JP2004115542A - Plum extract having medicinal virtue and composition containing the extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide plum extracts contributing to effective utilization of plum trees and giving various medicines. <P>SOLUTION: The extracts are prepared from plum leaf stems, plum kernels and plum flowers by using 5-times volumetric methanol. The extracts have antioxidative effect, gastric mucosal injury inhibition effect, aldose reductase inhibiting effect, blood sugar increase suppressing effect, platelet aggregation promoting effect, alcohol absorption suppressing effect, anti-inflammatory effect and the like. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a plum extract having a medicinal effect and a composition containing the same.

Numerous plum trees have been cultivated to obtain edible plums. However, plum trees other than plum meat and ome extract are currently treated as industrial waste. For example, in the cultivation of plum trees, prunus branches are pruned every fall, which is a huge amount of thousands to tens of thousands of tons per year. The pruned branches are incinerated as waste. Most of the seeds and shells after collecting the plum extract and plum meat are also discarded. This is not desirable from the viewpoint of effective use of resources and environmental conservation.

On the other hand, in the field of medicine and the like, medicines derived from natural organisms (including so-called herbal medicines and crude drugs) have attracted more attention than chemically synthesized medicines. This is because even though the synthetic drug has excellent efficacy, there is a problem of side effects. On the other hand, it is considered that substances isolated from organisms existing in nature, particularly organisms that have been eaten by humans since ancient times, have excellent safety. In addition, organisms are diverse depending on their living environment and the like, and the substances separated from them have unknown potential as medicines.In particular, plum has been considered to be good for human health since ancient times, Since it is a sacred plant that is used for religious events, it can be expected to have some medicinal properties. This will contribute to society if pharmaceuticals can be developed for diseases that are currently an important problem.

Atsuko Nakamura et al., Search for antioxidants contained in plum meat extract, Bulletin of Tokyo Kasei Gakuin University, July 31, 1999, No. 39, pp23-28

Accordingly, an object of the present invention is to provide a plum extract having a medicinal effect and a composition containing the same, which can contribute to the effective use of plum.

The present inventors have conducted a series of studies on the medicinal effects of extracts from various parts of plum to achieve the above object. As a result, a plum extract having excellent medicinal effects as shown below could be obtained.

That is, the first plum extract of the present invention is at least selected from the group consisting of a plum tree trunk, a plum tree branch, a plum tree leaf, a plum tree stem, a plum tree root, a plum meat, a plum seed shell and a plum tree. A plum extract having a medicinal effect extracted from one, which is an antioxidant, a gastric mucosal damage inhibitor, a diabetic cataract preventive agent having an aldose reductase inhibitory activity, a diabetic neurological disease having an aldose reductase inhibitory activity It is a ume extract used as a prophylactic agent, a blood glucose level increase inhibitor, an alcohol absorption inhibitor, a platelet coagulation accelerator, a liver inflammation inhibitor, an anti-inflammatory agent, and a melanin pigment formation inhibitor having tyrosinase inhibitory activity.

As described above, the present invention provides ume extracts having various medicinal effects. Therefore, according to the present invention, it is possible to contribute to the effective use of plums and to provide a medicine useful for diseases that have become a social problem.

Among them, extracts from at least one of plum leaves and plum stems are antioxidants, diabetic cataract preventives, diabetic neurological disease preventives, platelet aggregation promoters, anti-inflammatory agents or melanin pigment formation suppression. It is extremely excellent as an agent and is preferable. In addition, the stem of a plum tree refers to a portion that supports a leaf existing between a leaf and a branch. Hereinafter, the leaf and the stem are collectively referred to as a leaf stem.

場合 When the first extract is used as a gastric mucosa damage inhibitor or an alcohol absorption inhibitor, the ume extract is preferably an extract from ume seeds. This is because the extract is excellent in the above-mentioned medicinal effects. In the first extract, when the ume extract is used as a blood sugar rise inhibitor or a liver inflammation inhibitor, the extract has excellent medicinal effects regardless of whether it is an extract of ume kernel or leaf stem. Show.

It is preferable that the first extract contains at least one substance selected from the group consisting of six substances represented by the following chemical formulas (Chemical Formula 1) to (Chemical Formula 6). This is because these substances are presumed to be involved in the above various drug effects. Hereinafter, the substance of the following chemical formula (chemical formula 1) is referred to as PM-1, the substance of the following chemical formula (chemical formula 2) is referred to as PM-2, the substance of the following chemical formula (chemical formula 3) is referred to as PM-3, and the substance of the following chemical formula (chemical formula 4) is referred to. PM-4, a substance of the following chemical formula (Chemical Formula 5) is referred to as PM-5, and a substance of the following chemical formula (Chemical Formula 6) is referred to as PM-6, respectively. The names of these substances are as follows.

PM-1: 3β-hydroxy-12-olean-28-oic
acid
PM-2: 2α, 3β-dihydroxy-12-ursen-28-
oic acid
PM-3: 3β, 19α-dihydroxy-2-oxo-12-
ursen-28-oic acid
PM-4: 3β-hydroxy-12-ursen-28-oic
acid
PM-5: 2α, 3β-dihydroxy-12-olean-28-
oic acid
PM-6: 2α, 3α-dihydroxy-12-olean-28-
oic acid

Next, the second extract of the present invention is an extract from plum blossoms, and is a diabetic cataract preventive agent having aldose reductase inhibitory activity, a diabetic neurological disease preventive agent having aldose reductase inhibitory activity , An anti-inflammatory agent, an antioxidant, a melanin pigment formation inhibitor having tyrosinase inhibitory activity, and a platelet aggregation inhibitor. In the present invention, the plum blossom refers to a reproductive organ of the plum, and refers to a portion composed of pistils, stamens, petals, sepals, floral patterns, bracts, and the like.

