JP5546040B2 - Plum extract and its production method and use - Google Patents

Description

  The present invention relates to the field of plant extracts, and more specifically, to an effective fraction group extracted from non-fruit parts (flowers, branches, leaves) of plum, and a production method and use thereof.

  Plum is a plant of Rosaceae and its scientific name is Prunus mume Sieb. Et Zucc. In horticultural studies, plum is divided into flower plum and real plum, and real plum is divided into white plum, green plum and red plum.

  WO00 / 39249 (PCT / JP99 / 07285) discloses a plum extract having medicinal properties and a composition containing the same, from plum stems and leaves, plum nuclei and plum flowers in five times the volume of methanol. It has been disclosed that the produced extract has an antioxidant action, gastric mucosa injury inhibiting action, aldose reductase inhibiting action, blood glucose level raising inhibiting action, platelet aggregation promoting action, alcohol absorption inhibiting action and inflammation inhibiting action. The plum extract is obtained by alcohol extraction method or dry distillation method. Plum stem and leaf methanol extract contains pentacyclic triterpenoid compounds, plum flower methanol extract contains flavone substances such as quercetin glycosides. However, not only is there a systematic search for the extraction process, but there is also no quantitative data on possible active ingredients of plum extract, and of course there is no specification of the effective fraction and standardization of the formulation. This crude methanol extract is not separated and purified, has a high impurity content, low active ingredient content, deep color, strong product hygroscopicity, difficult to store, and poor adaptability to processing. . In addition, the plum varieties used are not clear, and when applying the extract to the pharmaceutical field, the three basic requirements of drug efficacy, safety, and stability cannot be met without identifying the origin of the plant. .

  Therefore, in this field, focusing on the mass waste in the non-fruit part of Plum, adopting the modern process and means of Chinese medicine, separating and extracting effective fraction groups, developing standardized formulations, It is eager to use the plum forest resources sufficiently and to preserve the environment efficiently.

International Publication No. 00/39249

  An object of the present invention is to provide a plum extract.

  A second object of the present invention is to provide a method for producing the aforementioned plum extract.

  The third object of the present invention is to provide a product containing the aforementioned plum extract.

  The fourth object of the present invention is to provide use of the above-mentioned plum extract.

  The first of the present invention provides a plum extract containing squalene in an amount of 0.5-50 wt% based on the total weight of the extract.

  In another preferred example, the aforementioned extract is a fat-soluble effective fraction of plum flowers, branches and / or leaves.

  In another preferred example, the aforementioned extract further contains a water-soluble effective fraction of plum blossoms, branches and / or leaves.

In another preferred example, the aforementioned fat-soluble effective fraction is a supercritical CO ₂ fluid extract or a nonpolar / low polarity organic solvent extract.

In another preferred example, the aforementioned fat-soluble effective fraction further contains a long-chain alkane, a polyene, V _E , a plant sterol and a triterpenoid compound.

  The aforementioned water-soluble effective fraction is an alcohol-water extract and contains a flavone glycoside, a triterpenoid saponin and an organic acid.

  In another preferred example, the aforementioned water-soluble effective fraction contains 0.5-10 wt% of organic acid based on the total weight of the water-soluble effective fraction.

  In another preferred example, the above-described plum extract contains at least one selected from the following ten compounds.

  In another preferred example, the ume is a rose family ume, preferably ome.

According to the second aspect of the present invention, (i) using a supercritical CO ₂ fluid or a non-polar organic solvent, a plum raw material containing flowers, branches and / or leaves is extracted, a fat-soluble effective fraction is separated, and squalene is separated. A method for producing the above-described plum extract is provided, which includes the step of making the plum extract containing 0.5 to 50 wt%.

  In another preferred example, the ume is a rose family ume, preferably ome.

In another preferred example, the nonpolar / low polarity organic solvent is n-hexane, petroleum ether, chloroform, diethyl ether, acetone, 6 ^# solvent naphtha or mixtures thereof.

  In another preferred example, the method further comprises (ii) extracting the ume raw material extracted in step (i) with an alcohol-water solvent, and containing a flavone glycoside, triterpenoid saponin and an organic acid. Obtaining a fraction.

  In another preferred example, the alcohol-water solvent is a 0-50 v / v% ethanol solution or methanol solution.

  In another preferred example, the extraction method is heat reflux extraction, microwave assisted extraction, ultrasonic assisted extraction or a combination thereof.

  In another preferred example, the method further comprises a step (ii ') of purifying the water-soluble effective fraction by a purification method selected from membrane separation, column chromatography or a combination thereof.

In another preferred example, the aforementioned extraction method is supercritical CO ₂ fluid extraction under the conditions of extraction pressure 5-50 MPa, extraction temperature 20-90 ° C., separation temperature 20-80 ° C., separation pressure 2-10 MPa. In the extraction with a non-polar organic solvent, the leaching temperature is 10 to 70 ° C., the leaching time is 0.2 to 48 hours, and the raw material-solution ratio is W / V. A dipping method or a percolation method of 1: 3-30 is adopted.

  In another preferred example, the extraction temperature with the above-mentioned alcohol-water solvent is 60-100 ° C., the extraction time is 0.1-5 hours, and the raw material-solution ratio W / V is 1: 3-30.

  3rd of this invention provides the composition containing the plum extract which contains squalene 0.5-50 wt% with respect to the total weight of an extract.

  In another preferred example, the aforementioned extract is a fat-soluble effective fraction of plum flowers, branches and / or leaves.

  In another preferred example, the ume is a rose family ume, preferably ome.

  In another preferred example, the aforementioned composition further comprises an additional component selected from the group consisting of plum fruit extract, pine pollen, bamboo flavone and mixtures thereof.

  In another preferred example, the aforementioned composition comprises a drug composition, a food composition or a health food composition, a food formulation composition, a supplement composition, a natural drug ingredient composition or a cosmetic functional ingredient composition.

