JP2009234919A - Therapeutic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent which uses a cranberry extract, which is known to be useful as a prophylactic agent for periodontal diseases, a therapeutic agent for autoimmune diseases, etc., as an active ingredient and is markedly effective particularly against various diseases other than those above. <P>SOLUTION: An anti-allergic agent, an agent for preventing or ameliorating hyperuricemia, an anti-osteoporosis agent, an anti-depression-anti-stress agent, an adiponectin production promoter and a cholesterol-reducing agent include the cranberry extract and proanthocyanidine as active ingredients. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a therapeutic agent useful for various diseases comprising cranberry extract and proanthocyanidins as active ingredients.

Cranberry extract is known for its use as a preventive agent for periodontal disease (Patent Document 1), an autoimmune disease therapeutic agent (Patent Document 2), and the like.
However, there is no view that the combination of cranberry extract and proanthocyanidins is effective with a particularly remarkable effect on various diseases applied in the present invention described below.
JP 2007-91703 A International Publication WO2005 / 030200 Pamphlet

  An object of the present invention is to provide various therapeutic agents that are effective with a particularly remarkable effect on various diseases applied in the present invention described below.

The invention according to claim 1 is an antiallergic agent comprising cranberry extract and proanthocyanidins as active ingredients.
The invention according to claim 2 is a preventive or ameliorating agent for hyperuricemia characterized by comprising cranberry extract and proanthocyanidins as active ingredients.
The invention according to claim 3 is an anti-osteoporosis agent characterized by containing cranberry extract and proanthocyanidins as active ingredients.
The invention according to claim 4 is an antidepressant / antistress agent characterized by comprising cranberry extract and proanthocyanidins as active ingredients.
The invention according to claim 5 is an adiponectin production promoter characterized by comprising cranberry extract and proanthocyanidins as active ingredients.
The invention according to claim 6 is a cholesterol-lowering agent characterized by containing cranberry extract and proanthocyanidins as active ingredients.

  According to the present invention, there are provided various therapeutic agents that are particularly effective for various diseases applied in the present invention described below with remarkable effects.

  Hereinafter, the present invention will be described in more detail.

  Cranberry (Vaccinium macrocarpon Ait.) Used in the present invention is not limited in its variety, but for example, Crowley, Stevens, Benlear, Pilgrim, Early black , Bergman and the like. Commercially available cranberry powder can also be used. For example, Early Black is manufactured under the trade name PACran from Decas Botany Synergies, USA.

  Cranberry extracts include juice obtained from cranberry fruits, fruit skin, stems, seeds, concentrates, purified products, dried products and freeze-dried products, and cranberry fruits, fruit skins, stems, seeds as solvents. An extract obtained by contacting is mentioned.

  In the latter form, examples of the solvent to be used include a polar solvent and an ambipolar solvent. For example, water, an organic solvent, a mixed solution of water and an organic solvent, or the like can be used. Examples of the organic solvent include lower alcohols represented by ethanol, methanol, propanol and butanol, ethers represented by dimethyl ether and diethyl ether, halogenated hydrocarbons represented by chloroform, carbon tetrachloride, acetonitrile, and the like. Examples include ethyl acetate and acetone. Among these, water, an aqueous ethanol solution, an aqueous acetone solution, and ethyl acetate are preferable.

  In the case of purified cranberry product, dried product, or freeze-dried product, any means known in the art may be appropriately employed. For example, the method for purifying cranberry extract using column chromatography is described in the above-mentioned patent document. 1.

  The extraction time may be a time for sufficiently extracting soluble components from the extraction raw material, and may be set as appropriate according to the extraction temperature and the like. Preferably, it is 30 minutes to 48 hours. For example, when the extraction temperature is less than 50 ° C., it can be extracted for 6 hours to 48 hours, and when it is 50 ° C. or more, it can be extracted for 30 minutes to 24 hours.

  For example, in the case of a freeze-dried product of an aqueous extract of cranberry fruit, the intake amount of the cranberry extract is preferably in the range of 50 to 2000 mg as a total amount per day for an adult, but is not particularly limited.

