JP6030284B2 - GLP-1 activity enhancer - Google Patents

Description

  The present invention relates to a method for efficiently enhancing GLP-1 activity in vivo.

  In recent years, diabetes has increased explosively around the world, and it is said that there are 6 million diabetic patients in Japan and 12 to 15 million reserves. In diabetes, blood vessels are gradually damaged by hyperglycemia, and abnormalities occur in various organs. Diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy have been known for the past as three major complications, but in recent years, it is also known that the risk of developing arteriosclerosis is increased.

  Among diabetes mellitus, type I diabetes develops due to insulin deficiency caused by destructive lesions of pancreatic β cells, and type II diabetes occurs due to a decrease in insulin secretion ability due to dysfunction of pancreatic β cells and liver / muscle / adipose tissue, etc. It develops from a concomitant decrease in insulin sensitivity in the target tissue. Diabetes, which has increased rapidly in recent years, is derived from type II and is considered to account for 90 to 95% of diabetes. Type II diabetes is caused by lifestyle habits in modern society, such as stress, obesity, decreased basal metabolism due to lack of exercise, and intake of high-calorie foods, as it is said to be a “lifestyle-related disease”.

  Incretin, which is attracting attention as a drug discovery target for diabetes, is a gastrointestinal hormone and is known to have an action of increasing the number of pancreatic β cells. GLP-1 (glucagon-like peptide 1), which is a kind of incretin, is known to have an appetite-suppressing action and suppresses obesity, which is one of the causes of type II diabetes In particular, the GLP-1 enhancer has been reported several times (Patent Documents 1 to 3). Although human GLP-1 analog preparations have already been put into practical use, it is necessary to carry out subcutaneous injection at least once a day, and side effects such as nausea have been reported (Non-patent Document 1).

  Salacia oblonga (Salacia reticulata), Salacia chinensis; Salacia prinoides (same species as Salacia prinoides); It is a vine perennial plant that grows in the southern region. These plants of the genus Salacia have been used as natural drugs in traditional medicine in India, Sri Lanka, and Southeast Asian countries, and more recently, extracts of these plants have α-glucosidase inhibitory action, lipase inhibitory action, etc. (Patent documents 4 to 8).

JP 2006-199694 A JP 2006-117466 A JP 2009-517464 A Japanese Patent Laid-Open No. 9-301882 Japanese Patent Application Laid-Open No. 11-116496 JP-A-11-29472 Japanese Patent Laid-Open No. 2001-26169 JP 2005-8572 A

Diabetes, 52, 6, 427-431, 2009

  Incretin is known to have a glucose concentration-dependent insulin secretion promoting effect, a glucagon secretion inhibiting effect, a pancreatic β cell number increasing effect, a neuroprotective effect, a bone mass increasing effect, and the like. Furthermore, since GLP-1 has a function of suppressing appetite, it can be used as a means for fundamental cure or prevention of diabetes. However, none of the compounds or compositions that specifically increase the secretion of GLP-1 described above reach a level of practical use in terms of activity and safety.

  An object of the present invention is to provide a GLP-1 activity enhancer, particularly a GLP-1 production enhancer that is effective, safe and can be taken continuously. Furthermore, an object of the present invention is to provide an agent for preventing diabetes or a pancreatic protective agent that is effective, safe and can be taken continuously.

  As a result of diligent research to solve the above problems, the present inventor has found a good GLP-1 production enhancing action and a GLP-1 metabolism inhibiting action by a GLP-1 metabolism inhibiting action on an extract of a Salacia plant. The present invention has been completed.

  According to one aspect of the present invention, there is provided a GLP-1 activity enhancer comprising an extract of a Salacia plant, for example, an extract of a Salacia plant selected from Salacia chinensis, Salacia reticulata and Salacia oblonga as an active ingredient Is done. In one embodiment of the present invention, Salacia chinensis is used as a plant of the genus Salacia. In one embodiment of the present invention, the above GLP-1 activity enhancer is used for diabetes prevention and / or pancreas protection.

  According to another aspect of the present invention, there is provided the aforementioned GLP-1 activity potentiator having an inhibitory action on dipeptidyl peptidase IV (DPP4) activity in blood. According to still another aspect of the present invention, the aforementioned GLP-1 activity enhancer having an effect of lowering the blood concentration of DPP4 is provided.