In another preferred example, the aforementioned composition is
(I) capsules, soft capsules, powders, tablets, granules, liquids for internal use, sprays, creams, emulsions, liquids or ointments;
(Ii) with drinks or wine;
Is selected from the group consisting of

  The fourth of the present invention is anti-radical, antioxidant, antibacterial, desensitization, gastrointestinal function improvement, blood acidification prevention, blood circulation promotion, fatigue elimination, stress relief, immune function enhancement, lipid metabolism regulation, weight loss, blood pressure reduction The use of the above-described plum extract, which is applied to the manufacture of a composition for cardiovascular protection, cancer prevention, hair growth or hair loss prevention, is provided.

  In another preferred example, the aforementioned composition comprises a drug composition, a food composition or a health food composition, a food formulation composition, a supplement composition, a natural drug ingredient composition or a cosmetic functional ingredient composition.

In another preferred example, the aforementioned composition is
(I) capsules, soft capsules, powders, tablets, granules, liquids for internal use, sprays, creams, emulsions, liquids or ointments;
(Ii) with drinks or wine;
Is selected from the group consisting of

  Thereby, this invention isolate | separated and extracted the effective fraction group from the Umebayashi non-fruit part.

FIG. 1 is a GC-MS spectrum of a plum flower fat-soluble effective fraction (supercritical CO ₂ fluid extract). FIG. 2 is a GC-MS spectrum of an ume branch fat-soluble effective fraction (supercritical CO ₂ fluid extract). FIG. 3 is a GC-MS spectrum of ume leaf fat-soluble effective fraction (supercritical CO ₂ fluid extract). FIG. 4 is an infrared spectrum of a plum water-soluble effective fraction. In the infrared spectrum obtained by sandwiching between potassium bromide window plates, the corresponding extract has characteristic absorption peaks in the vicinity of 3404, 2929, 1606, 1516, 1403, 1270, 1078, 868, 818, 780, and 612 cm ^−1. It is shown that. FIG. 5 is an infrared spectrum of a plum branch water-soluble effective fraction. The infrared spectrum obtained by sandwiching with potassium bromide window plate shows that the extract has characteristic absorption peaks around 3406, 2926, 1609, 1519, 1447, 1394, 1284, 1070, 611 cm ^−1. Yes. FIG. 6 is an infrared spectrum of a plum leaf water-soluble effective fraction. In the infrared spectrum obtained by sandwiching between potassium bromide window plates, the corresponding extract has characteristic absorption peaks in the vicinity of 3386, 2932, 1596, 1516, 1404, 1314, 1074, 776, 721, 611, 527 cm ^−1. It is shown that. FIG. 7 is an ultraviolet spectrum of the plum water-soluble effective fraction. The result of dissolving the extract in spectrally pure methanol and scanning in the wavelength range of 190-700 nm indicates a strong absorption at 327 nm and a second strong absorption at 290 nm. FIG. 8 is an ultraviolet spectrum of a water-soluble effective fraction of plum branch. The result of dissolving the extract in spectrally pure methanol and scanning in the wavelength range of 190-700 nm indicates a strong absorption at 280 nm and a second strong absorption at 320 nm. FIG. 9 is an ultraviolet spectrum of a water-soluble effective fraction of plum leaves. The results of dissolving the extract in spectrally pure methanol and scanning in the wavelength range of 190-700 nm indicate a strong absorption at 322 nm and a second strong absorption at 285 nm.

  Hereinafter, the present invention will be specifically described.

The inventor has extensively and extensively studied and surprisingly found that plum extract, particularly non-fruit portion of plum, contains a large amount of squalene. The aforementioned squalene is present in a fat-soluble effective fraction obtained by supercritical CO ₂ extraction or nonpolar organic solvent extraction. Furthermore, the inventors extracted the non-fruit portion of plum with an alcohol-water solution to obtain a water-soluble effective fraction. These found effective fractions have a wide range of biological uses.

  As used herein, plum (Prunus mume Sieb. Et Zucc) is a plant of the Rosaceae family of cherry blossoms. Plums used in the present invention include white plum, green plum, and red plum, and preferably, “Luna green” (Prunus mume'Da Ye Qing '), “fine leaf blue” (Prunus mume'Xi) of Oyama Ome. Ye Qing '), “Prunus mume'Hong Feng'” and “Prunus mume'Hong Ding '”.

  Although the plum extract provided by the present invention may be derived from the whole plum, it is preferably derived from a non-fruit part. The aforementioned non-fruit portion includes a flower, a branch, a trunk, a root or a leaf, and among them, a flower, a branch or a leaf is preferable.

Plum extract The plum extract provided by the present invention contains 0.5 to 50% of squalene with respect to the extract (molecular formula I).

The plum extract provided by the present invention includes an effective fraction group of non-plum fruit portions, that is, an extract of a fat-soluble effective fraction and a water-soluble effective fraction, or a mixture thereof. Fat-soluble effective fraction described above containing long-chain alkanes, polyenes, V _E plant sterol and triterpenoid compounds. The content of the fat-soluble effective fraction is 1-15% on a dry matter basis (plum flowers, plum branches and / or plum leaves). The aforementioned water-soluble effective fraction contains flavone glycosides, triterpenoid saponins and organic acids. The content of the water-soluble effective fraction is 1-20% on a dry matter basis (plum flowers, plum branches and / or plum leaves). The content of flavone glycosides in the water-soluble effective fraction is 5-70% in rutin.

  The form of the plum extract provided by the present invention is not particularly limited, but may be, for example, a powder form, a cream form, or a liquid form (including a paste form).

Squalene is an oily liquid (C ₃₀ H ₅₀ ) that is obtained by extraction and production mainly from the deep sea coral liver, and contains carbon and hydrogen elements. It does not solidify in bulk and does not solidify even at extremely low temperatures. Many experiments have shown that squalene is only contained in deep-sea shark livers, and squalene components are hardly present in liver oils of other fish.

  Deep-sea ridges live on the seabed with little sunlight, water temperature of about 2 ° C, water pressure of 0.1 ton / cubic foot, and depth of 500-1000 meters. The lack of sunlight makes it difficult for plants and planktonic organisms to survive. Oxygen is very poor because there is no photosynthesis by plants. Deep sea bream can survive in such a harsh environment, but its stubborn vitality comes from squalene in its huge liver. Squalene can supply a large amount of oxygen to cells, restore cell activity, and improve the natural healing power of the living body.

