fresh”: loquat leaf, “M”: Nejimebiwa tea hot water. Extract M, “A”: Fraction A, “B”: Fraction B, “C”: Fraction C, “D”: Fraction D, “C2”: Fraction C2).

  In Table 2 below, “Leaves water extract (L)” indicates the water extract of loquat leaves, “Tea water extract (T)” indicates the hot water extract M of Nemebiwa tea, and “Fraction C "Represents fraction C, and" Fraction C2 "represents fraction C2.

  As shown in FIG. 29 and Table 2, four known main active components (3-caffeoylquinic acid (3-CQA), 5-caffeoylquinic acid (5-caffeoylquinic acid) present in loquat leaves acid: 5-CQA), (-)-epicatechin ((-)-epicatechin: EC) and procyanidin B2 (procyanidin B2: PCB2)) were analyzed as standards, and as a result, 3-CQA, 5-CQA, EC and PCB2 was abundant in the water extract (L) of loquat leaves, and only a small amount was present in the hot water extract M of Nemebiwa tea.

On the other hand, 3-CQA, 5-CQA, EC, and PCB2 were not detected in the fraction C and the fraction C2 obtained by purification. Instead, new components that were not present in the water extract of loquat leaves were detected (Compound 1 and Compound 2 shown in Table 2). As a result of FT-IR analysis of the new component, many OH and a plurality of CH, C = C, CC rings were found, and it was presumed to be a single substituted benzene ring. Furthermore, as a result of analysis by ¹ H-NMR, aromatic or olefin-like protons, methylene and alicyclic protons were recognized. Although the final structures of these compounds have not yet been identified, they were thought to be multiple phenolic compounds produced during the loquat tea manufacturing process that roasts loquat leaves. These compounds have antioxidant activity and the like, and are considered to be a part of the functional substance of Nemebiwa tea.

Claims (7)

(a) subjecting Nemebiwa tea to hot water extraction to obtain an extract, wherein the extract is a concentrate or a dry powder;
(b) The hot water extract of step (a) is subjected to column chromatography, and fractionated by extraction using a 30% to 70% aqueous ethanol solution or a mixture containing water and acetone in a ratio of 1: 1. Obtaining
A method for producing a lipase inhibitor, α-glucosidase inhibitor or anti-inflammatory agent.   The method according to claim 1, wherein the 30% -70% aqueous ethanol solution is a 30%, 50% or 70% aqueous ethanol solution. (a) subjecting Nemebiwa tea to hot water extraction to obtain an extract, wherein the extract is a concentrate or a dry powder;
(b) subjecting the hot water extract of step (a) to column chromatography to obtain a fraction by extraction with 50% -70% aqueous ethanol;
(c) The fraction obtained in step (b) was subjected to column chromatography, and a mixed solution containing water and methanol at a ratio of 80%: 20%, 70%: 30% or 50%: 50% was used. Obtaining a fraction by extraction;
A method for producing a lipase inhibitor, α-glucosidase inhibitor or anti-inflammatory agent.   The method according to claim 3, wherein the 50% to 70% aqueous ethanol solution is a 50% or 70% aqueous ethanol solution.   A lipase inhibitor, an α-glucosidase inhibitor or an anti-inflammatory agent containing the fraction obtained by the method according to any one of claims 1 to 4 as an active ingredient.   The anti-inflammatory agent according to claim 5, which is a prostaglandin E2 biosynthesis inhibitor.   The anti-inflammatory agent according to claim 5, which is a cyclooxygenase-2 (COX-2) inhibitor.

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