  According to another aspect of the present invention, there is provided an agent for preventing diabetes or a pancreatic protective agent comprising, as an active ingredient, an extract of a plant belonging to the genus Salacia, for example, a plant of the genus Salacia selected from Salacia chinensis, Salacia reticulata and Salacia oblonga. Provided. In one embodiment of the present invention, Salacia chinensis is used as a plant of the genus Salacia.

According to another aspect of the present invention, a food or drink containing the above GLP-1 activity enhancer, diabetes preventive agent or pancreatic protective agent is provided.
According to still another aspect of the present invention, there is provided a pharmaceutical composition comprising the above GLP-1 activity enhancer, diabetes preventive agent or pancreatic protective agent.

  The GLP-1 activity enhancer of the present invention has a good GLP-1 production enhancing action, inhibits the activity of dipeptidyl peptidase IV (DPP4), thereby suppressing the metabolism of GLP-1, Has the effect of enhancing GLP-1 activity. Furthermore, the present invention has an excellent effect as a GLP-1 activity enhancer in that it has an action of suppressing the activity of DPP4 in blood and an action of lowering the concentration of DPP4 in blood. The GLP-1 activity enhancer of the present invention is highly safe and suitable for continuous intake, and can be used as a diabetes preventive agent or pancreatic protective agent.

FIG. 1 is an example of a graph showing the transition of the increase in rat blood GLP-1 after sucrose loading. FIG. 2 is an example of a graph showing DPP4 inhibitory activity of Salacia plant extracts. FIG. 3 is an example of a graph showing the influence of a Salacia plant extract on blood DPP4 activity. FIG. 4 is an example of a graph showing the effect of a Salacia plant extract on the blood concentration of DPP4.

  As the plant of the genus Salacia used as an extraction raw material, for example, a trunk, a root, a leaf, a fruit dried after collection, or a bark of a trunk and a root part cut or pulverized can be used.

Extraction can be performed by appropriately using a conventional method, and for example, it may be performed by a method such as continuous extraction, immersion extraction, countercurrent extraction, supercritical extraction, or column extraction.
Although it does not specifically limit as an extraction solvent, It is preferable to use hydrophilic solvents, such as water, a lower alcohol (methanol and ethanol), acetone, or those mixed solvents, and it is preferable to use water especially. It is preferable to heat at the time of extraction, for example, extraction can be performed at the reflux temperature of the extraction solvent. When water is used as a solvent, the extraction can be performed at a temperature of 60 to 110 ° C., preferably 80 to 98 ° C., for example, for an extraction time of 1 to 24 hours, preferably 1 to 4 hours.

  As the extract used in the present invention, a solid obtained by drying a stock solution of the extract, a concentrate of the extract or the extract concentrate can be used, and the concentrate is solidified from the viewpoint of ingestion efficiency. Is preferably used. As a method for concentrating the extract, conventional techniques can be appropriately used. For example, a vacuum drying method, a freeze drying method, a spray drying method, or the like can be performed.

The extract of Salacia plant may be subjected to a purification treatment. Examples of the purification treatment include treatment with an adsorbent such as activated carbon and ion adsorption resin, and liquid-liquid countercurrent distribution treatment.
As the extract of the genus Salacia, a commercially available product may be used. Further, for the purpose of ingesting a substantial extract of a plant belonging to the genus Salacia, a ground product of a Salacia plant or the like may be used as it is as a component of the present invention.

  The intake of the GLP-1 activity enhancer of the present invention can be appropriately adjusted depending on the body type, age, physical condition, etc. of the subject. For example, for an adult weighing 60 kg, the extract of Salacia plant is administered at a dose of 300 mg / day or more, preferably 450 mg / day or more, more preferably 900 mg / day or more, and for example 3000 mg / day or less, preferably 2400 mg. / Day or less, more preferably 1800 mg / day or less.

  The GLP-1 activity enhancer of the present invention can be provided as a pharmaceutical with some processing or as it is. According to another aspect of the present invention, a GLP-1 activity enhancer comprising, as an active ingredient, an extract of a Salacia plant, for example, an extract of a Salacia plant selected from Salacia chinensis, Salacia reticulata and Salacia oblonga A pharmaceutical composition is provided.