The effect of squalene is mainly
(1) To promote blood circulation, that is, to contribute to the prevention and treatment of lesions caused by blood circulation disorders such as heart disease, high blood pressure, low blood pressure and stroke,
(2) Activating functional cells in the body, which contributes to the prevention and treatment of lesions caused by oxygen deficiency in functional cells such as gastric ulcer, duodenal ulcer, enteritis, hepatitis, liver cirrhosis, pneumonia, etc. Reduce aging, improve immunity, increase resistance to diseases (including cancer),
(3) It may contribute to anti-inflammation / sterilization, that is, to diseases caused by bacteria such as colds, skin diseases, and otolaryngitis. It may be used externally to treat wounds or burns. For external use, break the capsule with a needle and apply directly.

  To obtain squalene, it is necessary to hunt a lot of deep-sea sharks, which is not only difficult, but also destroys marine ecosystems. The present invention provides a novel source of plant-derived squalene by obtaining a large amount of squalene for the first time from the non-fruit portion of the genus Rosaceae. Plum is a traditional cultivated plant in China, has a long history, and is now cultivated in large quantities. However, conventionally, the non-fruit portion of plum has not yet been effectively developed and used, and most of it is discarded, which not only greatly wastes resources but also adversely affects the environment. The present invention extracts squalene having an excellent biological effect from the non-fruit portion of plums, and can effectively solve the above problems.

Manufacturing method of plum extract Although the plum extract provided by this invention may be derived from the whole plum, it is preferable to derive from the non-fruit part of them. The aforementioned non-fruit parts include flowers, branches, trunks, roots or leaves. Plum blossoms are mainly non-fruited flowers, which are artificially collected and then dried by being exposed, fried or baked, and then prepared for use. The plum branch may be a nutrient branch cut in spring or a branch pruned in autumn, or it may be a branch after the plum tree has been cut down, but after exposure to dryness, cutting and pulverization Prepare for use. Plum leaves are usually collected twice in summer and autumn, exposed to dryness and ready for use. Plum trunks and plum roots are derived from plum trees after they are cut down.

  The plum raw material employed in the present invention may be pretreated or may not be pretreated. A preferred raw material is a 10-20 mesh piece having a water content of 10% or less.

The plum extract containing 0.5-50% of squalene provided by the present invention can be obtained by extraction with a supercritical CO ₂ fluid or a nonpolar organic solvent. Extraction with supercritical CO ₂ fluid or non-polar organic solvent also provides other fat-soluble effective fractions in plum extract.

In a preferred example, the supercritical extraction is performed by dynamic circulation extraction for 0.5-7 hours under extraction conditions of an extraction pressure of 5-50 MPa, an extraction temperature of 20-90 ° C., a separation temperature of 20-80 ° C., and a separation pressure of 2-10 MPa. Preferably, the dynamic circulation extraction is performed for 1-5 hours under the extraction conditions of an extraction pressure of 15-35 MPa, an extraction temperature of 40-70 ° C., a separation temperature of 40-60 ° C., and a separation pressure of 4-8 MPa. In supercritical CO ₂ fluid extraction, an entrainer may or may not be used.

Non-polar / low polarity organic solvents well known in the art, preferably n-hexane, petroleum ether, chloroform, diethyl ether, acetone, 6 ^# solvent naphtha or mixtures thereof may be used and are conventional methods in the art. Extraction temperature 10-70 ° C., extraction time 0.2-48 hours, raw material-solution ratio W / V: 1: 3-30 immersion method or percolation method, more preferably extraction temperature 20-50 ° C., extraction You may apply the immersion method or the percolation method of the raw material-solution ratio 1: 5-20 (W / V) for time 0.5-36 hours.

The water-soluble effective fraction in the plum extract provided by the present invention can be obtained by extraction with an alcohol-water solution. In a preferred example, the ume raw material is volatilized from one extracted with supercritical CO ₂ or leached with a polar organic solvent and leached with an alcohol-water solution. The alcohol-water solution described above is an alcohol solution having a volume ratio of 0-50%. Alcohols commonly used in this field such as ethanol and methanol may be used, and ethanol is preferably used. The extraction method described above is heat reflux extraction, microwave assisted extraction, ultrasonic assisted extraction or a combination thereof.

  In another preferred example, the process parameters of microwave assisted extraction are microwave radiation power 100-6000W, radiation time 2 minutes-5 hours, raw material-solution ratio 1: 3-30 (W / V), preferably Is a microwave radiation power of 200-4000 W, a radiation time of 5 minutes to 2 hours, and a raw material-solution ratio of 1: 5-20 (W / V).

  In another preferred example, the ultrasound assisted extraction has a raw material-solution ratio of 1: 3-30 (W / V), an ultrasonic power of 50-800 W, an ultrasonic action time of 0.2-5 hours, preferably Raw material-solution ratio 1: 5-20 (W / V), ultrasonic power 100-500 W, ultrasonic action time 0.5-3 hours.

  In another preferred example, the water-soluble effective fraction obtained by extraction with an alcohol-water solution may be further purified in combination with other high-speed separation means. Separation and purification may be performed by applying ordinary methods in this field, preferably membrane separation or column chromatography.

  In another preferred example, the membrane separation employs a spiral ultrafiltration membrane system, the operating pressure is 0.3-3 MPa, the operating temperature is 10-70 ° C., preferably spiral ultrafiltration. A membrane system is employed, the operating pressure is 0.6-1 MPa, and the operating temperature is 20-50 ° C.

  The above-mentioned oil-soluble effective fraction of flowers, branches and / or leaves and the water-soluble effective fraction may be merged and mixed uniformly to obtain an effective fraction group of plum flowers, plum branches and / or plum leaves.