  The proanthocyanidins used in the present invention have a degree of polymerization of flavan-3-ol and / or flavan-3,4-diol as a structural unit of 2 or more, preferably 2 to 10-mer, more preferably 2 to 2. A compound group consisting of a tetramer condensation polymer, a derivative, and a stereoisomer thereof are designated. Among the proanthocyanidins, a condensation polymer having a polymerization degree of 2 to 4 having flavan-3-ol and / or flavan-3,4-diol as a structural unit is referred to as OPC (oligomeric proanthocyanidin). OPC is a powerful antioxidant and is abundant in plant leaves, bark, fruit peel or seed parts. Specifically, it is contained in grapes, pine bark, peanut skin, ginkgo, false acacia fruit, cowberry, blueberry, strawberry, avocado, barley, wheat, soybean, black soybean, cacao and the like. It is also known that OPC is contained in the roots of cola nuts in West Africa and Latania in Peru. OPC is a substance that cannot be produced in the human body.

  With regard to proanthocyanidins used as the active ingredient of the present invention, the origin of the raw material or the utilization part of the raw material, the production method, and the purification method are not limited at all, but the bark, fruit or seed pulverized product, or the extract of these extracts Such food ingredients can be used. In particular, it is preferable to use an extract of pine bark, more preferably French coast pine bark rich in OPC. French coastal pine bark is preferably used as a raw material for proanthocyanidins.

  Proanthocyanidins can be obtained by known methods [for example, extraction from pine bark; RW Hemingway et al., Phytotochemistry, 1983, Vol. 22, p. 275-281] or a method based thereon can be easily obtained from the above-mentioned various plants.

  Hereinafter, a method for preparing proanthocyanidins will be described by taking an extract of pine bark rich in OPC as an example.

  The pine bark extract is obtained by extracting pine bark with water or an organic solvent. When water is used, warm water or hot water is used. As the organic solvent used for extraction, an organic solvent that is acceptable for food or pharmaceutical production is used, for example, methanol, ethanol, propanol, butanol, butane, acetone, hexane, cyclohexane, propylene glycol, hydrous ethanol, hydrous propylene glycol, Examples include ethyl methyl ketone, glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible fats and oils, 1,1,1,2-tetrafluoroethane, 1,2-trichloroethene and the like. These water and organic solvent may be used alone or in combination. In particular, hot water, hydrous ethanol, and hydrous propylene glycol are preferably used.

  A method for extracting proanthocyanidins from pine bark is not particularly limited, and for example, a warm extraction method, a supercritical fluid extraction method, or the like is used.

  The supercritical fluid extraction method is a method of performing extraction using a supercritical fluid that is a fluid that exceeds the critical point (critical temperature, critical pressure) of a gas-liquid substance. As the supercritical fluid, carbon dioxide, ethylene, propane, nitrous oxide (laughing gas) or the like is used, and carbon dioxide is particularly preferably used.

  The supercritical fluid extraction method includes an extraction step of extracting a target component with a supercritical fluid and a separation step of separating the target component and the supercritical fluid. In the separation step, any one of extraction separation by pressure change, extraction separation by temperature change, or extraction separation using an adsorbent and an absorbent may be performed.

  Moreover, you may perform supercritical fluid extraction by the entrainer addition method. In this method, for example, about 2 to 20% of ethanol, propanol, n-hexane, acetone, toluene, or the like is added to the supercritical fluid, and supercritical fluid extraction is performed with the obtained extraction fluid, so that OPC or the like is performed. This is a method for dramatically increasing the solubility of the extract in the extraction fluid, or for enhancing the separation selectivity, and for efficiently obtaining a pine bark extract.

  Extraction from pine bark may be performed by a liquid carbon dioxide batch method, a liquid carbon dioxide reflux method, a supercritical carbon dioxide reflux method, or the like, in addition to the above method.

