JP2015034149A - Aquaporin 3 expression enhancer and tissue repair promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new use of ursolic acid.SOLUTION: This invention provides an aquaporin 3 expression enhancer and tissue repair promoter comprising ursolic acid as an active ingredient.

Description

  The present invention relates to an aquaporin 3 expression enhancer and a tissue repair promoter.

  Aquaporin (hereinafter sometimes abbreviated as AQP) is a six-transmembrane protein discovered by Agre in 1992, and is an important protein that functions as a water channel that regulates the amount of water in cells. Aquaporins exist universally from bacteria to mammals, and so far, 13 types of aquaporins from AQP0 to AQP12 have been confirmed in mammals.

Aquaporin 3 (AQP3), which is a type of water channel, is abundant in skin keratinocytes and is known to exhibit water and glycerol transport promoting action, and is an important component for physiological skin moisturization.
In addition, the expression of aquaporin 3 is significantly reduced at the site of skin inflammation, and it is suggested that the change in the expression level of aquaporin 3 is closely related to the formation of dry symptoms associated with various skin diseases. Further, aquaporin 3 is also involved in the promotion of keratinocyte proliferation or migration ability and is considered to be involved in promoting healing at the time of wounding (for example, see Patent Document 1).

  Ursolic acid is a kind of pentacyclic triterpenic acid. Ursolic acid is known to exhibit a melanin production inhibitory action (for example, see Patent Document 2) and an antioxidant action (for example, see Patent Document 3).

JP 2011-32191 A JP 2013-32300 A JP 2012-51940 A

However, it has not been known that ursolic acid exhibits an action of enhancing the expression of aquaporin 3 or promoting tissue repair.
An object of the present invention is to provide a new use of ursolic acid.
Another object of the present invention is to provide a novel aquaporin 3 expression enhancer and a novel bioactive agent based on the aquaporin 3 expression enhancing action.

The present invention is as follows.
<1> An aquaporin 3 expression enhancer containing ursolic acid as an active ingredient.
<2> A tissue repair promoter containing ursolic acid as an active ingredient.
<3> The tissue repair promoter according to <2>, wherein the tissue repair promotion is at least one granulation formation promotion selected from the group consisting of ulcers, pressure sores, trauma and surgical wounds.
<4> Obtaining an alcohol extract extracted from a plant using alcohol, mixing and dispersing the obtained alcohol extract in a mixture of alcohol, hydrocarbon and water to obtain an alcohol layer A method for producing ursolic acid, comprising: mixing and dispersing the alcohol layer in water to obtain a water-insoluble layer.

According to the present invention, a new use of ursolic acid can be provided.
Furthermore, according to the present invention, a novel aquaporin 3 expression enhancer and a novel bioactive agent based on the aquaporin 3 expression enhancing action can be provided.

It is a photograph which shows the effect | action of ursolic acid with respect to the expression level of aquaporin 3 concerning Example 2. 3 is a graph showing the action of ursolic acid on the expression level of aquaporin 3 according to Example 2. It is a graph which shows the effect | action of ursolic acid with respect to the migration ability of the keratinocyte concerning Example 3.

The aquaporin 3 expression enhancer and tissue repair promoter of the present invention are bioactive agents containing ursolic acid as an active ingredient.
The aquaporin 3 expression enhancer of the present invention has an action of enhancing the expression of aquaporin 3 and a tissue repair promoting action based on the action of enhancing the expression of aquaporin 3 on cells.
In the present specification, unless otherwise specified, aquaporin 3 expression enhancers and tissue repair promoters are collectively referred to as “bioactive agents”.

In this specification, the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. . In the present specification, a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In the present invention, when referring to the amount of each component in the composition, when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. Means the total amount.
In the present specification, the “cell” is not particularly limited as long as it is a cell expressing aquaporin 3. Specific examples include airway epithelial cells, renal epithelial cells, gastrointestinal epithelial cells, and skin cells. Skin cells include epidermal cells (keratinocytes), pigment cells (melanocytes), Langerhans cells, α-dendritic cells, and Merkel cells, among which epidermal cells (keratinocytes) are preferred.
The present invention will be described below.