It is preferable that the second extract contains at least one substance selected from the group consisting of eight kinds of substances represented by the following chemical formulas (Chem. 7) to (Chem. 14). This is because these substances are presumed to be involved in the above various drug effects. Hereinafter, the substance of the following chemical formula (Chemical formula 7) is referred to as PM-7, the substance of the following chemical formula (Chemical formula 8) is referred to as PM-8, the substance of the following chemical formula (Chemical formula 9) is referred to as PM-9, and the substance of the following chemical formula (Chemical formula 10). PM-10, the substance of the following chemical formula (Chemical formula 11) is PM-11, the substance of the following chemical formula (Chemical formula 12) is PM-12, the substance of the following chemical formula (Chemical formula 13) is PM-13, and the following chemical formula (Chemical formula 14). The substance is referred to as PM-14, respectively. The names of these substances are as follows.

PM-7: 2 '''-O-Acetylrutin
PM-8: Isohamnetin 3-rhamnoside
PM-9: Rutin
PM-10: Qurcetine 3-O-rhamnopyranos
yl (1 → 6) galactoside
PM-11: Quercetin 3-O-neohesperidoshi
de
PM-12: Eugenylglucoside
PM-13: Benzyl Glucopyranoside
PM-14: Bezyl alcohol xylosyl (1 → 6)
glucoside

に お い て In the present invention, the ume extract is preferably an organic solvent extract, and particularly preferably an alcohol extract. The alcohol extract is preferably at least one of an ethanol extract and a methanol extract, and particularly preferably a methanol extract. In addition, the ume extract is preferably a dry distillation extract.

に お い て In the present invention, the form of the ume extract is not particularly limited, and may be, for example, a powder form or a liquid form (including a paste form).

Next, the composition of the present invention contains the ume extract of the present invention. If this composition contains the ume extract of the present invention as a main component or an active ingredient, other components are not particularly limited, and are appropriately determined according to the use. For example, when the composition is a medicament, its dosage form is a solid or liquid excipient containing the ume extract as a main component or an active ingredient, and in the case of an internal medicine, powder, tablets, capsules, There are tea, granules, and liquids (alcoholic, tincture, liquid extract, syrup, etc.). In addition, injections, ointments, solutions, compresses, crude drugs, sprays, nutritional enemas, emulsions and the like are available. As the excipient, those known in the art can be used. For example, as excipients for powders for internal use such as powders, granules, capsules and tablets, lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic or natural aluminum silicate, magnesium oxide, dry aluminum hydroxide, Examples include magnesium stearate, sodium bicarbonate, and dried yeast. Examples of excipients for external powders include zinc oxide, talc, starch, kakirin, boric acid powder, zinc stearate, magnesium stearate, bismuth hypogallate, and potassium aluminum sulfate powder. Excipients in the liquid preparation include water, glycerin, propylene glycol, syrup, ethanol, fatty acid, ethylene glycol, polyethylene glycol, sorbitol and the like. As excipients in ointments, fats, fatty oils, lanolin, petrolatum, glycerin, beeswax, mocro, paraffin, liquid paraffin resins, higher alcohols, plastics, glycols, water, surfactants, etc. Bases or hydrophilic bases (including emulsion bases, water-soluble bases, and suspending bases).

When the composition of the present invention is used as a health food or a food additive, it can be used in the form of tablets, capsules, granules, powders, liquids, etc., but in a form that can be clearly distinguished from pharmaceuticals. It is preferred to use. In this case, the above-mentioned excipients can be used.

に お い て In the composition of the present invention, the content ratio of the ume extract is, for example, in the range of 1 to 60% by weight, and preferably in the range of 10 to 40% by weight, based on the whole composition.

Next, the ume extract of the present invention can be produced, for example, as follows.

First, when extracting with an organic solvent, the branches and leaf stems of the plum tree are processed into flakes. This processing can be performed using a wood cutter or the like for hard parts such as the trunk, branches and roots of the plum tree, and using a cooking cutter or mixer for soft parts such as plum blossoms and leaf stems. Can be done.

Then, flake-shaped plum tree branches and the like are immersed in an organic solvent for extraction. Examples of the organic solvent include methanol, ethanol, hexane, acetone, ethyl acetate, glycerin, propylene glycol, and n-butanol. Among them, as described above, methanol or ethanol is preferable, and methanol is particularly preferable. In addition, these organic solvents may be used alone or in combination of two or more. The amount of the organic solvent used is usually 5 to 10 times the volume of the flake, and preferably about 5 times the volume. The immersion (extraction process) may be performed once, but is preferably performed twice or more. In addition, the extraction is preferably heated under reflux. In this case, the time required for the extraction is usually 1 hour to 3 hours, preferably about 3 hours. The temperature of the organic solvent is appropriately determined depending on the type of the solvent, but the temperature at which the reflux of the extraction solvent occurs Is preferable, and specifically, the range of about 50 to 80 ° C. is preferable. When extraction is performed at room temperature, the extraction time is preferably all day and night. By this extraction, a medicinal component is extracted into the organic solvent from the branches, leaves and stems of the plum tree, the flowers of the plum, and the like. Then, flakes such as plum tree branches are separated with a filter or the like, and the extract is recovered.

The extract may be used as it is, or may be used as a powder or paste by evaporating (eg, drying under reduced pressure) the organic solvent.

Next, in the case of dry distillation, first, the branches and leaf stems of the plum tree are processed into flakes in the same manner as described above. Then, this is subjected to heat treatment (dry distillation) while shutting off the air. The heating temperature at this time is usually 90 to 300 ° C., and the heating time is usually 1 to 3 hours. For this dry distillation, for example, a retort pot or a dry distillation pot can be used. The liquid flowing out by the carbonization is separated and collected as the extract from the branches and the like. This dry distillation extract may be used as it is, or may be dried and used in the form of powder or paste.