  The proanthocyanidins obtained as described above can be obtained in a liquid or semi-solid form. If the extraction solvent is removed from this product by a known method such as distillation under reduced pressure, spray drying, freeze drying, or the like, it remains as it is. It can be used as a proanthocyanidin-containing concentrate or dried product. For further purification, a known purification means such as column chromatography or countercurrent distribution method can be employed to achieve the object.

  Proanthocyanidins dissolve well in water and have high absorbability to living bodies. The stability is high in any of acidic, neutral and alkaline conditions.

  The dose of proanthocyanidins varies depending on the type, dosage form, age, weight, indication, etc. of the patient, but for example, adults once to several times a day, about 1 to 500 mg, preferably 3 times a day. It is better to administer ~ 300mg.

  The therapeutic agent of the present invention can be appropriately prepared by a known method in the form of a solid or solution (hereinafter, also referred to as a preparation) such as a tablet, pill, capsule, granule, powder, powder, or liquid. That is, the solid preparation or liquid preparation useful in the present invention is produced by using a well-known preparation method that has been well established. Examples of additives include excipients, pH adjusting agents, cooling agents, suspending agents, diluents, antifoaming agents, thickeners, solubilizers, disintegrating agents, binders, lubricants, antioxidants. Agents, coating agents, coloring agents, flavoring agents, surfactants, plasticizers or fragrances.

  Examples of the excipient include sugar alcohols such as D-sorbitol, D-mannitol or xylitol, sugars such as glucose, sucrose, lactose or fructose, crystalline cellulose, carmellose sodium, calcium hydrogen phosphate, wheat starch, rice Examples thereof include starch, corn starch, potato starch, dextrin, β-cyclodextrin, light anhydrous silicic acid, titanium oxide, and magnesium aluminate metasilicate.

Examples of the pH adjuster include citric acid, malic acid, sodium hydrogen phosphate, and dipotassium phosphate.
Examples of the refreshing agent include l-menthol or mint water.
Examples of the suspending agent include kaolin, carmellose sodium, xanthan gum, methylcellulose, and tragacanth.
Examples of the diluent include purified water, ethanol, vegetable oil, and emulsifier.
Examples of the antifoaming agent include dimethylpolysiloxane or silicon antifoaming agent.

Examples of the thickener include xanthan gum, tragacanth, methylcellulose, and dextrin.
Examples of the solubilizer include ethanol, sucrose fatty acid ester, and macrogol.
Examples of the disintegrant include low-substituted hydroxypropylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, hydroxypropyl starch, or partially pregelatinized starch.

  Examples of the binder include methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gelatin, gum arabic, ethylcellulose, polyvinyl alcohol, pullulan, pregelatinized starch, agar, tragacanth, sodium alginate, or propylene glycol alginate. Can be mentioned.

Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, polyoxyl stearate, cetanol, talc, hydrogenated oil, sucrose fatty acid ester, dimethylpolysiloxane, beeswax and white beeswax.
Examples of the antioxidant include ascorbic acid, dibutylhydroxytoluene (BHT), propyl gallate, butylhydroxyanisole (BHA), tocopherol, ascorbic acid, and citric acid.

Examples of the coating agent include hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, cellulose acetate phthalate, polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate. Copolymer, hydroxypropyl methylcellulose acetate succinate, methacrylic acid copolymer, polyvinyl acetate diethylaminoacetate or shellac.
Examples of the colorant include turmeric extract, riboflavin, titanium oxide, or carotene solution.

Examples of the flavoring agents include citric acid, adipic acid, ascorbic acid, fructose, D-sorbitol, glucose, sodium saccharin, simple syrup, sucrose, honey, amacha, licorice, citric acid, adipic acid, ascorbic acid, orange oil, Spruce tincture, fennel oil, mint or menthol.
Examples of the surfactant include polyoxyethylene hydrogenated castor oil, glyceryl monostearate, sorbitan monostearate, sorbitan monolaurate, polyoxyethylene polyoxypropylene, polysorbates, sodium lauryl sulfate, macrogol or sucrose. Examples include fatty acid esters.
Examples of the plasticizer include triethyl citrate, polyethylene glycol, triacetin, and cetanol.
As said fragrance | flavor, synthetic fragrance | flavors, such as natural fragrance | flavors, such as an animal fragrance | flavor or a vegetable fragrance | flavor, or an isolated fragrance | flavor etc. are mentioned, for example.

  You may mix | blend the therapeutic agent of this invention with food-drinks material. Examples of such materials include solid foods such as bread, chewing gum, cookies, chocolate and cereal, jams such as jam, ice cream, yogurt and jelly, cream or gel foods, juice, coffee, cocoa and green tea. And beverages such as oolong tea and black tea. Moreover, it can also mix | blend with a seasoning, a food additive, etc.

  Furthermore, in the present invention, for example, royal jelly, propolis, vitamins (A, C, D, E, K, folic acid, pantothenic acid, biotin, derivatives thereof, etc.), minerals (iron, magnesium, calcium, zinc, etc.), Selenium, lecithin, carotenoids (lycopene, astaxanthin, zeaxanthin, lutein, etc.), saponins (gymnemic acid, soybean saponin, ginseng saponins, etc.), fatty acids, proteins (collagen, elastin, etc.), oligosaccharides (isomalto-oligosaccharides, cyclic oligosaccharides, etc.) ), Phospholipids and derivatives thereof (phosphatidylcholine, sphingomyelin, ceramide, etc.), sulfur-containing compounds (eg, alliin, sepaene, taurine, glutathione, methylsulfonylmethane), sugar alcohols, lignans (sesamin, etc.), Contains animal and plant extracts Root vegetables (turmeric, ginger, etc.), green leaves of gramineous plants such as young wheat powder, green leaves of cruciferous plants such as kale, dried leaves of cactus, terminaria berylica extract, whole potato stems and leaves, soybean Contains ceramide-like ingredients extracted from herbs, herbs such as Chancapiedra, etc.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these. The therapeutic agent of the present invention is extremely useful as an antiallergic agent, an agent for preventing or improving hyperuricemia, an anti-osteoporosis agent, an antidepressant / antistress agent, an adiponectin production promoter, and a cholesterol lowering agent. Hereinafter, the various medicinal effects will be described with examples.

Example 1 (Usefulness as an antiallergic agent)
Cranberry hot water extract 1 was obtained by adding 20 times the amount of hot water (80 ° C.) to the dried product of cranberry fruit and stirring for 2 hours. Cranberry hot water extract 1 was concentrated under reduced pressure and freeze-dried to obtain powder 1.

  On the other hand, 7.2 L of purified water was added to 900 g of pine bark, crushed with a blender (Waring Blender), and then extracted by heating at 100 ° C. for 10 minutes. Then, it filtered immediately and obtained the filtrate. The residue after filtration was washed with 1.8 L of purified water, and the filtrate and the washing solution were combined to obtain 9 L of a crude hot water extract of pine bark. When 1 mL of this extract was freeze-dried, the dry weight was 8 mg.

  1 L of the above crude extract (powder dry weight 8 g) was allowed to cool to 25 ° C., sodium chloride was added to 100% saturation concentration and stirred well, and then allowed to stand at 4 ° C. for 24 hours. After standing, this solution was filtered to obtain 910 mL of filtrate. This filtrate was further purified by the steps shown below. First, the filtrate was passed through a 30 × 300 mm column packed with 100 mL of an aromatic synthetic resin swollen with water (Diaion HP-20: manufactured by Mitsubishi Chemical Corporation), and further washed with 1 L of purified water. . Next, the column was eluted with a 15% (V / V) ethanol-water mixed solvent to obtain 200 mL of a pine bark hot water extraction purified product (hereinafter referred to as pine bark hot water extract 1). The obtained purified product was freeze-dried and powdered. This operation was repeated to obtain 7.51 g of dry powder 1 of pine bark hot water extract 1 from 9 L of pine bark hot water extract (hereinafter referred to as powder 2).

  As a result of measuring the content of proanthocyanidins and OPC in the powder 2 by the method described in JP-A-2005-23032, 40% by weight of proanthocyanidins was contained in the powder 2. Moreover, 20 wt% of OPC was contained in the powder 2 as a dimer to tetramer.