<Bioactive agent>
Ursolic acid which is an active ingredient in the bioactive agent according to the present invention is a compound represented by the chemical formula C ₃₀ H ₄₈ O ₃ (molecular weight 456.7, CAS registration number 77-52-1), and has the following structural formula It is represented by

As ursolic acid, generally used synthetic materials, plant-derived extracts, and the like can be used.
As ursolic acid, plant-derived extracts such as apple, basil, bilberry, cranberry, elderflower, peppermint, rosemary, lavender, oregano, thyme, hawthorn and prune can be used.
Moreover, a commercial item can be used for ursolic acid. Examples of commercially available products include Ursolic acid (Sigma Ardrich), Ursolic acid (Santa Cruz), Ursolic acid (Tokyo Chemical Industry), ursolic acid (Wako Pure Chemical Industries), and the like.

Since ursolic acid has an action of enhancing the expression of aquaporin 3, it is useful as an active ingredient of a medicament for the treatment of a disease state caused by an abnormal function of aquaporin 3.
Abnormal function of aquaporin 3 refers to the state of aquaporin 3 in tissue cells under pathological conditions, which is different from the state of aquaporin 3 in healthy tissue cells, a decrease in aquaporin 3 function, a decrease in the number of aquaporin 3 in cells Etc.
An aquaporin 3 expression enhancer containing ursolic acid as an active ingredient can be specifically used as a tissue repair promoter, but is not limited to these uses, and is not limited to tissues or organs that express aquaporin 3. It can be used for prevention and treatment of diseases.

(Aquaporin 3 expression enhancer)
The aquaporin 3 expression enhancer according to the present invention contains ursolic acid as an active ingredient. Thereby, the expression enhancer of aquaporin 3 can enhance the expression of aquaporin 3 in the cell, compared to when ursolic acid is not used. Further, the aquaporin 3 expression enhancer has an action of promoting tissue repair.
Further, the aquaporin 3 expression enhancer can be expected to have an effect of improving skin moisture retention function, improving skin turnover, preventing dry skin, improving rough skin by drying, improving pruritus, and beautifying skin.

The agent that enhances the expression of aquaporin 3 only needs to have an action of enhancing the expression of aquaporin 3 in the cell as compared with when ursolic acid is not used.
The action of enhancing the expression of aquaporin 3 in the cell is not particularly limited. For example, it can be evaluated by measuring the expression level of aquaporin 3 in the cell.
Specifically, as an evaluation method, aquaporin expression levels of cultured cells, commercially available three-dimensional epidermis models, cells derived from the stratum corneum collected by tape stripping or the like, or skin-derived epidermis cell lines expressing aquaporin 3 are obtained by Western blotting. Examples include a measurement method, a method for measuring the expression level of aquaporin 3 mRNA by PCR method and Northern blot method, a method for measuring cell membrane water permeability by the stopped flow method, a method for measuring cell membrane permeability of labeled glycerol, and the like.

Preferably, the aquaporin 3 expression enhancer increases the expression level of aquaporin 3 to 1.5 to 4 times when the intracellular expression level of aquaporin 3 is measured, compared to when ursolic acid is not used. It is more preferable to increase 2 times to 4 times, and it is further preferable to increase 3 times to 4 times.
In addition, the measuring method of the expression level of aquaporin 3 is Horie I et al. , Biochem Biophys Res Commun, 387, 564-8, 2009.
Examples of cultured cells include keratinocyte strain DJ-M1 cells (RIKEN Cell Bank), Primary Human Keratinocyte Cells (Life Technologies), normal human epidermal keratinocytes (Kurabo), and the like.
Examples of commercially available three-dimensional skin models include LaboCyte Epimodel (Japan Tissue Engineering Co., Ltd.), EPI-200 (Kurabo Co., Ltd.), TESTSKIN (TOYOBO Co., Ltd.), and the like.

(Tissue repair promoter)
The tissue repair promoter according to the present invention contains ursolic acid as an active ingredient. Thereby, the tissue repair promoter can promote the repair of a wound generated in a tissue which is an aggregate of cells as compared with when ursolic acid is not used.
Examples of tissues that are aggregates of cells include the digestive tract such as skin, airways, stomach, and small intestine, and preferably skin.