The type of plum used in the present invention is not particularly limited, for example, Minamikoume, Kozoku Minamitaka, Kojo, Improved Uchida, Shirakaga, Yosei, Linzhou, Uguisuku, Koshu Koume, Beni-sashi, Minahira, etc. Can be used.

Next, embodiments of the present invention will be described. The plums used in the following examples are Minamitaka plums.

(Example 1)
Example 1 This example is an example in which the methanol extract of plum kernel and leaf stem was confirmed to have an inhibitory effect on gastric mucosal damage.

(Preparation of leaf stem extract)
5.1 kg of the finely ground plum leaf stem was extracted by heating and refluxing for 3 hours with 5 times the volume of methanol. After the extract was filtered, the residue was again heated and refluxed with methanol in the same manner as above to perform extraction. Then, the obtained extracts were combined and dried under reduced pressure to obtain 338 g of an extract. The physical properties are shown below.

(Appearance and properties)
Blackish brown paste with slight peculiar smell.

(Thin layer chromatography)
(1) Condition carrier: silica gel (60F254, manufactured by Merck)
Developing solvent: chloroform: methanol: water = 10: 3: 1 mixture (volume ratio)
Color development: heating with 10% cerium sulfate and 10% sulfuric acid aqueous solution (2) Rf value spot 1: 0.70 (red)
Spot 2: 0.63 (brown)

(Infrared absorption spectrum)
(1) Measuring equipment: Shimadzu FT-IR DR-8000 spectrometer
(2) Measurement results (unit: cm ^-1 )
3432 (hydroxyl group), 2936 (methyl group, methylene group, methane group), 1736 (carbonyl group), 1655 (unsaturated bond), 1383 (methyl group, methylene group, methane group), 1109, 1082, 1037 (hydroxyl group, (Ether bond), 792, 760 (unsaturated bond). The results are shown in the chart of FIG.

(Preparation of umenin extract)
2.5 kg of finely ground ume seeds were heated and refluxed with 5 times the volume of methanol for 3 hours to perform extraction. After the extract was filtered, the residue was again heated and refluxed with methanol in the same manner as above to perform extraction. Then, the obtained extracts were combined and dried under reduced pressure to obtain 223 g of an extract. The physical properties are shown below.

(Appearance and properties)
It is brownish paste and has a slight peculiar smell.

(Thin layer chromatography)
(1) The conditions are the same as above.
(2) Rf value spot 1: 0.10 (brown)
Spot 2: 0.95 (brown)

(Infrared absorption spectrum)
(1) Measuring equipment: Same as above.
(2) Measurement results (unit: cm ^-1 )
3453 (hydroxyl group), 2928 (methyl group, methylene group, methane group), 2500 to 2000 (phenolic hydroxyl group, carboxyl group), 1746 (carbonyl group), 1655 (unsaturated bond), 1072, 1051 (hydroxyl group, ether bond) ) Has a characteristic absorption. The results are shown in the chart of FIG.

(Confirmation of gastric mucosal damage inhibitory effect)
After the male SD rats (body weight: about 250 g) were fasted for about 24 hours, the methanol extract was orally administered. One hour later, methanol was orally administered at a rate of 1.5 ml / animal, and one hour later, the stomach was removed. The stomach was subjected to formalin treatment, and the maximum diameter (damage coefficient (mm)) of the injured portion generated in the glandular stomach and the score (0 to 9) according to the following criteria were determined. As a control, the damage coefficient (mm) and the score (0 to 9) were similarly obtained for rats to which the methanol extract was not administered. The suppression rate (%) was determined by the following equation. The results are shown in Table 1 below. In addition, in the same table, the value is shown by an average value ± standard error (significant difference: * P <0.05, ** P <0.01), and the other tables are also the same.

Inhibition rate (%) = 100 − [(C / A) × 100]
A: Damage factor of methanol-administered rats C: Damage factor of control rats

(Score)
0: No damage 1: Less than 5 slight damages with a total length of 5 mm or less and 2 mm or less 2: 5 or more slight damages with a total length of 5 mm or less and 2 mm or less 3: 5 with moderate damage of a total length of 5 mm or more and 2 mm or less in width Less than 4 pieces: 5 or more moderate damages with a total length of 5 mm or more and 2 mm or less 5: 1 to 3 bleeding bands of moderate damage with a total length of 5 mm or less and 2 mm or more 6: Medium length of 5 mm or less and a width of 2 mm or more 4 or more hemorrhagic bands of damage 7: 1 to 3 severely injured hemorrhagic bands having a total length of 5 mm or more and 2 mm or more 8: 4 to 6 9 of severely injured hemorrhagic bands having a total length of 5 mm or more and 2 mm or more 9 : 7 or more hemorrhagic bands with severe damage of 5 mm or more in total length and 2 mm or more in width

(Table 1)

Plum extract type Dose N Damage coefficient Inhibition rate Score
(Mg / kg) (animal) (mm) (%)
Control −8 173.4 ± 16.9 −7.8 ± 0.2
Plum leaf stem 250 6 92.8 ± 22.8 ** 46.5 5.7 ± 0.7 *
Plum leaf stem 500 6 24.0 ± 12.4 ** 86.2 2.2 ± 0.7 **
Umenin 250 6 46.1 ± 4.4 ** 73.4 3.5 ± 0.8 **
Umenin 500 6 25.8 ± 11.7 ** 85.1 1.7 ± 0.7 **
(* P <0.05, ** P <0.01)

明 ら か As is clear from Table 1 above, the gastric mucosal damage was suppressed by giving the ume methanol extract, and this effect was better with umenin.