  The GLP-1 activity enhancer of the present invention is a food (health food (nutrient functional food, food for specified health use, supplement, etc.), food for patients etc.) or beverage (tea, refreshment) with some processing added or as it is. Can be used as a beverage). Therefore, according to still another aspect of the present invention, enhancement of GLP-1 activity comprising an extract of a Salacia plant, for example, a Salacia plant selected from Salacia chinensis, Salacia reticulata and Salacia oblonga as an active ingredient A food or drink comprising the agent is provided.

  According to still another aspect of the present invention, a GLP-1 activity enhancer comprising, as an active ingredient, an extract of a plant selected from Salacia plants, for example, Salacia chinensis, Salacia reticulata and Salacia oblonga. An ingestible composition is provided. The composition for oral intake of the present invention includes the pharmaceutical composition of the present invention and food and drink as defined herein.

The said pharmaceutical composition and food-drinks can also mix | blend and prepare components, such as a conventionally well-known coloring agent, a preservative, a fragrance | flavor, a flavor agent, and a coating agent, as needed.
Moreover, you may mix | blend the pharmaceutical composition and food-drinks of this invention, mix | blending one or more additional components. Examples of additional components include hypoglycemic agents, anticholesterol agents, immunostimulants, antioxidants, vitamins, amino acids, peptides, proteins, minerals (iron, zinc, magnesium, iodine, etc.), fatty acids (EPA, DHA, etc.) ) And the like.

  Here, examples of the hypoglycemic agent are not particularly limited, but include indigestible dextrin, α-linolenic acid, bean extract, wheat albumin, L-arabinose, and plant-derived components (eg, guava leaf, mulberry leaf) , Ginger, sweet potato, barley, yellow aloe vera, dandelion, red daisies, daffodil, garlic, pearl barley, banaba, bilberry, black cohosh, makomo, chufu, evening primrose, caiapo, bitter gourd, madeglucil or extracts thereof).

  Examples of anticholesterol agents include, but are not limited to, soy protein, phospholipid-bound soy peptide, chitosan, plant sterol, plant sterol ester, plant stanol ester, resistant digestive dextrin, sodium alginate, psyllium seed coat, astaxanthin, inositol , Coenzyme A, calcium, magnesium, carnitine, silk protein, taurine, methionine, α-linolenic acid, guar gum, chondroitin sulfate, soy saponin, and plant-derived ingredients (eg, macaque, alfalfa, ginkgo, plantain, barley, oat, Olive, Gadju, Gymnema, Cat's Claw, Cuco, Chlorella, Spirulina, Hawthorn, Pepper, Garlic, Bilberry, Safflower, Yucca, Rahma, Agariku , Red yeast or extracts thereof) and the like.

  Examples of immunostimulants include, but are not limited to, lactoferrin, arginine, tryptophan, valine, leucine, chitin, chitosan, and plant-derived components (eg, Agaricus, Cordyceps, Aloe, Kidachi aloe, Echinacea, Ogi, Cats claw, Wolfberry, spirulina, pearl barley, safflower, maca, makomo, luffa, or an extract thereof).

  Examples of antioxidants include, but are not limited to, dry yeast, glutathione, lipoic acid, quercetin, catechin, coenzyme Q10, enzodinol, proanthocyanidins, anthocyanidins, anthocyanins, carotenes, lycopene, flavonoids, resaveratrol, Examples include isoflavones, zinc, melatonin, and plant-derived components (for example, ginkgo biloba, moon peach leaf, hibiscus, or an extract thereof).