Composition The composition provided by the present invention comprises a plum extract component as a main component or an active component. The above-mentioned plum extract is a plum non-fruit part fat-soluble effective fraction, plum non-fruit part water-soluble effective fraction, plum non-fruit part alcohol extract, plum non-fruit part water extract, plum fruit part extract or their Mixtures may be included. It preferably contains a fat-soluble effective fraction of ume non-fruit part, a water-soluble effective fraction and a plum fruit extract, and more preferably contains a fat-soluble effective fraction and a water-soluble effective fraction of ume non-fruit part. .

  The aforementioned plum extract in the composition contains at least one selected from the following ten compounds.

  The composition according to the present invention further contains other necessary or beneficial components for the human body, such as pine pollen, bamboo flavone or mixtures thereof.

  In the present invention, various compositions are prepared by methods well known in the art such as mixing the active ingredient with a “pharmaceutically acceptable” or “food acceptable” ingredient. May be. “Pharmaceutically acceptable” or “foodically acceptable” ingredient means that there is no excessive side reaction (eg toxicity, irritation or allergic reaction) even when administered to humans and / or animals, ie rational It has a material profit-risk ratio.

  “Pharmaceutically acceptable carrier” refers to a carrier used for administration of a therapeutic agent, and includes various excipients and diluents. The term refers to a pharmaceutical carrier that is not itself a necessary active ingredient and that is not excessively toxic after administration. In Remington's Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991, a thorough discussion of pharmaceutically acceptable excipients can be found. Pharmaceutically acceptable carriers in the composition may include liquids such as water, saline, glycerin and ethanol. In addition, auxiliary substances such as fillers, disintegrating agents, lubricants, fluidizing agents, boiling agents, wetting or emulsifying agents, corrigents, pH buffering substances may be present in these carriers.

  In another preferred form of the invention, the food-acceptable carrier or excipient described above is a filler, a disintegrant, a lubricant, a fluidizing agent, a boiling agent, a corrigent, a coating material, an edible It is selected from the group consisting of products or slow / controlled release agents.

  The dosage form of the composition according to the present invention is not particularly limited and may be any dosage form that can be administered to mammals. Preferably, capsules, soft capsules, powders, tablets, granules, liquids for internal use, sprays, It may be selected from the group consisting of creams, emulsions, liquids, ointments and the like.

  The composition according to the present invention includes a drug composition, a food composition, a health food composition, a food composition, a supplement composition, a natural drug ingredient composition or a cosmetic functional ingredient composition, but a health drink, wine It may be. These may be those containing plum extract or substantially consisting of plum extract.

Use of Plum Extract Plum extract provided by the present invention is anti-radical, antioxidant, antibacterial, desensitization, gastrointestinal function improvement, blood acidification prevention, blood circulation promotion, fatigue relief, stress relief, immune function enhancement, lipid metabolism It can effectively exert the effects of regulation, weight loss, blood pressure reduction, cardiovascular protection, cancer prevention, hair growth or hair loss prevention effect. Therefore, the present invention also provides a method for preventing / ameliorating the above-mentioned diseases by administering an effective amount of the above-mentioned plum extract to a subject in need.

  At the time of administration, the effective dosage of the plum extract used varies depending on the dosage form and the severity of the disease to be treated. The fact that the specific situation is determined by the individual situation of the subject is included in a range that can be determined by a skilled doctor.

  As used herein, the term “effective amount” refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals.

The main advantages of the present invention are:
1. A large amount of squalene was extracted from non-fruit parts of plum, especially plum.

  2. An efficient extraction process can be established to identify the effective fraction group of plum non-fruit parts and the standardization of the formulation related thereto.

  3. It can be ensured that the three basic requirements of drug effectiveness, safety and stability are satisfied.

  4). We will make full use of the conventional non-fruit portion of ume to reduce waste of resources and contribute to environmental conservation.

  5. Improve the utilization rate of plum resources, improve the economic benefits of plum forests, and contribute to increasing farmers' income.

  Hereinafter, the present invention will be further described with reference to specific examples. It should be understood that these examples are only used to illustrate the invention and do not limit the scope of the invention. Experimental methods that do not describe specific conditions in the following examples are performed under normal conditions or conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

  Unless otherwise defined, all technical and scientific terms used in the sentence have the same meanings as are well known to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. The preferred embodiments and materials described in the text are for listing only.

Measuring method GC-MS analysis method Detection equipment: 6890-5933 gas chromatography mass spectrometry system (purchased from Agilent, USA).

  Chromatographic conditions: HP5-MS chromatography column (5% phenylmethylsiloxane, 30 m × 250 μm × 0.25 μm), sample introduction amount 1 μL, sample introduction port temperature 270 ° C., program temperature rising method, column temperature 80 ° C. For 3 minutes, and then the temperature was raised from 80 ° C. to 270 ° C. at 10 ° C./min.

  Mass analysis conditions: EI ion source, source temperature 230 ° C., electron energy 70 eV, multiplier voltage 1376 eV, mass spectrometer connection port temperature 280 ° C., scanning range 50.0 to 600.0 m / z.

two. Total flavone measurement method (aluminum nitrate-sodium nitrite colorimetric method, calculation with rutin)
This method is referred to “Health Food Functional Component Detection Method” edited by Wang Amitsu (Chugoku Light Industry Publishing Co., Ltd., 2002, pp 29-31).

  Precisely weigh a predetermined amount of sample or reference product (rutin), dissolve in 30% ethanol solution, make a constant volume, and dilute to an appropriate concentration to record the total volume. Take a predetermined volume of the solution to be measured, add 0.3 mL of 5% sodium nitrite solution, shake, leave for 5 min, add 0.3 mL of 10% aluminum nitrate solution, shake and leave for 6 min. Add 2 mL of 0.0 mol / L sodium hydroxide, adjust to a scale with 30% ethanol, shake uniformly with a blank tube as a blank, and then measure the absorbance at a wavelength of 510 nm using a 1 cm colorimetric dish. A calibration curve is created with the absorbance as the ordinate and the concentration as the abscissa, and the content of all flavones in the sample is calculated.