(Example 2)
This example is an example in which the antioxidant effect of the methanol extraction part of the plum stem was confirmed. The methanol extract of the leaf stem was prepared in the same manner as in Example 1.

(Method of confirming antioxidant action)
An ethanol solution of the stable radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) has an absorption at a blue color of 517 nm, and utilizes the fact that when a radical scavenger is added, the color fades according to the amount added. First, 1 ml of ethanol, 1 ml of an ethanol solution of the above extract (concentration 0 to 100 μg / ml), 2 ml of 0.2 M acetate buffer (pH 5.5) and a DPPH solution (2.0 × 10 ⁻⁷ mol / ml ethanol solution) 1 ml was mixed and left for 30 minutes. Thereafter, the absorbance at 517 nm of this mixture was measured. On the other hand, a mixture using 1 ml of ethanol instead of the DPPH solution was used as a blank, and the absorbance at 517 nm was measured in the same manner as described above. Then, the amount of the extract necessary for reducing the absorbance to 1/2 was calculated and used as an index of antioxidation. In addition, as a comparative example, α-tocopherol and green tea were used in place of the extract, and the antioxidant power was also examined. The results are shown in Table 2 below.

(Table 2)

Sample Antioxidant power
Plum leaf stem extract 10μg
α-tocopherol 21 μg
Green tea 20μg
(Antioxidant power: 1.0 × 10 ^-7 mol Amount required to capture DPPH)

As is apparent from Table 2, the plum leaf stem extract has excellent antioxidant activity, which is twice as strong as α-tocopherol used as an antioxidant in foods and the like. Met.

(Example 3)
This example is an example in which the effect of suppressing the increase in blood glucose level of the plum kernel and leaf stem was confirmed. In this example, the umenin methanol extract and the ume leaf stem methanol extract were prepared in the same manner as in Example 1.

(Confirmation of blood glucose elevation suppression effect)
The extract (500 mg / kg) was orally administered to male Wistar rats (body weight 150 to 180 g) that had been fasted for about 20 hours, and 30 minutes later, sucrose (1.0 g / kg) was orally administered. Thirty minutes, one hour and two hours later, about 0.2 ml of blood was collected from the orbital vein. The collected blood was centrifuged at 3000 rpm to obtain serum, and the blood glucose level was measured by a glucose oxidase method using a commercially available kit (Glucose CII Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). Further, as a normal group, the blood glucose level of the rats to which the extract and sucrose were not administered were similarly measured, and the blood glucose level of the rats to which only the sucrose was administered without administration of the extract was used as a control group. It was measured. The results are shown in Table 3 below. The number of rats subjected to the experiment was 6 in each treatment group.

(Table 3)

Treatment group Blood sugar level (mg / 100ml)
0.5h 1.0h 2.0h
Normal group 73.1 ± 4.6 ** 81.9 ± 6.8 ** 80.2 ± 5.3 **
Control group 182.0 ± 5.7 163.1 ± 4.5 109.4 ± 3.5
Umejin 172.7 ± 4.8 155.8 ± 9.5 107.8 ± 4.4
Plum stem 174.5 ± 9.7 157.5 ± 4.5 109.8 ± 4.6

As is clear from Table 3, the administration of the extract of plum kernel and leaf stem suppressed the increase in blood sugar level. In addition, regarding this effect, it was considered that there was not much difference between the kernel and the leaf stem.

(Example 4)
This example is an example in which the methanol extract of umenin and the methanol extract of the stem portion were confirmed to have an effect of suppressing alcohol absorption. Each methanol extract was prepared in the same manner as in Example 1.

(Confirmation of alcohol absorption suppression effect)
The extract (500 mg / kg) was orally administered to male Wistar rats (body weight: about 210 to 240 g) which had been fasted for about 20 hours, and 30 minutes later, ethanol (20% (v / v), 5 ml / kg) was administered. It was administered orally. Thirty minutes, one hour and two hours later, about 0.5 ml of blood was collected from the orbital vein, and the blood ethanol concentration was measured by an enzyme method (a blood alcohol test "BMY", a commercially available measurement kit manufactured by Boehringer Mannheim). )). In addition, as a control group, the ethanol concentration in the blood was similarly measured for rats to which ethanol was administered without administering the extract. The results are shown in Table 4 below. The number of rats subjected to the experiment was 5 in each treatment group.

(Table 4)

Treatment group Blood ethanol concentration (mg / ml)
0.5h 1.0h 2.0h
Control group 0.825 ± 0.051 0.671 ± 0.022 0.337 ± 0.003
Umenin 0.571 ± 0.154 0.586 ± 0.079 0.332 ± 0.029
Leaf stem 0.783 ± 0.147 0.760 ± 0.086 0.512 ± 0.067

(4) As is clear from Table 4, the administration of the umenin methanol extract and the leaf stem methanol extract suppressed the alcohol absorption. In addition, this effect was superior in the extract of umenin.

(Example 5)
In this example, the aldose reductase inhibitory activity of a rat lens-derived aldose reductase of a umenin methanol extract and a leaf stem methanol extract was confirmed. Aldose reductase is an enzyme involved in diabetic cataract and diabetic neuropathy. Each methanol extract was prepared in the same manner as in Example 1.

(Preparation of aldose reductase solution)
5 g of a lens of a Wistar male rat (6 weeks old) was homogenized in 20 ml of a phosphate buffer (135 mM, pH 7.0, containing 10 mM mercaptoethanol), and centrifuged at 100,000 × g for 30 minutes. Was used as an enzyme solution.