  Examples of vitamins include, but are not limited to, vitamins belonging to the vitamin A group [eg, retinal, retinol, retinoic acid, carotene, dehydroretinal, lycopene, and pharmacologically acceptable salts thereof (eg, retinol acetate). , Retinol palmitate, etc.), vitamins belonging to vitamin B group [for example, thiamine, thiamine disulfide, dicetiamine, octothiamine, chicotiamine, bisbutiamine, bisbenchamine, prosultiamine, benfotiamine, fursultiamine, Riboflavin, flavin adenine dinucleotide, pyridoxine, pyridoxal, hydroxocobalamin, cyanocobalamin, methylcobalamin, deoxyadenocobalamin, folic acid, tetrahydrofolic acid, dihydrofolic acid, nicoti Acids, nicotinamide, nicotinic alcohol, pantothenic acid, panthenol, biotin, choline, inositol, pangaminic acid and their pharmaceutically acceptable salts (eg thiamine hydrochloride, thiamine nitrate, dicetiamine hydrochloride, fursulfuric acid hydrochloride) Thiamine, riboflavin butyrate, sodium flavin adenine dinucleotide, pyridoxine hydrochloride, pyridoxal phosphate, calcium pyridoxal phosphate, hydroxocobalamin hydrochloride, hydroxocobalamin acetate, calcium pantothenate, sodium pantothenate, etc.)], vitamins belonging to the vitamin C group [ Ascorbic acid and its derivatives, erythorbic acid and its derivatives, and pharmacologically acceptable salts thereof (for example, sodium ascorbate, sodium erythorbate) Etc.], vitamins belonging to the vitamin D group (for example, ergocalciferol, cholecalciferol, hydroxycholecalciferol, dihydroxycholecalciferol, dihydrotaxosterol, and pharmacologically acceptable salts thereof), Vitamins belonging to the vitamin E group [for example, tocopherol and derivatives thereof, ubiquinone derivatives and pharmacologically acceptable salts thereof (such as tocopherol acetate, tocopherol nicotinate, tocopherol succinate, tocopherol calcium succinate, etc.), etc. Vitamins [eg carnitine, ferulic acid, γ-oryzanol, orotic acid, rutin (vitamin P), eriocitrin, hesperidin, and pharmacologically acceptable salts thereof (such as carnitine chloride). ] And the like.

  Examples of amino acids include, but are not limited to, leucine, isoleucine, valine, methionine, threonine, alanine, phenylalanine, tryptophan, lysine, glycine, asparagine, aspartic acid, serine, glutamine, glutamic acid, proline, tyrosine, cysteine, histidine. Ornithine, hydroxyproline, hydroxylysine, glycylglycine, aminoethylsulfonic acid (taurine), cystine, or pharmacologically acceptable salts thereof (eg, potassium aspartate, magnesium aspartate, cysteine hydrochloride, etc.) Can be mentioned. Preferred examples include branched chain amino acids such as valine, leucine and isoleucine, glutathione, cysteine, glutamic acid, glycine, serine, tryptophan, tyrosine, phenylalanine, histidine, methionine, threonine, lysine, cystine, arginine, alanine, aspartic acid, proline, Aminoethylsulfonic acid.

  The GLP-1 activity enhancer of the present invention is a granule (including dry syrup), for example, so as to facilitate continuous ingestion as a pharmaceutical composition or a food or drink (especially functional food, health food, supplement, etc.) Various solid preparations such as capsules (soft capsules, hard capsules), tablets (including chewables), powders (powder), pills, or liquids for internal use (solutions, suspensions, syrups) In the form of liquid preparations. A capsule or tablet form is preferable from the viewpoint of stability of ingredients and ease of ingestion, but is not particularly limited.

  The GLP-1 activity enhancer of the present invention in the form of a capsule or a tablet can be produced using a known additive acceptable as a medicine or food, and is usually used in the field of medicine or food. Formulation techniques can be applied. For example, a tablet can be prepared by adding and blending each component according to the formulation, crushing, granulating, drying, sizing and mixing, and tableting the resulting preparation mixture.

  Additives for formulation are not limited, but include, for example, excipients, lubricants, binders, disintegrants, fluidizers, dispersants, wetting agents, preservatives, thickeners, pH adjustments. Agents, coloring agents, flavoring agents, surfactants, solubilizing agents and the like. Moreover, when making it into the form of a liquid agent, thickeners, such as pectin, xanthan gum, and guar gum, can be mix | blended. Moreover, it can also be set as a coating tablet using a coating agent, or it can also be set as a paste-form glue. Furthermore, even if it is a case where it prepares in another form, what is necessary is just to follow the conventional method.

  Moreover, when this invention is food-drinks, this invention can be implemented with forms, such as a functional food, health food, food for specific health, food for special uses, and a supplement. According to a preferred embodiment of the present invention, there is provided a food or drink which is a food for specified health use or a special purpose food containing an amount of a plant belonging to the genus Salacia or an extract thereof having a GLP-1 activity enhancing action, a diabetes preventing action, or a pancreatic protecting action. Is done. Here, description regarding the action effect (GLP-1 activity enhancement action, diabetes prevention action, pancreas protection action) may be attached to the packaging, package, package insert or advertisement of the food.