Total flavone content (%) = (m1 × V2) / (m × V1 × 10 ⁶ ) × 100
In the formula: m1--the total content of flavone (μg) in the solution to be measured calculated from the calibration curve;
m2—amount of sample collected (g);
V1--preparative volume (mL) of liquid to be measured;
V2—Total volume of the solution to be measured (mL).

three. Method for measuring total triterpenoids Precisely weigh a predetermined amount of sample or reference sample (Ginsenoside Re), dissolve in dichloromethane, make a constant volume, put each in a 10 mL stoppered test tube, volatilize the solvent, and ice with 5% vanillin Add 0.2 mL of acetic acid solution and 0.8 mL of perchloric acid, place in a (60 ± 1) ° C. water bath, heat for 15 min, take out immediately, cool with ice water, add 5 mL of glacial acetic acid and shake uniformly. According to the spectrophotometry, the absorbance at a wavelength of 560 nm is measured, a calibration curve is prepared with the absorbance as the ordinate and the concentration as the abscissa, and the content of all flavones in the sample is calculated.
Triterpenoid saponin content (%) = (m1 × V2) / (m × V1 × 10 ⁶ ) × 100
In the formula: m1—Total content of triterpenoid saponin (μg) in the solution to be measured calculated from the calibration curve;
m2—amount of sample collected (g);
V1--preparative volume (mL) of liquid to be measured;
V2—Total volume of the solution to be measured (mL).

Four. Total acid titration method Take an appropriate amount of sample, add a small amount of carbon dioxide-free distilled water, dissolve the sample in a 250 mL constant volume bottle, heat in a 75-80 ° C. water bath for 0.5 hours, cool, Filter at a constant volume and dry with filter paper, discard the first liquid and collect the filtrate for use. Accurately take 50 mL of the prepared filtrate, add 2-3 drops of phenolphthalein indicator, and titrate with 0.1 mol / L standard alkaline solution until it turns slightly red and does not fade in 30 seconds. Record and perform a blank experiment. The acid content of the sample is calculated by the following formula.

Total acidity (%) = C × (V1−V2) × K × V3 × 100 / (m × V4)
In the formula: C—concentration mol / L of reference sodium hydroxide solution;
V1—mL of reference alkaline solution consumed by titration;
V2—mL of reference alkaline solution in which the blank is consumed;
V3—total volume of sample diluent mL;
V4—mL volume of sample solution taken during titration;
M—Sample mass or volume (g or mL)
K = 0.064
Five. HPLC measurement method with organic acid Equipment conditions: Waters 2695 high performance liquid chromatography, Waters 2996 diode array detector and C18 chromatography column;
Chromatographic conditions: mobile phase is 3% methanol and 0.01 mol / LKH ₂ PO ₄ aqueous solution (pH value = 2.85), flow rate 1 mL / min, column temperature 30 ° C., detection wavelength 210 nm, sample introduction amount 20 μL;
Pre-treatment method: Take 2 g each of Ome flower, branch, and leaf extract powders, dissolve in 50 mL of ultrapure water, filter and load, tartaric acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, The contents of ascorbic acid, maleic acid, succinic acid, fumaric acid and chlorogenic acid are measured.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.

[Example 1]
Plum flower extract Prunus mume 'Da Ye Qing' flowers are collected, dried, crushed to about 10 mesh coarse powder, 1.5 kg taken, and CO ₂ supercritical extraction clave And extracted.

  Dynamic circulation extraction was performed for 2 hours under the extraction conditions of an extraction pressure of 35 MPa, an extraction temperature of 60 ° C., a separation temperature of 40 ° C., and a separation pressure of 4 MPa, and 72 g of a fat-soluble extract of plum flowers was obtained in a separation clave (Dietmate® ) (Trademark of Hangzhou Yumi Special Technology Co., Ltd.)-F01).

According to GC-MS analysis, this extract contains mainly long-chain alkanes, polyenes, V _E , plant sterols, and triterpenoid compounds, of which squalene (triacontahexaene) is a fat-soluble compound. The content in the fraction was 1.04% by mass fraction. The results are shown in Table 1 and FIG.

  The extraction residue was taken out from the extraction clave and subjected to hot reflux extraction with a 30% ethanol-water solution.

  Thermal reflux extraction was performed for 2 hours under the extraction conditions of a temperature of 80 ° C. and a raw material-solution ratio of 1:10 (W / V) to obtain 114 g of a water-soluble extract of plum flowers (Dietmate®-F02).

  Analysis showed that the total flavone content was 18.84%, the total triterpenoid saponin content was 7.08%, and the total acid was 5.64%. The results are shown in Table 2.

According to the infrared spectrum, this extract had characteristic absorption peaks in the vicinity of 3404, 2929, 1606, 1516, 1403, 1270, 1078, 868, 818, 780, and 612 cm ^{−1 as} shown in FIG. According to the ultraviolet spectrum, this extract showed a strong absorption at 327 nm and a second strong absorption at 290 nm as shown in FIG.

  F01 and F02 were merged to obtain an effective fraction group of plum flowers (Dietmate (registered trademark) -F0).

[Example 2]
Plum branch extract Prunus mume 'Xi Ye Qing' branch is collected, dried, crushed to a coarse powder of about 10 mesh, and 1.5 kg is taken into a supercritical extraction clave. Extracted.

  Dynamic circulation extraction was performed for 2 hours under the extraction conditions of extraction pressure of 30 MPa, extraction temperature of 55 ° C., separation temperature of 45 ° C., and separation pressure of 4 MPa to obtain 64.5 g of fat-soluble extract of plum branch (Dietmate®-B01 ).

According to GC-MS analysis, this extract contains mainly long-chain alkanes, polyenes, V _E , plant sterols, and triterpenoid compounds, of which squalene (triacontahexaene) is a fat-soluble compound. The content in the fraction was 4.87% by mass fraction. The results are shown in Table 1 and FIG.

  The extraction residue was taken out from the extraction clave and subjected to hot reflux extraction with a 30% ethanol-water solution.

  Reflux extraction was performed for 2 hours under extraction conditions of a temperature of 80 ° C. and a raw material-solution ratio of 1:15 (W / V) to obtain 24 g of a water-soluble extract of plum branch (Dietmate (registered trademark), B02).