(Confirmation of aldose reductase inhibitory activity)
135 mM phosphate buffer (pH 7) containing 1 mM DL-glyceraldehyde, 0.03 mM NADPH, 0.1 M lithium sulfate, the aldose reductase solution from the rat lens and the extract DMSO solution (concentration 0 to 30 μg / ml) .0) was incubated at 30 ° C. for 30 minutes. The reaction was started by adding NADPH and stopped by adding hydrochloric acid. After the reaction solution in which the reaction was stopped was treated with a strong alkali, the solution was heated at 60 ° C. for 10 minutes and allowed to reach room temperature. Thereafter, the fluorescence intensity of the reaction solution was measured (excitation wavelength: 360 nm, emission wavelength: 460 nm). The aldose reductase inhibitory activity was evaluated as the amount of the extract that reduced the activity to 50% (IC ₅₀ (μg / ml)).

As a result, the plum leaf stem extract had an IC ₅₀ of 4.96 μg / ml, and showed an inhibitory activity in a trace amount. Also, the umenin extract showed 29.3% inhibitory activity at a concentration of 30 μg / ml.

(Example 6)
This example is an example of confirming the platelet aggregation-promoting effect of the methanol extract of plum buds. The plum stem methanol extract was prepared in the same manner as in Example 1.

First, washed platelets (5 × 10 ⁵ cells / ml) were prepared from whole blood of a Japanese white male rabbit by a conventional method, and at 37 ° C., in the presence of 1 ml Ca ²⁺ , a methanol extract (100 μg / ml) of plum leaf stems was prepared. And the change in light transmittance at that time was measured (platelet aggregometer: Model PAT-4A, manufactured by Nikko Bioscience). In addition, as a positive control, stimulation with platelet activating factor (PAF) instead of the extract was performed, and the change in light transmittance was measured in the same manner. Then, the platelet aggregation promoting action of the methanol extract of the plum leaf stem was expressed as a relative ratio with respect to the aggregation rate of the positive control as 100%. The results are shown in the chart of FIG. In this chart, Ca indicates the time when Ca ²⁺ was added, and S indicates the time when the aggregation stimulus was started.

As is clear from FIG. 1, the addition of the methanol extract of the stem and plum of the plum caused platelet aggregation to occur immediately, and after 1 minute, the relative aggregation rate increased to about 45%.

(Example 7)
This example is an example of confirming the liver inflammation-suppressing action of the umenin methanol extract and the leaf stem methanol extract. Each methanol extract was prepared in the same manner as in Example 1.

(Confirmation of suppression of D-galactosamine / LPS-induced acute liver inflammation)
The ume extract was orally administered at a rate of 1000 mg / kg to ddY male mice (10 mice, body weight 25 to 30 g) which had been fasted for about 20 hours, and 1 hour later, D-galactosamine and lipopolysaccharide (LPS) were administered. Intraperitoneal administration was performed at a rate of 350 mg / kg and 10 μg / kg, respectively. Ten hours later, blood is collected, serum is obtained by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the transaminase (s-GPT, s-GOT) activity in the serum is determined using a commercial kit (S, T.A. (Yakusha). In addition, mice (10 mice) treated in the same manner except that the ume extract was not administered were used as a control group. Then, the ratio of the transaminase activity of the ume extract-administered mouse to the control group was calculated as the inhibition rate (%). The results are shown in Table 5 below.

(Table 5)
s-GPT s-GOT
Inhibition rate (%) 26.6 ± 15.8 32.6 ± 11.7

(D-galactosamine-induced acute hepatitis inhibitory action in primary cultured rat hepatocytes)
Liver parenchymal cells collected from a Wistar male rat (body weight 120 to 150 g) by a collagenase perfusion method are suspended in William's medium containing 10% calf serum (CG) and seeded at 4 × 10 ⁴ cells / ml in a 96-well flat bottom microplate. And incubated for 4 hours. Then, the medium was replaced with a medium containing 1 mM D-galactosamine and the ume extract (concentration: 3, 10, 30, 100, 300 μg / ml), incubated for 44 hours, and then incubated at 5 mg / ml 3- (4,4). 5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) was added in 10 μl portions and incubated for 4 hours. Next, the medium was removed, formazan was extracted with 2-propanol containing 0.04N HCl, and the absorbance was measured (measurement wavelength: 570 nm, reference wavelength: 655 nm). A control obtained by performing the same operation as described above except that the ume extract was not added to the medium was used as a control. Then, the ratio of the measured value when the ume extract was added to the control was calculated as the suppression rate (%). The results are shown in Table 6 below.

(Table 6)
Extract concentration
(Μg / ml) 3 10 30 100 300
Inhibition rate (%) 5.2 ± 0.5 9.4 ± 0.8 15.4 ± 1.0 7.9 ± 0.7 2.8 ± 0.2

急性 As is clear from Tables 5 and 6, acute hepatitis was suppressed by administration of the ume extract.

(Example 8)
This example is an example in which the inflammation-suppressing action of the methanol extract of plum stem was confirmed. The plum stem methanol extract was prepared in the same manner as in Example 1.

(Confirmation of anti-inflammatory effect)
The anti-inflammatory effect was evaluated by examining NO production from intraperitoneal macrophages of mice by LPS (10 μg / ml) stimulation.

(Measurement of macrophage NO)
Macrophages collected from the abdominal cavity of ddY strain male mice (body weight about 30 g) were suspended in RPMI-1640 medium containing 10% fetal calf serum (FCS), and seeded at 5 × 10 ⁵ cells / ml in a 96-well flat bottom microplate. Incubated for hours (37 ° C., 5% CO ₂ ). Subsequently, the medium was replaced with a medium containing 10 μg / ml LPS and the ume extract (concentration: 3, 10, 30, 100, 300 μg / ml) and incubated for 20 hours. Since NO is unstable and difficult to directly measure, NO ₂ which is a metabolite of NO was quantified using a Griess reagent (Griess method). That is, the same amount of a grease reagent (1% sulfanilamide / 0.1% N-1-naphthylethylenediamine / 5% phosphoric acid) as that of the culture supernatant was mixed, and the mixture was allowed to stand at room temperature for 10 minutes. Measurement was performed (measurement wavelength: 570 nm, reference wavelength: 655 nm), and quantification was performed using NaNO ₂ diluted with the medium as a standard. The control was the same as above except that the ume extract was not added. Then, the ratio of the measured value when the ume extract was added to the control was calculated as the inhibition rate (%). The results are shown in Table 7 below.

(Table 7)
Extract concentration
(Μg / ml) 3 10 30 100 300
Inhibition rate (%) 26.8 ± 2.2 2.7 ± 1.7 5.7 ± 1.6 13.5 ± 1.4 30.5 ± 0.9

炎症 As is clear from Table 7, administration of the plum extract suppressed inflammation.

(Example 9)
This example is an example in which the melanin pigment production inhibitory effect of the plum stem stem methanol extract was confirmed. The plum stem methanol extract was prepared in the same manner as in Example 1.

(Confirmation of melanin pigment formation inhibitory action)
It was evaluated by its inhibitory activity against mushroom-derived tyrosinase. That is, 0.25 mg was added to a mixture (1.9 ml) containing the ume extract (concentration: 30, 100, 300 μg / ml) and a substrate (L-dopa) in a 40 mM phosphate buffer (pH 6.8). 0.1 ml / ml tyrosinase (from mushrooms) was added and incubated at 25 ° C. for 5 minutes. Thereafter, the absorbance at 475 nm was measured. The control was the same as above except that the ume extract was not added. Then, the ratio of the measured value when the ume extract was added to the control was calculated as the inhibition rate (%). The results are shown in Table 8 below.

(Table 8)
Extract concentration
(Μg / ml) 30 100 300
Suppression rate (%) 3.4 10.5 34.3

明 ら か As is clear from Table 8, the administration of the ume extract suppressed the production of melanin pigment.

(Example 10)
This example is an example of purification of a substance which is presumed to contribute to the above various medicinal effects.

First, a methanol extract (338.0 g) of a plum leaf stem was prepared in the same manner as in Example 1. Then, 326.7 g of this extract was partitioned and extracted using 10 liters of ethyl acetate (AcOEt) and 10 liters of distilled water (H ₂ O), and the AcOEt-soluble fraction (93.4 g) and the water-soluble fraction (233 .3 g). Then, the AcOEt-soluble fraction (80.0 g) was fractionated into eight fractions (Fr. 1 to Fr. 8) using silica column chromatography. In this fractionation, a mixed solution of hexane (Hex) -AcOEt (volume ratio: 10: 1 → 5: 1 → 3: 1) was used first, and then a mixed solution of CHCl ₃ -methanol (MeOH) (volume ratio, 10: 1 → 5: 1 → 3: 1) and finally a MeOH solution. As a result, Fr. 4 (CHCl ₃ : MeOH = 10: 1) gave PM-1 (49 mg), PM-2 (61 mg), PM-3 (25 mg) and PM-4 (81 mg), and Fr. In the 5 (CHCl ₃ : MeOH = 5: 1) fraction, PM-5 (38 mg) and PM-6 (20 mg) were obtained. FIG. 2 shows these purification steps.

(Example 11)
This example is an example in which the aldose reductase inhibitory activity of a methanol extract of a plum flower derived from a rat lens was confirmed. Aldose reductase is an enzyme involved in diabetic cataract and diabetic neuropathy. The methanol extract of the plum blossom was prepared as follows, and the aldose reductase inhibitory activity was examined using the same as described below.

(Preparation of Plum Flower Methanol Extract)
3.0 kg of the finely ground plum blossom was extracted by heating and refluxing for 5 hours with 5 times the volume of methanol. After the extract was filtered, the residue was again heated and refluxed with methanol in the same manner as above to perform extraction. Then, the obtained extracts were combined and dried under reduced pressure to obtain 250.1 g of an extract. The physical properties are shown below.

(Appearance and properties)
Blackish brown powder with slight peculiar smell.

(Thin layer chromatography)
(1) Condition carrier: silica gel (60F254, manufactured by Merck)
Developing solvent: chloroform: methanol: water = 10: 3: 1 mixture (volume ratio)
Color development: heated with 10% cerium sulfate and 10% sulfuric acid aqueous solution (3) Rf value spot 1: 0.70 (yellow)
Spot 2: 0.63 (yellow)

(Preparation of aldose reductase solution)
5 g of a lens of a Wistar male rat (6 weeks old) was homogenized in 20 ml of a phosphate buffer (135 mM, pH 7.0, containing 10 mM mercaptoethanol), centrifuged at 100,000 × g for 30 minutes, and the obtained supernatant was obtained. Was used as an enzyme solution.

(Confirmation of aldose reductase inhibitory activity)
1 mM DL-glyceraldehyde, 0.03 mM NADPH, 0.1 M lithium sulfate, the rat lens-derived aldose reductase solution and the extract DMSO solution (concentration: 1, 3, 10, 30 μg / ml), 135 mM phosphate buffer The solution (pH 7.0) was mixed and incubated at 30 ° C. for 30 minutes. The reaction was started by adding NADPH and stopped by adding hydrochloric acid. After treating the reaction solution in which the reaction was stopped with a strong alkali, heating at 60 ° C. for 10 minutes, NADP produced by a fluorescence method (excitation wavelength: 360 nm, emission wavelength: 460 nm) was quantified, and the obtained value was calculated as follows. The inhibition rate of aldose reductase was calculated by substituting into the equation. The results are shown in Table 9 below.