Examples of the wound include at least one selected from the group consisting of burns, frostbite, ulcers, pressure sores, trauma and surgical wounds.
Examples of the tissue repair include promoting granulation of the wound site, promoting healing of the wound site (wound healing), promoting regeneration of the airway and gastrointestinal mucosal epithelium, and the like.
Preferably, the tissue repair promotion is at least one granulation promotion selected from the group consisting of ulcers, pressure sores, trauma and surgical wounds.

Specifically, the tissue repair promoting action can be evaluated by measuring the migration ability of keratinocytes and the repair ability of experimental trauma of animals.
For example, the evaluation of the migration ability of keratinocytes may be performed according to the method described in paragraph [0037] of JP 2011-32191 A.

When the bioactive agent according to the present invention is used in the form of a composition, the content of ursolic acid in the composition varies depending on the dosage form.
Generally, it is preferably 0.0000001% by mass to 1% by mass, more preferably 0.0001% by mass to 0.05% by mass, and particularly preferably 0.001% by mass to the total mass of the composition. 0.01% by mass.

When the bioactive agent according to the present invention is used, the form of the bioactive agent is not particularly limited. In addition to ursolic acid, it may be in the form of a composition containing other components described later (carriers acceptable as pharmaceuticals and cosmetics, and other optional components as required).
Examples of the form of the composition include an oil composition, an emulsified composition, and a powder composition. The oil composition, emulsion composition, and powder composition can be prepared according to a known method.

The bioactive agent according to the present invention can be administered orally or parenterally. From the viewpoint of the action of enhancing the expression of aquaporin 3, it is preferably administered parenterally. Specifically, local administration capable of directly administering ursolic acid to skin cells is preferred, and transdermal administration is more preferred.
The dose of the bioactive agent according to the present invention varies depending on the dosage form and the like, but in general, it can be 1 mg to 15 mg as an active ingredient per kg body weight per day, preferably 2 mg to 10 mg. More preferably 3 mg to 5 mg.

  Other components contained in the composition containing the bioactive agent according to the present invention can be appropriately selected according to the form, purpose, and the like of the composition of the present invention. Preferable examples of other components include emulsifiers and other additive components.

  As other additive components, in addition to the above-described components, additive components usually used in the fields of pharmaceuticals, functional foods, cosmetics, and the like may be appropriately added to the composition of the present invention according to the form. The other additive component can be contained in the composition of the present invention as an oil-soluble or water-soluble additive component depending on its characteristics.

For example, other additive components include polyhydric alcohols such as glycerin and 1,3-butylene glycol; glucose, fructose, lactose, maltose, sucrose, pectin, copper carrageenan, locust bean gum, guar gum, hydroxypropyl guar gum, xanthan gum , Karaya gum, tamarind seed polysaccharide, gum arabic, gum tragacanth, hyaluronic acid, sodium hyaluronate, sodium chondroitin sulfate, dextrin, etc .; sugar alcohols such as sorbitol, mannitol, maltitol, lactose, maltotriitol, xylitol Inorganic salts such as sodium chloride and sodium sulfate; casein, albumin, methylated collagen, hydrolyzed collagen, water-soluble collagen, gelatin, etc. Protein: glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, cystine, methionine, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, acetylhydroxyproline, etc. Amino acids and derivatives thereof; synthetic polymers such as carboxyvinyl polymer, sodium polyacrylate, polyvinyl alcohol, polyethylene glycol, ethylene oxide / propylene oxide block copolymer; water-soluble cellulose derivatives such as hydroxyethyl cellulose and methyl cellulose; Based on the function, for example, a functional component, an excipient, a viscosity modifier, a radical scavenger and the like may be included.
In addition, in the present invention, for example, various other medicinal ingredients, pH adjusters, pH buffering agents, ultraviolet absorbers, preservatives, fragrances, coloring agents, and other additives that are usually used in the application are used in combination. be able to.

The bioactive agent can be used as a pharmaceutical, a cosmetic, a functional food, or the like.
The bioactive agent can be administered to humans and livestock. Examples of domestic animals include dogs, monkeys, cats, cows, horses and sheep.