  Analysis revealed that the total flavone content was 40.59%, the total triterpenoid saponin content was 19.07%, and the total acid was 3.70%. The results are shown in Table 2.

According to the infrared spectrum, this extract had characteristic absorption peaks in the vicinity of 3406, 2926, 1609, 1519, 1447, 1394, 1284, 1070, and 611 cm ^{−1 as} shown in FIG. According to the ultraviolet spectrum, this extract showed a strong absorption at 280 nm and a second strong absorption at 320 nm as shown in FIG.

  B01 and B02 were merged to obtain an effective fraction group of Umeda (Dietmate (registered trademark) -B0).

[Example 3]
Plum leaf extract Prunus mume 'Hong Ding' leaf, which is a cultivar of Ulsan Dai Ome, is collected, dried, ground to a coarse powder of about 10 mesh, and 1.5 kg is taken for supercritical extraction. Extracted in a clave.

  Dynamic circulation extraction was performed for 2 hours under the extraction conditions of extraction pressure of 35 MPa, extraction temperature of 55 ° C., separation temperature of 40 ° C., and separation pressure of 6 MPa to obtain 97.5 g of a fat-soluble extract of plum leaves (Dietmate (registered trademark) − L01).

According to GC-MS analysis, this extract contains mainly long-chain alkanes, polyenes, V _E , plant sterols, and triterpenoid compounds, of which squalene (triacontahexaene) is a fat-soluble compound. The content in the fraction was 44.15% by mass fraction. The results are shown in Table 1 and FIG.

  The extraction residue was taken out from the extraction clave and subjected to hot reflux extraction with a 30% ethanol-water solution.

  Extraction was performed for 3 hours under the extraction conditions of a temperature of 70 ° C. and a raw material-solution ratio of 1:12 (W / V) to obtain 168 g of a water-soluble extract of plum leaves (Dietmate®-L02).

  According to the analysis, the total flavone content was 30.46%, the total triterpenoid saponin content was 4.93%, and the total acid was 5.96%. The results are shown in Table 2.

According to the infrared spectrum, this extract had characteristic absorption peaks in the vicinity of 3386, 2932, 1596, 1516, 1404, 1314, 1074, 776, 721, 611, and 527 cm ^{−1 as} shown in FIG. According to the ultraviolet spectrum, this extract showed a strong absorption at 322 nm and a second strong absorption at 285 nm as shown in FIG.

  L01 and L02 were merged to obtain an effective fraction group of plum leaves (Dietmate (registered trademark) -L0).

[ Reference Example 4]
Plum flower extract Prunus mume 'Hong Feng', which is a cultivar of Ulsan Dai Ome, is collected, dried, crushed to a coarse powder of about 10 mesh, 1.5 kg is taken into an extraction tank, Leaching was performed by adding n-hexane.

  Leaching was carried out for 4 hours under a leaching condition of a temperature of 25 ° C. and a raw material-solution ratio of 1:10 (W / V). After an extract was obtained, n-hexane was recovered and 85 g of plum flower fat-soluble extract was obtained. Obtained (Dietmate®-F11).

  After volatilizing the solvent from the plum flower residue, pure water was added and microwave assisted extraction was applied.

  Extraction was performed under the conditions of microwave radiation power 2000 W, radiation time 30 min, raw material-solution ratio 1:12 (W / V), and the extract was concentrated and dried to obtain 102 g of plum flower water-soluble extract (Dietmate (registered) Trademark) -F12).

  F11 and F12 were merged to obtain Dietmate®-F1.

[ Reference Example 5]
Plum branch extract The branch of Prunus mume 'Da Ye Qing', the cultivar of Ulsan Dai Ome, is collected, dried, crushed to a coarse powder of about 10 mesh, and 1.5 kg is taken into the extraction tank. Leaching was performed by adding petroleum ether.

  Leaching was conducted for 3 hours under a leaching condition of a temperature of 20 ° C. and a raw material-solution ratio of 1:15 (W / V) to obtain an extract, and then petroleum ether was recovered to obtain 65 g of ume branch fat-soluble extract. (Dietmate®-B11).

  After volatilizing the solvent from the plum branch residue, hot reflux extraction was performed with 50% ethanol.

  Extraction was performed for 2 hours under conditions of a raw material-solution ratio of 1:13 (W / V) and a temperature of 80 ° C., and the extract was subjected to membrane separation, concentration, and microwave vacuum drying to obtain 20.7 g of plum plum water-soluble extract Obtained (Dietmate®-B12).

  B11 and B12 were merged to obtain Dietmate®-B1.

[ Reference Example 6]
Plum leaf extract The leaves of Prunus mume 'Xi Ye Qing', which is a cultivar of Ulsan Dai Ome, are collected, dried, crushed to a coarse powder of about 10 mesh, and 1.5 kg taken to extract tank And leached by adding diethyl ether.

  Leaching was conducted for 1.5 hours under a leaching condition of a temperature of 20 ° C. and a raw material-solution ratio of 1:16 (W / V) to obtain an extract, and then diethyl ether was recovered, 102.6 g was obtained (Dietmate®-L11).

  After volatilizing the solvent from the plum leaves residue, pure water ultrasonic assisted extraction was applied.

  Extraction is performed under the conditions of raw material-solution ratio 1:10 (W / V), ultrasonic power 500 W, ultrasonic action time 1 hour, and the extract is filtered, macropore resin adsorption analysis, concentration, spray drying, plum leaf water solution 178.4 g of sex extract was obtained (Dietmate®-L12).

  L11 and L12 were merged to obtain Dietmate (registered trademark) -L1.

[ Reference Example 7]
The Dietmate prepared in Examples 1, 2 and 3 (TM) -F02, radical B02 and L02 (DPPH ·, · OH and O · ₂ -) were determined for scavenging activities.
The method was as follows.