Inhibition rate (%) = [(AB) / A] × 100
A: Amount of NADP produced when no extract is added B: Amount of NADP produced when an extract is added

(Table 9)
Extract concentration (μg / ml) 13 10 30
Inhibition rate (%) 29.9 51.2 76.8 89.6

As can be seen from Table 9, the ume flower methanol extract had excellent aldose reductase inhibitory activity (IC ₅₀ = 3 μg / ml).

(Example 12)
This example is an example in which the inflammation-suppressing action of a plum flower methanol extract was confirmed. A plum flower methanol extract was prepared in the same manner as in Example 11.

(Confirmation of anti-inflammatory effect)
The anti-inflammatory effect was evaluated by examining NO production from intraperitoneal macrophages of mice by LPS (10 μg / ml) stimulation.

(Measurement of macrophage NO)
Macrophages collected from the abdominal cavity of a ddY male mouse (body weight about 30 g) were suspended in RPMI-1640 medium containing 10% fetal calf serum (FCS), and seeded at 5 × 10 ⁵ cells / ml in a 96-well microplate. Incubated for hours (37 ° C., 5% CO ₂ ). Subsequently, the medium was replaced with a medium containing 10 μg / ml LPS and the ume extract (concentration: 3, 10, 30, 100, 300 μg / ml) and incubated for 20 hours. Since NO is unstable and difficult to directly measure, NO ₂ which is a metabolite of NO was quantified using a Griess reagent (Griess method). That is, the same amount of a grease reagent (1% sulfanilamide / 0.1% N-1-naphthylethylenediamine / 5% phosphoric acid) as that of the culture supernatant was mixed, and the mixture was allowed to stand at room temperature for 10 minutes. Measurement was performed (measurement wavelength: 570 nm, reference wavelength: 655 nm), and quantification was performed using NaNO ₂ diluted with the medium as a standard. The control was the same as above except that the ume extract was not added. Then, the ratio of the measured value when the ume extract was added to the control was calculated as the inhibition rate (%). The results are shown in Table 10 below.

(Table 10)
Extract concentration
(Μg / ml) 3 10 30 100 300
Inhibition rate (%) 8.0 ± 4.5 1.7 ± 4.8 5.0 ± 3.8 51.6 ± 6.0 * 94.8 ± 1.2 *

As can be seen from Table 10, the methanol extract of plum blossom exhibited an excellent inflammation inhibitory effect (IC ₅₀ = 100 μg / ml).

(Example 13)
This example is an example in which the melanin pigment formation inhibitory effect of a methanol extract of plum blossoms was confirmed. A plum flower methanol extract was prepared in the same manner as in Example 11.

(Confirmation of melanin pigment formation inhibitory action)
It was evaluated by its inhibitory activity against mushroom-derived tyrosinase. That is, a 40 mM solution containing the ume extract in a DMSO solution (concentrations: 1, 3, 10, 30, 100, 300, and 1000 μg / ml), L-dopa at 0.1 mg / ml, and 500 U / ml tyrosinase (derived from mushroom). Phosphate buffer (pH 6.8) was incubated at 25 ° C. for 5 minutes. Thereafter, the absorbance at 475 nm was measured. The control was the same as above except that the ume extract was not added. Then, the ratio of the measured value when the ume extract was added to the control was calculated as the inhibition rate (%). The results are shown in Table 11 below.

(Table 11)
Extract concentration
(Μg / ml) 13 10 30 100 300 1000
Suppression rate (%) 2.3 7.5 11.3 24.1 46.3 63.5 77.5

明 ら か As is clear from Table 11, the administration of the ume extract suppressed the production of melanin pigment.

(Example 14)
This example is an example in which the antioxidant effect of the methanol extract of plum blossoms was confirmed. A plum flower methanol extract was prepared in the same manner as in Example 11.

(Method of confirming antioxidant action)
1.0 ml of 0.1 M acetic acid-sodium acetate buffer (pH 5.5), 0.5 ml of ethanol, 0.5 ml of 2.0 × 10 ⁻⁴ M ethanol solution of DPPH and 0. After mixing 5 ml and leaving the mixture at room temperature for 30 minutes, the absorbance at 517 nm was measured. From the obtained value, the amount of the extract necessary to reduce the DPPH radical of 2.0 × 10 ⁻⁷ M by 50% was calculated. As a result, the amount of the extract required to reduce the DPPH radical by 50% was 44 μg. From these results, it can be said that the plum flower extract has excellent antioxidant power.

(Example 15)
This example is an example of confirming the platelet aggregation inhibitory effect of a methanol extract of plum blossom. A plum flower methanol extract was prepared in the same manner as in Example 11.

First, whole blood was collected from the auricular artery of a Japanese white male rabbit, and then platelet-rich plasma was obtained by centrifugation. This was washed with a Tyrode-Hepes solution containing 0.4 mM EGTA to prepare washed platelets (5 × 10 ⁵ cells / ml). Then, the cells were stimulated with 0.05 U / ml thrombin for 5 minutes in the presence of the ume flower ethanol extract, and the change in light transmittance at that time was measured by the platelet aggregometer. The measured values were substituted into the following equation to calculate the platelet aggregation inhibition rate (%). The results are shown in Table 12 below and the chart of FIG.

Inhibition rate (%) = [(AB) / A] × 100
A: maximum agglutination rate when no extract is added B: maximum agglutination rate when an extract is added

Further, the methanol extract of the plum blossom was partitioned between ethyl acetate and water, and the platelet aggregation inhibitory effects of the ethyl acetate phase and the aqueous phase were examined by the method described above. The results are shown in Table 12 below and the chart of FIG. Then, the aqueous phase was further transferred to n-butanol, and the platelet aggregation inhibitory action of the n-butanol phase was examined by the above method. The results are shown in Table 12 below and the chart of FIG. In FIG. 5, FIG. 6, and FIG. 7, the term “sample” indicates the time when the extract or each phase is added, and the term “thrombin” indicates the time when thrombin is added.

(Table 12)

Concentration (μg / ml) 10 30 100
(Inhibition rate (%))
Methanol extract 0 10 28
Ethyl acetate phase --5
Aqueous phase 11 27 89
n-butanol phase 96 87 96

よ う As can be seen from the above Table 12 and the charts of FIGS. 5, 6 and 7, it was confirmed that the methanol extract of plum blossom inhibited platelet aggregation. When this methanol extract was partitioned between ethyl acetate and water, no platelet aggregation inhibitory action was confirmed in the ethyl acetate phase, but a significant platelet aggregation inhibitory action was confirmed in the aqueous phase. Furthermore, when the aqueous phase was extracted with n-butanol, concentration of activity was observed in the n-butanol phase. On the other hand, the ume flower methanol extract hardly suppressed platelet aggregation by platelet activating factor (PAF) (not shown). From the above results, it was confirmed that the plum flower extract specifically inhibited thrombin aggregation. From these results, it can be said that the plum flower extract has a preventive and ameliorating effect on pathological conditions caused by thrombosis such as myocardial infarction and cerebral infarction.

(Example 16)
This example is an example of the purification of the various substances in a plum flower methanol extract.

(Preparation of Plum Flower Methanol Extract)
3.0 kg of the finely ground plum blossom was extracted by heating and refluxing for 5 hours with 5 times the volume of methanol. After filtering the extract, the residue was again heated and refluxed with methanol in the same manner as described above, and extraction was performed, followed by filtration. Then, the obtained filtrates were combined and dried under reduced pressure to prepare 250.1 g of a methanol extract of plum blossom. Then, 250.1 g of this extract was partitioned and extracted using 10 liters of ethyl acetate (AcOEt), 10 liters of butanol (BuOH) and 10 liters of distilled water (H ₂ O), and an AcOEt soluble fraction (39.2 g) was obtained. And a BuOH-soluble fraction (51.2 g) and a water-soluble fraction (150.0 g). Then, the BuOH-soluble fraction (25.1 g) was fractionated into 16 fractions (Fr. 1 to Fr. 16) using normal phase silica gel column chromatography. In this fractionation, first, a mixed solution of chloroform (CHCl ₃ ) -MeOH (volume ratio: 10: 1 → 5: 1) was used as a solvent, and then a mixed solution of CHCl ₃ -MeOH-H ₂ O (volume ratio) , 6: 4: 1) and finally a MeOH solution. As a result, Fr. 7 (CHCl ₃ : MeOH = 5: 1), PM-12 (Eugenylglucoside: 141 mg) and PM-13 (Benzyl glycopyranoside: 54 mg) were obtained, and Fr. In the fractions of 10 (CHCl ₃ : MeOH: H ₂ O = 6: 4: 1), PM-7 (2 ′ ″-O-Acetylrutin: 63 mg), PM-8 (isorhamnetin: 36 mg), PM-10 (Quercetine 3-O-rhamnopyranosyl (1 → 6) galactoside: 48 mg) and PM-14 (Bezyl alcohol xylosyl (1 → 6) glucoside: 14 mg) were obtained, and Fr. _{12 (CHCl 3: MeOH: H} 2 O = 6: 4: 1) in fractions of, PM-9 (Rutin: 20mg ) and PM-11 (Quercetin 3-O -neohesperidoside: 69mg) was obtained. FIG. 8 shows these purification steps.

As described above, the present invention provides ume extracts having various medicinal effects. Therefore, according to the present invention, it is possible to contribute to the effective use of plums and to provide a medicine useful for diseases that have become a social problem.

It is the chart which confirmed the platelet aggregation promotion effect of the ume extract of one Example of this invention. It is a figure which shows the refinement | purification process of the medicinal component in the ume extract in another Example of this invention. It is the chart which measured the infrared absorption spectrum of the ume extract in still another Example of this invention. It is the chart which measured the infrared absorption spectrum of the ume extract in still another Example of this invention. It is the chart which confirmed the platelet aggregation promotion effect of the ume extract of further another Example of this invention. It is the chart which confirmed the platelet aggregation promotion effect of the ume extract of further another Example of this invention. It is the chart which confirmed the platelet aggregation promotion effect of the ume extract of further another Example of this invention. It is a figure which shows the refinement | purification process of the medicinal component in the ume extract in still another Example of this invention.

Claims (11)

A medicinal plum extract extracted from at least one selected from the group consisting of a plum tree trunk, a plum tree branch, a plum tree leaf, a plum tree stem, a plum tree root, a plum meat, a plum seed shell and a plum kernel. Ume extract used as a platelet aggregation promoter. The plum extract according to claim 1, wherein the plum extract is an extract from at least one of leaves of a plum tree and stems of a plum tree. The ume extract according to claim 1, wherein the ume extract contains at least one substance selected from the group consisting of six substances represented by the following chemical formulas (1) to (6).

The ume extract according to any one of claims 1 to 3, wherein the ume extract is an organic solvent extract. The ume extract according to claim 4, wherein the organic solvent extract is an alcohol extract. The ume extract according to claim 5, wherein the alcohol extract is an extract of at least one of an ethanol extract and a methanol extract. The ume extract according to claim 5, wherein the alcohol extract is a methanol extract. The ume extract according to any one of claims 1 to 7, wherein the ume extract is a dry distillation extract. The ume extract according to any one of claims 1 to 8, wherein the ume extract is in a powder form. The ume extract according to any one of claims 1 to 8, wherein the ume extract is in a liquid form. A composition containing the plum extract according to any one of claims 1 to 10.

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