  20 patients (10 males and 10 females aged 20-22 years) with food allergen positive chronic urticaria by the RAST method were administered 300 mg and 1 month of the powder 1 and powder 2, respectively, with one meal. . The results are shown in Table 1 below.

Example 2
20 patients with atopic dermatitis by RAST method (10 males and 10 females 20 to 22 years old) were administered 300 mg and 1 month of Powder 1 and Powder 2 respectively with a meal. The results are shown in Table 2 below.

Example 3 (improvement effect of hyperuricemia)
Experimental Method As test animals, Wistar rats (8 weeks old, body weight of about 180 g) were used in groups of 6 animals.
Adenine was added to the test feed at a concentration of 0.75% and fed to rats to cause inhibition of uric acid excretion into the urine from the kidney, thereby giving a model animal of hyperuricemia.
The control group was the above 0.75% adenine feed alone, and the drug administration group was the feed containing 0.75% adenine and powder 1 and powder 2 of Example 1. The feed was freely consumed, but the concentrations of Powder 1 and Powder 2 in the test diet of the drug administration group were adjusted so that the amount of intake was 1 mg / kg body weight, respectively. The uric acid level in the blood was measured on the test start day and 24th day.
As a result, the blood uric acid concentration on the test start day of the control group was 0.57 mg / ml, and it was 2.33 mg / ml on the 24th day, whereas the blood uric acid concentration on the 24th day of the drug administration group was The uric acid concentration was 0.78 mg / ml.
As is clear from this result, the blood uric acid concentration in the control group was significantly increased, whereas in the drug administration group, the concentration was not increased. Therefore, it has been shown that a drug containing cranberry extract and proanthocyanidins as active ingredients is useful as an agent for preventing or improving hyperuricemia.

Example 4 (Anti-osteoporosis effect)
Osteoporosis Improvement Effect Test SD rat (22 weeks old) Female ovaries were surgically removed to create osteoporosis model rats. Ovariectomized rats were divided into 6 groups of 7 animals, and the intake of powder 1 and powder 2 of Example 1 was 1 mg / kg every other day (total 17 times) during the 35-day test period. Then, 2 ml of a physiological saline-dissolved liquid was orally administered. The feed was the solid feed CRF-1 for mice, rats and hamsters from Oriental Yeast Co., Ltd. There was no difference in food intake between groups during the study period. On the 35th day after the start of the test, the weight of the rat was measured, and then the femur was removed. The femur was used for analysis with the adhesive tissue and muscle removed. After measuring the volume of the femur, it was washed three times with ethanol, then washed three times with acetone, dried overnight, and then weighed to determine the dry weight of the femur. From the volume and dry weight, the bone density (dry weight g / volume mm ³ ) was measured. In addition, as a control experiment, Table 3 shows the result together with an example (comparative example) in which the above experiment was repeated except that physiological saline not containing the powder 1 and the powder 2 was administered to the rat.

  When Example 4 and the comparative example were compared, Example 4 showed a significant difference at a risk rate of p <0.05.

Example 5 (Antidepressant / Anti-stress effect)
The therapeutic effect of powder 1 and powder 2 of Example 1 above was examined.
Evaluation of tranquilizing effect by forced mouse swimming test The therapeutic agent of the present invention was evaluated by the forced mouse swimming test developed by Porsolt in 1977. This is one of the most frequently used animal model experiments for depression. In this test, the mouse is forced to swim in a limited space to cause “immobility”. This immobility state is thought to reflect a kind of “despair state” in which stressed animals abandon their escape from water, and is associated with depression and stress in humans. In fact, antidepressants have been found to specifically reduce the duration of immobility in this situation, and this shortening has been found to have a significant correlation with clinical titer.

The test method is as follows.
Mice are forced to swim in a plastic cylinder containing 25 ° C. water to a depth of 15 cm. After forced swimming for 5 minutes, dry in a dryer at 30 ° C. for 15 minutes and return to the home cage. On the next day, the test sample is intraperitoneally administered to the mouse, one hour later, forced swimming for 5 minutes is imposed again, and the duration of the immobility that appears is measured using a stopwatch. A state in which the mouse floats on the water and is stationary is determined as an immobile state. For the duration of stationary state, a significant difference test is performed, and a statistically significant difference is tested. For the experiment, male ddY mice are used, and there are 6 mice per group. All tests are conducted between 1pm and 6pm. A test using imipramine, an antidepressant, as a positive control will also be conducted.

  As a result, the duration of immobility in mice administered with 30 mg / kg of powder 1 and powder 2 was 172.5 ± 3.7 seconds. The control (saline only) was 220.0 ± 2.2 seconds. The duration of immobility in positive control mice (30 mg / kg dose) was 176.5 ± 4.0 seconds. The duration of immobility in this example and the positive control is significantly different at a risk rate of 1%. In addition, even if it used 2-3 times amount of powder 1 and powder 2, the same result was obtained.

Example 6
Adiponectin production increase confirmation test Normal human preadipocytes were used and seeded in a 96-well microplate at 1.0 × 10 ⁴ cells. Human preadipocyte basal medium was used as the seeding medium. After 24 hours, the medium was replaced with a growth medium supplemented with a differentiation-inducing additive and powders 1 and 2 of Example 1, and further cultured for 1 week. Thereafter, the amount of adiponectin produced in the culture supernatant was quantified by ELISA. The evaluation results of each sample are shown below as relative values when the amount of adiponectin in the blank (sample not added) is 100. The added powder 1 and powder 2 concentrations were 10 μg / ml, respectively.

  Test result: relative value = 373. This value has a significant difference at a risk rate of 1%.

Example 7 (cholesterol lowering action)
ICR male mice weighing about 20 g (5 per group) were given a high cholesterol-cholate diet (71.9% standard diet, 15% sucrose, 2% salt, 10% coconut oil, 0.6% cholesterol, 0.2% cholic acid, 0.3% choline chloride) was fed (free intake) from the first day to the seventh day of the test. On the 6th and 7th day of the test, 5 mg of each of the powder 1 and the powder 2 of Example 1 was dissolved in distilled water and orally administered. Thereafter, fasting was performed for 24 hours, and blood was collected from the mice on the 8th day of the test, and the serum was separated.

  In addition, heparin was added to a portion of the collected serum and precipitated to obtain heparin-precipitated lipoprotein as low density lipoprotein (LDL). The total cholesterol level in serum and the cholesterol level in LDL were reported by C. C. Allain et al. (Clinical Chemistry, 1974, 20, 470-475). ) And measured.

  The value obtained by subtracting the LDL cholesterol value from the total cholesterol value in the serum was calculated as a high density lipoprotein (HDL) cholesterol value. The control group is a group to which the powder 1 and the powder 2 are not administered.

  The results are shown in Table 5. As is clear from Table 5, an obvious effect of lowering serum total cholesterol was observed.

In each of the above examples, when the cranberry hot water extract 1 of Example 1 was prepared, the extract was prepared using a 20 to 100% by volume ethanol aqueous solution or acetone aqueous solution without using hot water. The same result as above was obtained.
Moreover, in this invention, when a cranberry extract and proanthocyanidin are used together, it has been confirmed that a synergistic effect of both occurs.

  The therapeutic agent of the present invention is useful as a pharmaceutical, food or feed form.

Claims (6)

  An antiallergic agent comprising cranberry extract and proanthocyanidins as active ingredients.   A preventive or ameliorating agent for hyperuricemia characterized by comprising cranberry extract and proanthocyanidins as active ingredients.   An anti-osteoporosis agent comprising cranberry extract and proanthocyanidins as active ingredients.   An antidepressant / antistress agent characterized by comprising cranberry extract and proanthocyanidins as active ingredients.   An adiponectin production promoter comprising cranberry extract and proanthocyanidins as active ingredients.   A cholesterol-lowering agent comprising cranberry extract and proanthocyanidins as active ingredients.