(1) Erase action on DPPH radicals Exactly 20 mg of DPPH was weighed and weighed into a 250 mL constant volume bottle with absolute ethanol to obtain a DPPH solution having a concentration of 2 × 10 ⁻⁴ mol / L. A ₅₁₇ after the sample (plum extract series) and its control (bamboo leaf antioxidant) were added using a characteristic absorption peak at 517 nm wavelength of the DPPH solution using a 721 UV / visible spectrophotometer. The drop value was measured to show the strength of their scavenging ability for DPPH radicals. The total volume of the reaction was set to 3 mL, and a sample was introduced as shown in Table 4. After uniformly mixing, the mixture was reacted for 30 mins, and the change in absorbance at a wavelength of 517 nm was measured. The inhibition rate of antioxidants against DPPH · was calculated according to the following formula.

Suppression rate (%) = 1− [DPPH ·] _t / [DPPH ·] _{t = 0} × 100
In the formula: [DPPH ·] _{t = 0} −Initial concentration of diphenylpicrylhydrazyl radical in the reaction system at the zero point.

[DPPH ·] The starting concentration of diphenylpicrylhydrazyl radical in the reaction system at the point of time _t −t.

  The results are shown in Table 4.

From the results, the Ome-eda extract has stronger DPPH / erasing ability than bamboo leaf antioxidant, and its IC ₅₀ (sample concentration when the inhibition rate reaches 50%) is 19.18 μg / mL. It was.

(2) Scavenging action on hydroxy radicals
In the Vc-Cu ²⁺ -H ₂ O ₂ -yeast polysaccharide system, OH is generated and suppressed by different concentrations of samples 0 ^# and 1 ^# , measured by chemiluminescence method, and luminescence intensity integration ( CP6s) was recorded. As a blank, PBS of 0.05 mol / L pH 7.8 was used instead of the sample. The sample showed its OH erasing ability due to the inhibition rate with respect to the emission intensity. The calculation method of the inhibition rate (I) was as follows.

Inhibition rate (%) = (CP6s blank−CP6s sample) × 100 / CP6s blank The sample concentration at the time when the inhibition rate reached 50% was regarded as a half-suppression concentration, and described as IC ₅₀ . The smaller the value, the stronger the antioxidant capacity. IC ₅₀ was determined from a linear reaction linear regression equation between the sample concentration and the inhibition rate, and the strength of the OH-erasing ability of the sample and the control was compared.

  The results are shown in Table 4.

(3) Erase action on superoxide anion radical O · ₂ -is generated in pyrogallol-luminol luminescence system, suppressed by different concentrations of samples 0 ^# and 1 ^# , measured by chemiluminescence method, and luminescence every 36 seconds The intensity integral (CP36s) was recorded. As a blank, PBS of 0.05 mol / L pH 7.8 was used instead of the sample. The sample showed its O • ₂ -erasability depending on the inhibition rate with respect to the emission intensity. The calculation method of the inhibition rate (I) was the same as the measurement of the OH erasing ability. IC ₅₀ was obtained from the linear reaction regression equation between the sample concentration and the inhibition rate, and the strength of O · ₂ -elimination ability of the sample and the control was compared.
The results are shown in Table 4.

(4) Measurement of total antioxidant capacity There are many antioxidant substances in vivo, these can reduce Fe ³⁺ to Fe ²⁺ , the latter can form a strong complex with phenanthroline-based substances, Depending on the colorimetry, the strength of their antioxidant ability can be measured. Using the kit for measuring the total antioxidant capacity in serum (serum) marketed by the Nanjing Kensei Institute of Biotechnology, the total antioxidant capacity in the serum of the Ome branch extract was detected.

  The results are shown in Table 5.

As can be seen from the results, Dietmate (registered trademark) -F02, Dietmate (TM) -B02 and Dietmate (TM) -L02 are relatively good _{DPPH / · OH / O · 2} - has a scavenging ability Compared to the blank control, Dietmate (registered trademark) -F02, Dietmate (registered trademark) -B02 and Dietmate (registered trademark) -L02 can significantly improve the total antioxidant capacity of serum (p < 0.05).

[ Reference Example 8]
The promoting effect of Dietmate (registered trademark) -F02 and L02 produced in Examples 1, 2, and 3 on the growth of human isolated hair follicles was measured.

  The method was as follows.

  Human isolated hair follicles are separated by microdissection method, inoculated with 1-2 isolated hair follicles per well in a 48 well plate, 8 overlapping wells are provided per group, cultured in M199 medium, sample And a medium were prepared at a ratio of 1:19 and applied to the culture, and the medium was changed every other day. From the first day of inoculation of the isolated hair follicle, the total length of the isolated hair follicle and the total length of the hair shaft is measured 5 times with an eyepiece micrometer every other day, and the length of the hair follicle and hair shaft on the 9th and 1st day. The difference in thickness (mm) was calculated.

  The results are shown in Table 6.

  As is apparent from the results, Dietmate®-F02 low dose group (5.45 μg / mL) has a significant promoting effect on the growth of isolated hair follicles, and Dietmate®-L02 The medium dose group (50 μg / mL) has a relatively significant promoting effect on the growth of isolated hair follicles.

[ Reference Example 9]
The anti-ultraviolet radiation action of Dietmate (registered trademark) -F02, B02 and L02 produced in Examples 1, 2, and 3 was measured.

  The method was as follows.

  Dietmate (registered trademark) -F02, B02, and L02 were taken, adjusted to different concentrations with ethanol, and each was subjected to UV scanning in the 100-400 nm wavelength range.

  As is apparent from the results, Dietmate (registered trademark) -F02 (1 mg / mL) has a very strong ultraviolet absorbing ability in the 290-370 nm wavelength range, and Dietmate (registered trademark) -B02 (1 mg / mL) has a 290-355 nm wavelength. Ultraviolet absorption ability was very strong in the wavelength range, and Dietmate (registered trademark) -L02 (1 mg / mL) had very strong ultraviolet absorption ability in the 290-350 nm wavelength range.

[ Reference Example 10]
The whitening effect of Dietmate (registered trademark) -F02, B02 and L02 produced in Examples 1, 2 and 3 was measured.

  The method was as follows.