  Furthermore, the GLP-1 activity enhancer of the present invention is, for example, as various foods and beverages such as beverages, confectionery, breads and soups or as additive components thereof; or as various pet foods such as dog foods and cat foods or additive components thereof. Can be used. The method for producing these foods and drinks is not particularly limited as long as the effects of the present invention are not impaired, and may follow the methods used by those skilled in the art for each application.

EXAMPLES Hereinafter, although the preferable Example of this invention is described in detail, this invention is not limited to these Examples.
The Salacia kinensis water extract used for the test was prepared by the following method. Hot water (20 kg) was added to a chip (1 kg) obtained by cutting the trunk portion of Salacia chinensis into 5 mm square, and the mixture was stirred and extracted at 98 ° C. for 120 minutes. The obtained extract was concentrated under reduced pressure using a rotary evaporator (concentration temperature 45 ° C., until Brix = 30), and the concentrate was freeze-dried to extract the Salacia chinensis extract (98.5 g, hereinafter). , Also referred to as Salacia extract).
[Test Example 1] Postprandial blood GLP-1 concentration measurement test 1) Purchase SD male rats (Japan SLC Co., Ltd., 6 weeks old) and reserve for approximately 1 week under free food intake until the start of the experiment. Raised. Then, fasted for about 20 hours, divided into 4 groups (6 animals each) based on body weight (weight range in use 202.0 g to 260.0 g), 1 group administered only sucrose (control group), The other three groups were designated as a group to which sucrose and a test substance were administered (Salasia extract group, tea extract group, and tea extract group). Tea extracts and tea tea extracts obtained by purchase were used (tea extract: Sunfood (registered trademark) 100, manufactured by Mitsubishi Chemical Foods Corporation; tea tea extract: tea tea extract M powder, manufactured by Maruzen Pharmaceutical Co., Ltd. ). Group composition and administered substances are shown in the table below.

A single dose of sucrose (1 g / kg) was orally administered to the rat together with the test substance, and blood was collected at the initial value (0 minute), 60 minutes and 120 minutes after administration, and blood GLP-1 concentration was measured.
Blood was collected by anesthetizing rats with diethyl ether and then inserting a hematocrit tube into the fundus. The collected blood was immediately transferred to a blood collection tube containing a DPP4 inhibitor to prevent degradation of GLP-1 in the blood. Treated blood was centrifuged and serum was separated. The amount of GLP-1 in the serum was measured by ELISA using a commercially available kit [Levis (registered trademark) GLP-1 (active) kit, Shibayagi Co., Ltd.]. The results are shown in Table 2 below and FIG.

From this test example, it was confirmed that the concentration of GLP-1 in the blood after 2 hours was increased about twice by ingesting the Salacia extract together with sucrose. On the other hand, such increase in GLP-1 concentration was not observed in the tea extract administration group and the tea extract administration group.
Test Example 2 DPP4 Inhibitory Activity Measurement Test The DDP4 inhibitory activity of Salacia extract was measured using a commercially available DPP4 inhibitory activity measurement kit (DPPIV Drug Discovery Kit-AK-499, manufactured by Enzo Life Science, Cosmo Bio Inc.) The DPP4 inhibitory activity of the Salacia extract was measured according to the assay method described in the instruction manual attached to the kit.

  The Salacia extract (2.5 mg) is suspended in the assay buffer (50 mM Tris, pH 7.5, 1 mL) attached to the kit, centrifuged, and the supernatant is recovered to obtain a sample solution (Salacia 2.5 mg / mL). ). The solution was diluted to prepare sample solutions of 1.3 mg / mL Salacia and 0.6 mg / mL Salacia. Using a measurement plate (kit accessory), DPP4 enzyme (15 μL, kit accessory, SE434-9090 DPPIV enzyme (human, recombinant product)) and well containing sample solution (35 μL) substrate (50 μL, kit accessory) Product, P188-9090 AMC substrate (H-Gly-Pro-AMC)) was added to initiate the reaction. The enzyme reaction was allowed to stand at 37 ° C. for 10 minutes, and the fluorescence intensity after 10 minutes (excitation wavelength: 380 nm, fluorescence wavelength: 460 nm) was measured with a plate reader (Multifunctional Reader Genios, Wako Pure Chemical Industries, Ltd.). . The same measurement was performed for wells to which the same amount of assay buffer was added instead of the sample solution, and the DPP4 activity rate of the well containing each sample solution was calculated with the measured value in the well as 100. The results are shown in Table 3 and FIG.