  The bioactive agent can be used in a method for enhancing the expression of aquaporin 3 containing ursolic acid as an active ingredient. The bioactive agent can also be used in a tissue repair promoting method containing ursolic acid as an active ingredient.

<Method for producing ursolic acid>
A method for producing ursolic acid includes obtaining an alcohol extract extracted from plants using alcohol (hereinafter referred to as “extraction step”), and mixing the obtained alcohol extract with alcohol, hydrocarbon and water. An alcohol layer is obtained by mixing and dispersing in a liquid (hereinafter referred to as “separation step”), and an alcohol layer obtained is mixed and dispersed in water to obtain a water-insoluble layer (hereinafter referred to as “isolation step”). ").
Thereby, highly pure ursolic acid can be obtained.
Moreover, the manufacturing method of ursolic acid may include processes other than an extraction process, a separation process, and an isolation process.

An extraction process is a process of obtaining the extract extracted from the plant using alcohol. Preferably, in the extraction step, a plant that has been appropriately cut or pulverized is dispersed in alcohol and extracted from room temperature under heating to obtain an alcohol extract.
Examples of the alcohol used for extraction include 50 w / w% to 100 w / w% methyl alcohol, 20 w / w% to 100 w / w% ethyl alcohol, 10 w / w% to 100 w / w% propanol, or 10 w / w%. Using ~ 100 w / w% butanol, 70 w / w% to 100 w / w% methyl alcohol or 50 w / w% to 100 w / w% ethyl alcohol is preferred. In addition, ketones such as acetone, liquid fatty acid esters such as glyceride linoleate and glycols such as ethylene glycol can be used.

The method of cutting or pulverizing the plant may be performed by a known method, for example, a method of cutting or pulverizing the plant using a pestle or a mortar until the plant becomes powdery.
The extraction temperature may be from room temperature to under heating. Specifically, the extraction temperature may be 0 ° C to 100 ° C, and preferably 20 ° C to 90 ° C.
The extraction time may be from several minutes to several days. For example, the extraction may be performed until the color of the plant moves to the extract.

In addition, the extraction step may include a stirring step for stirring the alcohol as necessary. The stirring speed is not particularly limited.
The alcohol extract can be obtained, for example, by a filtration step in which a plant is filtered to remove an extraction residue.

The separation step is a step of obtaining an alcohol layer by mixing and dispersing the obtained alcohol extract in a mixture of alcohol, hydrocarbon and water.
When the obtained alcohol extract is mixed and dispersed in a mixed liquid of alcohol, hydrocarbon and water (hereinafter referred to as “mixed liquid”), stirring to stir the alcohol extracted liquid and the mixed liquid as necessary. A process may be included. The stirring speed is not particularly limited.
The separation step may include an alcohol layer separation step in which the obtained alcohol extract is mixed and dispersed in the mixed solution and then left for a certain period of time until the alcohol layer is separated to separate the alcohol layer. The method for separating the alcohol layer is not particularly limited, and a known method can be adopted.

The isolation step is a step of obtaining a water-insoluble layer by mixing and dispersing the obtained alcohol layer in water.
When the obtained alcohol layer is mixed and dispersed in water, a stirring step of stirring the alcohol layer and water may be included as necessary. The stirring speed is not particularly limited.
The isolation step includes a water-insoluble layer fractionation step in which the obtained alcohol layer is mixed and dispersed in water and then left for a certain period of time until the water-insoluble layer separates, and the water-insoluble layer is fractionated. Also good.
The method for separating the water-insoluble layer is not particularly limited, and a known method can be adopted.

Moreover, the manufacturing method of ursolic acid may include a purification step of purifying ursolic acid from the obtained water-insoluble layer in addition to the extraction step, the separation step, and the isolation step.
The method for purifying ursolic acid from the water-insoluble layer is not limited, and a known method can be employed. Examples thereof include a method using column chromatography using silica gel, alumina and a hydrophobic chromatographic carrier.

As long as the plant contains ursolic acid, the plant may be either an undried plant or a dried plant.
Examples of undried plants include herbal medicines, herbs and fruits.
The dried plant may be either a sun-dried plant or a freeze-dried plant, but is preferably a sun-dried plant from the viewpoint of recovery efficiency.