  The main enzyme when melanosomes in pigment cells synthesize melanin is tyrosinase, and its whitening effect was evaluated by measuring the inhibitory effect on tyrosinase of the sample. Add 0.5 mL of tyrosinase solution and 0.9 mL of phosphate buffer to 1 mL of the test solution, incubate for 10 min at 25 ° C., add 1 mL of sample to the reaction mixture while maintaining the temperature at 25 ° C., and react for 5 min. The absorbance D1 at a wavelength of 475 nm is measured, and in another test, the same reaction is performed using an inactivated enzyme, the absorbance D2 at a wavelength of 475 nm is measured, and the absorbance D3 is further measured without a test solution. And the inhibition rate with respect to the tyrosinase activity of a sample was computed.

Inhibition rate (%) = (D3-D1) × 100 / (D3-D2), inhibition activity is usually expressed as IC ₅₀ , that is, a half-inhibition concentration.

  As is clear from the results, Dietmate (registered trademark) -F02, B02, and L02 all remarkably suppress tyrosinase activity, and their half-inhibitory concentrations are 6.59, 2.37, and 4.41 mg / mL, respectively. Met.

[Example 11]
Umeha supercritical extract (Dietmate (registered trademark) -L01, squalene content 44.15%) produced in Example 3 was made into soft capsules according to the formulation shown below (Table 7).

  In the present invention, 10 volunteers were given 2 capsules of the aforementioned soft capsule per day. As is clear from the results, the capsules are remarkable, improve immunity, strengthen constitution, improve hypoxia, relieve cerebral and systemic fatigue, rejuvenate, lower cholesterol, It has functions such as prevention and treatment of heart and cerebrovascular diseases, acceleration of cell metabolism, promotion of ulcer healing, skin protection and beauty.

[Example 12]
Dietmate (registered trademark) -B0 produced in Example 2 and broken wall pine pollen were used as main raw materials to produce tablets according to the formulation shown below, and the amount was 2 g per tablet. The formulation is shown in Table 8.

  As is apparent from the results of administration of 2 tablets per day to 20 volunteers, the produced tablets have a significant effect on enhancing human immunity.

[Example 13]
Dietmate prepared in Example 3 (R) -L0 and bamboo flavone as a starting material (1: 1), what not even the addition other ingredients, per grain in net weight was fabricated 0 ^# capsule 300 mg.
As is apparent from the result of administration of 2 capsules per day to 6 volunteers, this capsule can remarkably enhance human immunity.

[ Reference Example 14]
The dietmate (registered trademark) -B02 produced in Example 2 was used as a raw material to prepare a drink by compounding with plum essence, sugar, natural molasses, citric acid and the like. Its weight formulation is 2-6 grams Dietmate®-B02, 1-3 grams plum plum, 20-40 grams edible sugar, 1-3 grams natural molasses and 1-2 citric acid per 1000 grams of drinking water. Grams are blended. The blending process includes raw material blending, filtration, homogenous sterilization, seal filling and quality inspection.

  As is clear from the results of 12 volunteers drinking 500 mL a day, the drink is a healthy drink that can be enjoyed by both men and women, belongs to alkaline foods, prevents blood acidification, and enhances the immunity of the body. Has the effect of improving sub-health and raising intelligence.

[ Reference Example 15]
Dietmate (registered trademark) -F02 (100 g) produced in Example 1 is used as a raw material and added as it is to 1000 L of Shaoxing liquor with an alcohol content of 18%, and is sufficiently dissolved, mixed uniformly, and placed in a can. Umebana health liquor has a health effect of prolonging age.

[Example 16]
The perfume according to the present invention produced by applying the Dietmate (registered trademark) -F01 produced in Example 1 by a normal process has a delicate scent of elegant plum blossoms and has antibacterial and anti-inflammatory effects.

[ Reference Example 17]
Applying Dietmate (registered trademark) -F02 produced in Example 1 and applying the hair restorer according to the present invention by the normal process of hair restorer described in “Cosmetics” (Slow Gloss, Rose, Version 2003, Science and Technology Literature Publishers) Manufactured. Ten volunteers (aged 45-67 years old) tried, washed their heads with 5 mL every two days, and as evidenced by the continued results for 2 months, this hair growth agent prevents hair loss and hair Has the effect of promoting growth.

[ Reference Example 18]
Applying Dietmate (registered trademark) -B02 produced in Example 2 according to the present invention, the suntan cream normal process described in “Cosmetic Principle, Formulation, Production Process” (Wang Yam Sung, Version 1999, Chemical Industry Publishing Co., Ltd.) Suntan cream was produced. As seen from the results of 10 volunteers (women) who tried and applied to the back of their hands before going out every day and continued for a month, this suntan cream has a pronounced sunscreen and whitening effect.

  What has been described above is only a preferred embodiment of the present invention, and the scope of the substantial technical contents according to the present invention is not limited thereto. The substantial technical content of the present invention is broadly defined in the claims. Any technical entities and methods completed by others are exactly the same as those defined in the claims, or are included in the claims if the equivalent effect is changed.

Claims (3)

(I) a plum extract containing flowers, branches and / or leaves using a supercritical CO ₂ fluid, separating a fat-soluble effective fraction, and a plum extract containing 0.5-50 wt% squalene; the step of viewing including,
The extraction method described above is a method of performing dynamic circulation extraction for 0.5-7 hours under the conditions of an extraction pressure of 5-50 MPa, an extraction temperature of 20-90 ° C., a separation temperature of 20-80 ° C., and a separation pressure of 2-10 MPa. And characterized by
A method for producing a plum extract containing an extract obtained by supercritical CO ₂ fluid extraction and containing squalene in an amount of 0.5 to 50 wt% based on the total weight of the extract. The method further includes (ii) extracting the plum raw material extracted in step (i) with an alcohol-water solvent to obtain a water-soluble effective fraction containing flavone glycoside, triterpenoid saponin and organic acid. wherein the method of claim 1. The extraction temperature with the alcohol-water solvent is 60-100 ° C, the extraction time is 0.1-5 hours, and the raw material-solution ratio is W / V: 1: 3-30. 2. The production method according to 2 .

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