It was confirmed that the Salacia extract inhibits the enzyme activity of DPP4 in a concentration-dependent manner.
[Test Example 3] Blood DPP4 activity and concentration measurement test Genetic obesity model mouse (B6.V-Lep ^ob / J male mouse (Lep ^ob / Lep ^ob ), hereinafter ob / ob mouse) whose phenotype is obese ( Charles River Japan Co., Ltd., 5 weeks old) and mice that do not exhibit obesity in phenotype (male mice (Lep ^ob / + or + / +), hereinafter? / + Mice) (Nihon Charles River Co., Ltd., 5 weeks old) And pre-bred for about 1 week under free feeding until the start of the experiment. After that, ob / ob mice were divided into 3 groups, 1 group was administered only with CE-2 powder feed (Nippon Claire Co., Ltd.) [control group (5 animals)], and the other 2 groups were the concentration of Salacia extract. Is a group in which a CE-2 powder feed and a Salacia extract are mixed so as to be 0.20% by weight and 0.50% by weight [0.20% Salacia extract group (5 animals), 0.50% % Salacia extract group (5 animals)]. ? / + Mice were used as a control group (5 mice) to which only CE-2 powder feed was administered. Group composition and administered substances are shown in the table below.

  Each feed was administered to mice by free feeding, blood was collected on day 23 of the breeding, and blood DPP4 inhibitory activity and blood DPP4 concentration were measured. For blood collection, the mouse was anesthetized with diethyl ether and then opened, and heart blood was collected. The collected blood (about 100 to 200 μL) was quickly added to and mixed with a 1.5 mL tube to which JP Pharmacopoeia (Mitsubishi Tanabe Pharma Corporation) (5 μL) was added to prevent blood coagulation. Treated blood was centrifuged and plasma was separated. Blood DPP4 activity was measured using an AMC substrate (H-Gly-Pro-AMC, manufactured by Enzo Life Science) as a substrate. Plasma (10 μL) was added to a measurement plate (Nunc-96 well Black plate), and a substrate solution (50 mM Tris, pH 7.5, 1 mg / mL BSA, 5.56 μM H-Gly-Pro-AMC, 90 μL). ) Was added to start the enzyme reaction. The enzyme reaction is performed at 32 to 34 ° C. for 60 minutes, and the fluorescence intensity (excitation wavelength 360 nm, fluorescence wavelength 465 nm) of the reaction solution is obtained at a 1 minute interval with a plate reader (Multifunctional Reader Geonis, Wako Pure Chemical Industries, Ltd.). It was measured. The fluorescence intensity and reaction time were plotted on a graph, and blood DPP4 activity was calculated from the slope of the approximate line. The blood DPP4 concentration was measured using a commercially available DPP4 concentration measurement kit (ELISA Kit for Mouse Dipeptidyl Peptidase IV, USCN life science) according to the assay method described in the instruction manual attached to the kit. The results are shown in Table 5 below and FIGS. 3 and 4.

From this test example, it was confirmed that both the blood DDP4 concentration and the blood DPP4 activity were reduced by administering Salacia extract to obese model mice.
[Prescription example]
The Salacia extract can be mixed with an excipient according to the formulation shown in Table 6 below, and then tableted to obtain a food (tablet).

Claims (7)

A GLP-1 activity enhancer comprising an extract of Salacia chinensis as an active ingredient. The GLP-1 activity enhancer according to claim 1 , wherein the extract is a water extract. The GLP-1 activity enhancer according to claim 1 or 2 , which is used for diabetes prevention or pancreatic protection. The GLP-1 activity enhancer according to any one of claims 1 to 3 , which has a blood concentration lowering action of dipeptidyl peptidase IV (DPP4). The pharmaceutical composition containing the GLP-1 activity enhancer of any one of Claims 1-4 . A method for enhancing GLP-1 activity comprising administering an extract of Salacia chinensis to a subject (except for medical practice for humans). The method for enhancing GLP-1 activity according to claim 6 , wherein the extract is a water extract.