As a kind of plant, any plant may be used as long as it contains ursolic acid. Examples include Lamiaceae, Rosaceae, Azalea, Honeysuckle, etc.
Specifically, the flower ears of Schizonepeta tenuifolia Briquet (var. Japonica Kitagawa) belonging to the family Lamiaceae, Rosemary of the genus Rosmarina and Rosmarinus officinalis L. Examples include apple skins belonging to the genus Apple.

Examples of the alcohol include lower alcohols (methanol, ethanol, propyl alcohol, isopropyl alcohol, etc.), polyhydric alcohols (glycerin, propylene glycol, etc.) and the like. These alcohols may be used alone or as a mixture of two or more alcohols.
Moreover, as alcohol, a lower alcohol is preferable and methanol is especially preferable.
The amount of the alcohol in which the plant is dispersed can be about 2 to 100 times, preferably 3 to 30 times the amount of alcohol with respect to the total mass of the plant.

As a mixed liquid of alcohol, hydrocarbon and water, alcohol: hydrocarbon: water is 1: 1: 1 to 5: 2: 1: 1, 2: 2: 1 to 1: 1: 2, on a volume basis. Or the liquid mixture which is 3: 2: 1-3: 2: 2 can be used.
Examples of the alcohol include lower alcohols (methanol, ethanol, propyl alcohol, isopropyl alcohol, etc.) and polyhydric alcohols (glycerin, propylene glycol, etc.). Lower alcohols are preferred, and methanol is more preferred. .
Examples of the hydrocarbon include benzene and hexane, but hexane is preferably used.
As the amount of the mixed solution for mixing and dispersing the alcohol extract, a mixed solution of about 2 to 100 times, preferably 10 to 50 times the total volume of the alcohol extract can be used.

  The amount of water used to mix and disperse the alcohol layer is about 0.1 to 2 times, preferably 0.5 to 1.5 times, the total volume of the alcohol layer. it can.

  Since the water-insoluble layer thus obtained contains ursolic acid, the water-insoluble component constituting the water-insoluble layer can be used as it is as the bioactive agent of the present invention.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them.

[Example 1: Production of ursolic acid]
Add 3000 ml of methanol to 300 g of dried mussel (manufactured by Uchida) and heat extract at 70 ° C. for 2 hours to obtain 30 g of an alcohol extract.
The obtained alcohol extract was mixed and dispersed in 1000 ml of a mixed solution of methanol, hexane and water (210: 90: 70 (volume basis)), and after 30 minutes, the methanol layer was separated by a separating funnel, Evaporate to dryness (methanol layer 24 g). The obtained methanol layer is mixed and dispersed in 500 ml of water, and after 20 minutes, the water-insoluble layer is separated by centrifugation (3.8 g of water-insoluble layer). The obtained water-insoluble layer was applied to a silica gel column having a loading volume of 200 mL, and chloroform: methanol: water (7: 2: 0.1) 200 mL, chloroform: methanol: water (7: 3: 0.5) 200 mL and methanol. The eluate was sequentially eluted with 200 mL, and the fourth fraction (the amount of the eluate 20 mL to 100 mL) was collected from the obtained third fraction and concentrated to dryness to obtain about 100 mg of ursolic acid.

[Example 2: Effect of ursolic acid on expression level of aquaporin 3]
The keratinocyte strain DJ-M1 cell (RIKEN Cell Bank) is 10 v / v% fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin, 1 v / v% MEM Non-Essential Amino Acids (GIBCO). Using the MEM medium containing, static culture was carried out at 37 ° C with 5 v / v% carbon dioxide.
After culturing keratinocyte strain DJ-M1 cells to confluency 80% to 90%, the MEM medium is replaced with a serum-free medium supplemented with 0.005 mg / ml ursolic acid (Sigma Aldrich) and left to stand for 24 hours. The cells treated with ursolic acid were obtained.
The cells treated with ursolic acid were washed with PBS, scraped using a cell scraper, and centrifuged to obtain a pellet.
Lysis buffer (50 mM Tris-HCl: pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 1% v / v protease inhibitor cocktail) 0.05 ml of (Sigma Aldrich) was added, solubilized by pipetting, and centrifuged again to obtain the supernatant as a cell lysate.
Using the cell lysate as a sample, polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as follows.
10 μl of the cell lysate was subjected to SDS-PAGE with 12 v / v% polyacrylamide gel, and various proteins contained in the cell lysate were separated into each band, and then transferred to a PVDF membrane (Thermo Science).
The transferred PVDF membrane was blocked by shaking in 5% skim milk solution at room temperature for 1 hour, and then the primary antibody reaction (4 ° C, overnight) using anti-aquaporin 3 (AQP3) antibody (Allomone) diluted 1: 1000. After washing with 0.1 v / v% Tween20 / PBS, secondary antibody reaction (room temperature, 1 h) using horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody (1: 20000) (Santa Cruz) )
Further, after the membrane was washed, antibody reaction was detected using ECL (registered trademark) advanced Western blotting analysis system (GE Healthcare Life Science).
In addition, it was verified by a similar experiment using an anti-β-actin antibody as the primary antibody that the amount of the sample loaded onto the polyacrylamide gel was uniform.

The measurement result of the expression level of aquaporin 3 obtained by the above method is shown in FIGS.
FIG. 1 is a photograph of a membrane (a band corresponding to aquaporin 3) obtained by Western blotting.
FIG. 2 is a graph obtained by quantifying the amount of protein in each band with an image analyzer (LAS-1000, Fujifilm) based on the results of Western blotting. In addition, the vertical axis | shaft of FIG. 2 has shown the ratio (%) of the expression level of aquaporin 3 with respect to the amount of (beta) -actin which is control, and a horizontal axis | shaft shows the addition amount (M) of ursolic acid used for the ursolic acid process. Show.

  As is clear from the results of FIGS. 1 and 2, the expression of aquaporin 3 was increased by the addition of ursolic acid.

[Example 3: Effect of ursolic acid on migration ability of keratinocytes]
The confluent culture MSM medium of keratinocyte strain DJ-M1 cells was replaced with a serum-free medium supplemented with 0.005 mg / ml ursolic acid and cultured for 24 hours.
The cells at the bottom of the culture dish were brought into contact with the tip of a 200 μl pipette tip, and the cells were detached by moving linearly while maintaining a constant pressure, and the scratch width was measured (Initial Wound).
The serum-free medium was replaced with a medium containing 1 v / v% FBS and 0.005 mg / ml ursolic acid, and further cultured for 24 hours, and the scratch width was measured again.
The scratch width was measured at 5 locations per dish, and the average value was taken as one data.
The cell migration ability was calculated as the difference between the Initial Wound and the final scratch width (Relative Wound Healing Activity).

  As is clear from the results of FIG. 3, the addition of ursolic acid promoted the migration ability of keratinocytes and promoted the recovery of experimental wounds in vitro.

[Example 4: Production of ursolic acid]
300 g of dried mussel (spear) was dispersed in methanol to obtain a methanol extract. Thereafter, the methanol extract was partitioned with hexane / water (7: 3), and a fraction insoluble in hexane and water was fractionated into 20 fractions by silica gel column chromatography (Merck).
About each obtained fraction, the effect | action with respect to the expression level of aquaporin 3 was confirmed like Example 1. FIG. As a result, a strong aquaporin 3 expression enhancing activity was recognized in the component constituting the third fraction eluted with CHCl ₃ : MeOH: H ₂ O = 7: 2: 0.2.
As a result of structural analysis of the component constituting the third fraction using NMR and FAB-MS, the component was identified as ursolic acid.

Claims (4)

  An aquaporin 3 expression enhancer containing ursolic acid as an active ingredient.   A tissue repair promoter containing ursolic acid as an active ingredient.   The tissue repair promoter according to claim 2, wherein the tissue repair promotion is at least one granulation promotion selected from the group consisting of ulcers, pressure sores, trauma and surgical wounds. Obtaining an alcohol extract extracted from plants with alcohol;
The obtained alcohol extract is mixed and dispersed in a mixture of alcohol, hydrocarbon and water to obtain an alcohol layer;
The obtained alcohol layer is mixed and dispersed in water to obtain a water-insoluble layer;
A process for producing ursolic acid, comprising: