CN104829678B - Asiatic acid salt, preparation method and application thereof - Google Patents

Abstract

The invention discloses a choline asiatic acid salt, a preparation method and use thereof, and a glucosamine asiatic acid salt, a preparation method and a use thereof. The asiatic acid choline salt and asiatic acid glucosamine salt solutions have high purity, wide range of concentration, rich optional administration routes, and can prepare various drugs and drugs for preventing or treating fibrosis, anti-scar and anti-aging cosmetic. Compared with asiatic acid free acid and other forms of salts, the water solubility of Asiatic acid salt of the present invention is greatly improved, and it is easy to make injections and various solutions, and through appropriate preparation means, effectively The bioavailability of the drug is improved, the efficacy of the drug is greatly improved, and it can effectively treat various fibrotic diseases.

Description

Centella asiatica and its preparation method and use

technical field

The invention belongs to the field of biotechnology, and in particular relates to asiatic acid salt and its preparation method and application.

Background technique

Asiatic acid (2α, 3β, 23, trihydroxyurea-12-ene-28-acid), also known as Asiatic acid, was first isolated from Centella asiatica by Bondmonds et al., and its pharmacology It has a wide range of effects, and is often used to treat burns or chronic ulcers, skin deformation caused by tuberculosis or leprosy, and has certain curative effects on cardiovascular diseases and liver poisoning. In addition, studies have shown that asiatic acid has anti-depressant, anti-fibrosis, anti-bacterial, anti-tumor and anti-oxidative effects.

Although there are 4 hydrophilic groups (3 alcoholic hydroxyl groups and 1 carboxyl group) in the molecular structure of asiatic acid, its wettability is relatively poor and it is almost insoluble in water. Formulation, especially hydrophilic formulations, requires the use of unique methods and special excipients. In addition, skin absorption takes place mainly in a transepidermal manner (intracellularly and through cells), and is mainly controlled by the action of the active ingredient on the stratum corneum, which is mainly composed of keratin and water. Therefore, in addition to the issue of formulation, the question of the proper bioavailability of Asiatic acid at the epidermal level remains unresolved.

Asiatic acid salt is a protonated salt formed by combining Asiatic acid with a medically acceptable base. The water solubility of Asiatic acid salt is larger than that of Asiatic acid, which makes the development of Asiatic acid salt very important.

At present, the asiatic acid salts that have been reported by patents include asiatic acid hemisuccinic acid and hemisuccinic acid salts and asiatic acid alkylaminoalkanolates and dialkylaminoalkanes announced in U.S. Patent No. 3,366,669. alkoxide. In Chinese patent CN1238330C, salts such as ethylenediamine, ethanolamine, diethanolamine, lysine, benzyltrimethylamine hydroxide and tetramethylamine hydroxide of Asiatic acid are announced, and Chinese patents 200980108867.6, 201110069862.6 and 201110164382.8 also Asiatic acid tromethamine salt and the like are disclosed. However, these salts all have a disadvantage, that is, their water solubility is very poor, and it is difficult to improve their bioavailability, so they are not helpful for the development of cosmetics and medicines.

Contents of the invention

Therefore, the technical problem to be solved by the present invention is to provide a centella asiatica salt for the existing problems such as poor solubility, extremely low bioavailability, poor transdermal absorption, and difficulty in the development of cosmetics and medicines. Choline oxalate and its preparation method and use, asiatic acid glucosamine salt and its preparation method and use.

In order to solve the above-mentioned technical problems, one of the technical solutions adopted by the present invention is: a kind of asiatic acid choline salt, its structural formula is as shown in formula I:

In order to solve the above-mentioned technical problems, the second technical solution adopted by the present invention is: a preparation method of asiatic acid choline salt, the preparation method comprising the following steps: in an organic solvent, asiatic acid and choline are carried out as follows: The neutralization reaction shown can be obtained.

Wherein, the methods and conditions of the neutralization reaction may be conventional methods and conditions in the art. The present invention particularly prefers the following methods and conditions: in the neutralization reaction, the molar concentration of the asiatic acid is preferably 0.5 to 5 mol/L, more preferably 0.5 to 2 mol/L, preferably 1 mol/L; the molar concentration of the choline is preferably 0.5-5 mol/L, more preferably 0.5-2 mol/L, preferably 1 mol/L.

In the neutralization reaction, the molar ratio of Asiatic acid to choline is preferably 2:1˜1:2, more preferably 1.5:1˜1:1.5, preferably 1:1.

Wherein the organic solvent is a conventional organic solvent in the art, preferably an alcohol solvent, more preferably ethanol, methanol or isopropanol, most preferably ethanol or isopropanol, most preferably absolute ethanol.

The temperature of the neutralization reaction is a conventional reaction temperature in the art, and the reaction temperature is preferably 60°C-70°C, more preferably 60°C-65°C, preferably 60°C.

The reaction time of the neutralization reaction is generally the end point of the reaction when Asiatic acid disappears, and the process of the neutralization reaction can be monitored by conventional testing methods in the art (such as TLC, HPLC or NMR). The reaction time for the reaction with the reaction is preferably 0.5-2 hours, more preferably 1-1.5 hours, preferably 1 hour.

Preferably, the reaction solution can also be stirred during the neutralization reaction, and the stirring is a conventional stirring method in the art, and the stirring speed is preferably 50-100 rpm, more preferably 50-100 rpm. 80 rpm, preferably 60 rpm.

The preparation method preferably also includes the step of purifying the obtained asiatic acid choline salt after the neutralization reaction is completed. This step is a method commonly used in the art, preferably including evaporating to remove the solvent in the reaction system, and The resulting product was dried. The drying method is a conventional drying method in the art, preferably vacuum drying or vacuum drying, preferably vacuum drying.

In order to solve the above technical problems, the third technical solution adopted by the present invention is: the use of the asiatic acid choline salt in the preparation of drugs for preventing or treating fibrosis.

Wherein said fibrosis is common fibrosis in this field, and fibrosis (fibrosis) is a kind of disease that occurs in various organs, and main pathological change is that the fibrous connective tissue in organ tissue increases, and parenchymal cell reduces, and continuous progress can cause Organ structure damage and function decline, even failure, seriously threaten human health and life. The fibrosis in the present invention preferably includes pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, myocardial fibrosis, etc., more preferably pulmonary fibrosis.

The "pulmonary fibrosis" mentioned in the present invention is a common pulmonary fibrosis in the art, and pulmonary fibrosis refers to pulmonary fibrosis characterized by pathological changes of idiopathic pulmonary fibrosis and caused by various factors. Wherein said pulmonary fibrosis preferably refers to human or animal pulmonary fibrosis, and the symptoms of said pulmonary fibrosis are more preferably: lung inflammation caused by pulmonary fibrosis, lung function caused by pulmonary fibrosis degradation. The cause of said pulmonary fibrosis is preferably: pulmonary fibrosis caused by lung injury, pulmonary fibrosis caused by dust or pulmonary fibrosis caused by drugs, wherein the drug is preferably bleomyces white.

The pulmonary fibrosis described herein is preferably primary (specific) pulmonary fibrosis, i.e. pulmonary fibrosis of unknown cause; or secondary pulmonary fibrosis, i.e. pulmonary fibrosis secondary to a pre-existing disease change. The diseases of pulmonary fibrosis preferably include chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, interstitial pneumonia and the like. The pulmonary fibrosis is preferably pulmonary fibrosis with degeneration of lung function, lung inflammation and lung injury.

The asiatic acid choline salt of the present invention can be used alone as an active ingredient or in combination with conventional anti-pulmonary fibrosis drugs as an active ingredient to prepare a drug for preventing or treating pulmonary fibrosis. The conventional anti-pulmonary fibrosis drug described in the present invention is the existing anti-pulmonary fibrosis drug in the prior art.

The "active ingredient" in the present invention refers to the compound that has the function of preventing or treating pulmonary fibrosis, that is, the anti-pulmonary fibrosis drug is prepared by using the asiatic acid choline salt and the pharmaceutical carrier in the present invention. In the medicament, the pharmaceutical carrier includes pharmaceutically acceptable excipients, fillers, diluents and the like. The asiatic acid choline salt of the invention can be used as an active ingredient for preparing anti-pulmonary fibrosis drugs. The "prevention" in the present invention refers to the prevention or reduction of pulmonary fibrosis after the use of drugs in the presence of possible pulmonary fibrosis factors. The "treatment" in the present invention refers to the use of drugs to reduce the degree of pulmonary fibrosis, or to cure pulmonary fibrosis to normalize it, or to slow down the process of pulmonary fibrosis.

When the asiatic acid choline salt described in the present invention is used to prepare a drug for preventing or treating pulmonary fibrosis, the dosage form of the drug is not particularly limited, preferably in liquid form, more preferably in aqueous solution, non-aqueous solution or Suspension, preferably injection or solution. The route of administration of the drug is preferably injection, and the route of injection preferably includes the following methods: intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

In order to solve the above technical problems, the fourth technical solution adopted by the present invention is: a pharmaceutical composition comprising the aforementioned asiatic acid choline salt, and the dosage form of the pharmaceutical composition is an injection or a solution.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and the dosage form is preferably an injection or a solution, and can also be a coating, a gel, a cataplasm, an ointment, a transdermal absorption agent or a biofilm agent.

In order to solve the above-mentioned technical problems, the fifth technical solution adopted by the present invention is: the use of the above-mentioned asiatic acid choline salt in the preparation of drugs or cosmetics for preventing or treating scar hyperplasia.

The scar hyperplasia described in the present invention is conventional scar hyperplasia in the field, and the scar hyperplasia is preferably scar hyperplasia caused by fibroblast proliferation. The scar hyperplasia described therein is more preferably the scar hyperplasia caused by various wounds, burns, acne and plastic surgery.

Wherein the asiatic acid choline salt is preferably used as a single active ingredient or in combination with conventional drugs for preventing or treating scar hyperplasia as an active ingredient in the preparation of drugs or cosmetics for preventing or treating scar hyperplasia. The conventional medicine for preventing or treating scar hyperplasia described in the present invention is the medicine that has the function of preventing or treating scar hyperplasia existing in the prior art.

The dosage form of the cosmetics described therein is not particularly limited, and is preferably a coating agent, a gel agent, a cataplasm agent, an ointment, a transdermal absorption agent or a biofilm agent.

In order to solve the above-mentioned technical problems, the sixth technical solution adopted by the present invention is: a kind of asiatic acid glucosamine salt, its structural formula is as shown in formula IV:

In order to solve the above technical problems, the seventh technical solution adopted by the present invention is: a preparation method of asiatic acid glucosamine salt, the preparation method comprising the following steps: in an organic solvent, the product shown in formula V The neutralization reaction of notic acid and glucosamine as shown in formula VI is carried out as shown below.

Wherein, the methods and conditions of the neutralization reaction may be conventional methods and conditions in the art. The present invention particularly prefers the following methods and conditions: in the neutralization reaction, the molar concentration of the asiatic acid is preferably 0.5 to 5 mol/L, more preferably 0.5 to 2 mol/L, preferably 1 mol/L; the molar concentration of the glucosamine is preferably 0.5-5 mol/L, more preferably 0.5-2 mol/L, preferably 1 mol/L.

In the neutralization reaction, the molar ratio of asiatic acid to glucosamine in the alcohol solvent is preferably 2:1-1:2, more preferably 1.5:1-1:1.5, preferably 1:1.

Wherein the organic solvent is a conventional organic solvent in the art, preferably an alcohol solvent, more preferably ethanol, methanol or isopropanol, most preferably ethanol or isopropanol, most preferably absolute ethanol.

The temperature of the neutralization reaction is a conventional reaction temperature in the art, and the reaction temperature is preferably 60°C-70°C, more preferably 60°C-65°C, preferably 60°C.

The reaction time of the neutralization reaction is generally the end point of the reaction when Asiatic acid disappears, and the process of the neutralization reaction can be monitored by conventional testing methods in the art (such as TLC, HPLC or NMR). The reaction time for the reaction with the reaction is preferably 0.5-2 hours, more preferably 1-1.5 hours, preferably 1 hour.

Preferably, the reaction solution can also be stirred while the neutralization reaction is being carried out, the stirring is a conventional stirring method in the art, and the stirring speed is preferably 50-100rpm, more preferably 50-80rpm, preferably 60rpm.

The preparation method preferably also includes a step of purifying the obtained asiatic acid glucosamine salt after the neutralization reaction is completed, which is a method commonly used in the art, and preferably includes evaporating and removing the solvent in the reaction system, and the resulting product was dried. The drying method is a conventional drying method in the art, preferably vacuum drying or vacuum drying, preferably vacuum drying.

In order to solve the above-mentioned technical problems, the eighth technical solution adopted by the present invention is: the use of the asiatic acid glucosamine salt in the preparation of drugs for preventing or treating fibrosis.

Wherein said fibrosis is common fibrosis in this field, and fibrosis (fibrosis) is a kind of disease that occurs in various organs, and main pathological change is that the fibrous connective tissue in organ tissue increases, and parenchymal cell reduces, and continuous progress can cause Organ structure damage and function decline, even failure, seriously threaten human health and life. The fibrosis in the present invention preferably includes pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, myocardial fibrosis, etc., more preferably pulmonary fibrosis.

The "pulmonary fibrosis" mentioned in the present invention is a common pulmonary fibrosis in the art, and pulmonary fibrosis refers to pulmonary fibrosis characterized by pathological changes of idiopathic pulmonary fibrosis and caused by various factors. Wherein said pulmonary fibrosis preferably refers to human or animal pulmonary fibrosis, and the symptoms of said pulmonary fibrosis are more preferably: lung inflammation caused by pulmonary fibrosis, lung function caused by pulmonary fibrosis degradation. The cause of said pulmonary fibrosis is preferably: pulmonary fibrosis caused by lung injury, pulmonary fibrosis caused by dust or pulmonary fibrosis caused by drugs, wherein the drug is preferably bleomyces white.

The pulmonary fibrosis described herein is preferably primary (specific) pulmonary fibrosis, i.e. pulmonary fibrosis of unknown cause; or secondary pulmonary fibrosis, i.e. pulmonary fibrosis secondary to a pre-existing disease change. The diseases of pulmonary fibrosis preferably include chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, interstitial pneumonia and the like. The pulmonary fibrosis is preferably pulmonary fibrosis with degeneration of lung function, lung inflammation and lung injury.

The asiatic acid glucosamine salt of the present invention can be used alone as an active ingredient or in combination with conventional anti-pulmonary fibrosis drugs as an active ingredient to prepare a drug for preventing or treating pulmonary fibrosis. The conventional anti-pulmonary fibrosis drug described in the present invention is the existing anti-pulmonary fibrosis drug in the prior art.

The "active ingredient" in the present invention refers to the compound that has the function of preventing or treating pulmonary fibrosis, that is, the anti-pulmonary fibrosis drug is prepared by using the asiatic acid glucosamine salt and a pharmaceutical carrier in the present invention. In the medicine, the asiatic acid glucosamine salt can be used as an active ingredient alone or together with other compounds. The pharmaceutical carrier mentioned therein includes pharmaceutically acceptable excipients, fillers, diluents and the like. The asiatic acid glucosamine salt of the present invention can be used as an active ingredient in the preparation of anti-pulmonary fibrosis drugs. The "prevention" in the present invention refers to the prevention or reduction of pulmonary fibrosis after the use of drugs in the presence of possible pulmonary fibrosis factors. The "treatment" in the present invention refers to the use of drugs to reduce the degree of pulmonary fibrosis, or to cure pulmonary fibrosis to normalize it, or to slow down the process of pulmonary fibrosis.

When the asiatic acid glucosamine salt described in the present invention is used to prepare a drug for preventing or treating pulmonary fibrosis, the dosage form of the drug is not particularly limited, preferably in liquid form, more preferably in aqueous solution or non-aqueous solution Or suspension, preferably injection or solution. The route of administration of the drug is preferably injection, and the route of injection preferably includes the following methods: intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

In order to solve the above technical problems, the ninth technical solution adopted by the present invention is: a pharmaceutical composition comprising the aforementioned asiatic acid glucosamine salt, and the dosage form of the pharmaceutical composition is an injection or a solution.

When the pharmaceutical composition of the present invention is used to prepare a drug for preventing or treating fibrosis, the dosage form of the pharmaceutical composition is not particularly limited, and the dosage form is preferably an injection or a solution.

In order to solve the above-mentioned technical problems, the tenth technical solution adopted by the present invention is: use of the above-mentioned asiatic acid glucosamine salt in the preparation of drugs or cosmetics for preventing or treating scar hyperplasia.

The scar hyperplasia described in the present invention is conventional scar hyperplasia in the field, and the scar hyperplasia is preferably scar hyperplasia caused by fibroblast proliferation. The scar hyperplasia described therein is more preferably the scar hyperplasia caused by various wounds, burns, acne and plastic surgery.

The asiatic acid glucosamine salt is preferably used as a single active ingredient or in combination with conventional drugs for preventing or treating scar hyperplasia as an active ingredient in the preparation of drugs or cosmetics for preventing or treating scar hyperplasia. The conventional medicine for preventing or treating scar hyperplasia described in the present invention is the medicine that has the function of preventing or treating scar hyperplasia existing in the prior art.

In the application of the asiatic acid glucosamine salt of the present invention in the preparation of medicines or cosmetics for preventing or treating scar hyperplasia, the dosage form of the medicine is not particularly limited, and the dosage form is preferably a coating agent, a gel poultice, poultice, ointment, transdermal or biofilm agent.

The asiatic acid glucosamine salt of the present invention is a true solution in the state of an aqueous solution with a concentration of 0.1-20 mg/ml, and is suitable for preparing external solutions, such as injections and the like.

The asiatic acid glucosamine salt of the present invention may form a gel in an aqueous solution with a concentration of 20mg-30mg/ml, and can be used as a special preparation.

The asiatic acid glucosamine salt of the present invention can form a gel in an aqueous solution with a concentration greater than 30 mg/ml, and can be used as a gel in cosmetics and medicines.

In order to solve the above technical problems, the eleventh technical solution adopted by the present invention is: the use of the asiatic acid glucosamine salt as mentioned above in the preparation of anti-aging drugs or cosmetics:

The aging described in the present invention is the conventional aging in the field, preferably subacute aging or photoaging. Wherein the photoaging can be due to the damage caused by the skin being exposed to sunlight for a long time, manifested as rough skin, thickening, sagging, deep and thick wrinkles, excessive pigmentation or telangiectasia in the local area, and even various Benign or malignant tumors (such as solar keratosis, squamous cell carcinoma, malignant melanoma, etc.). Wherein the aging is preferably skin aging, the skin aging is conventional skin aging in the field, and the skin aging may be a skin aging phenomenon caused by natural factors or unnatural factors. Wherein said skin aging can be caused by the factor that causes skin aging conventionally in this field, and described factor that causes skin aging can comprise: age factor, health factor, mental factor, nutritional factor, living habit, environmental factor, endocrine disorder, One or more of improper skin care and improper medication.

The symptoms of skin aging described in the present invention are conventional symptoms in the art, which may include skin tissue deterioration or skin physiological function decline. The decline of the skin tissue can be manifested as an increase in melanin, hardening of the skin surface, loss of luster, reduction of subcutaneous fat, and generation of skin laxity and wrinkles; the low physiological function of the skin is manifested as: sebaceous glands, sweat glands function decline, sweat With the reduction of sebum removal, the skin gradually loses its luster and becomes dry, and the skin wounds are difficult to heal.

The skin folds described in the present invention are conventional skin folds in the art, and the skin folds are preferably wrinkles, which means that the skin is affected by the external environment to form free radicals, and free radicals destroy normal cell membrane tissue. Collagen, active substances, small fine lines and wrinkles formed by oxidized cells. Wrinkles are preferably divided into two types: atrophic wrinkles and hypertrophic wrinkles. The wrinkles mentioned therein preferably include: one or more of natural wrinkles, dynamic wrinkles, gravity wrinkles and mixed wrinkles.

Preferably, the application of the present invention is the application of the asiatic acid glucosamine salt as a single active ingredient or in combination with a conventional anti-aging compound as an active ingredient in the preparation of anti-aging drugs or cosmetics. The conventional anti-aging compounds in the present invention refer to the existing anti-aging compounds in the prior art. The "active ingredient" in the present invention refers to a compound with an anti-aging function, that is, an anti-aging drug is prepared by using the asiatic acid glucosamine salt and a pharmaceutical carrier in the present invention. In the drug, preferably, a pharmaceutical carrier is also included. The pharmaceutical carrier mentioned therein includes pharmaceutically acceptable excipients, fillers, diluents and the like. The "prevention" in the present invention refers to preventing or slowing down the occurrence of aging phenomenon after using the obtained medicine in the presence of factors that may cause aging. "Treatment" in the present invention refers to the use of drugs to reduce the degree of aging or to slow down the process of skin aging or skin wrinkles.

The medicine of the present invention can be utilized in the form of a pharmaceutical composition comprising the aforementioned asiatic acid glucosamine salt, and the dosage form of the medicine or pharmaceutical composition is not particularly limited, preferably in a liquid form or a solid form, so The dosage forms in liquid form are more preferably aqueous solutions, non-aqueous solutions or suspensions. The dosage form of the solid form is preferably a film-coating agent, a gel, an ointment, a transdermal absorption agent or a biofilm agent, preferably a transdermal absorption agent. The route of administration of the asiatic acid glucosamine salt is preferably smear administration or injection administration, wherein the route of injection administration preferably includes: intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or by subcutaneous injection.

The dosage form of the cosmetics described in the present invention is not particularly limited, and the dosage forms of the cosmetics are conventional dosage forms in the art, and the dosage forms preferably include: film coating agents, gels, lotions, creams, transdermal absorption agents or biofilm agent.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention lies in: the water solubility of Asiatic acid salt obtained in the present invention is greatly improved compared with asiatic acid free acid or other forms of Asiatic acid salt, effectively improving the performance of Asiatic acid salt. The problems of extremely low drug bioavailability and poor transdermal absorption have greatly improved the efficacy and safety of Asiatic acid salt. The asiatic acid choline salt of the invention can effectively treat pulmonary fibrosis, relieve pulmonary fibrosis symptoms, and effectively treat scar hyperplasia. The asiatic acid glucosamine salt of the present invention can also effectively treat pulmonary fibrosis, alleviate the symptoms of pulmonary fibrosis, and effectively treat scar hyperplasia. At the same time, the drug or cosmetic for preventing or treating skin aging or anti-skin wrinkling prepared by using the asiatic acid glucosamine salt of the present invention has a significant alleviating effect on skin aging and can significantly reduce the generation of skin wrinkling.

detailed description

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. The "room temperature" mentioned in the examples refers to the temperature in the operating room where the test is carried out, which is generally 20°C to 35°C.

In the following examples, the manufacturer of the raw material Asiaticosides is Guangxi Nanning Lichuang Biotechnology Co., Ltd.

Preparation and identification of embodiment 1 asiatic acid choline salt

1. Preparation of asiatic acid

Weigh 10 g of total asiaticosides, of which the content of asiaticoside is 35.5%, add 50% ethanol solution containing 4% NaOH, reflux extraction for 4 hours; add hydrochloric acid to adjust the pH value to 2-3, and let stand overnight. Centrifuge the sample, discard the filtrate, wash the solid with water and 30% ethanol in turn until the color of the filtrate is light to transparent; add absolute ethanol to dissolve the solid after washing, add granular activated carbon, and reflux for 1 hour for decolorization; filter while it is hot, and add Deionized water, let it stand overnight; Centrifuge to get Centella asiatic acid (containing asiatic acid, brahmic acid, isobrahmic acid and betulinic acid) tulinic acid) and other triterpene acids), i.e. intermediate I, wherein the asiatic acid content measured by HPLC method is 55%; an appropriate amount of intermediate I is taken and separated by silica gel column chromatography, and the eluent is petroleum ether: ethyl acetate Gradient elution (the volume ratio of petroleum ether and ethyl acetate in the eluent is 10:1 ~ 1:10), to obtain the crude product I of asiatic acid, wherein the content of asiatic acid is 83%; through HPLC semi-preparative column (cosmosil C18 Flow rate 5mL/min mobile phase is acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76) prepares the pure product of asiatic acid, wherein the content of asiatic acid is 98.2%, and the yield is 49% .

2. HPLC analysis of asiatic acid

HPLC analysis conditions: Chromatographic column: Agela Promosil C18 4.6×250mm, 5μm, detection wavelength: 205nm; column temperature: 30°C; flow rate: 1ml/min;

Mobile phase: A, asiaticoside: acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76 isocratic elution;

B. Asiatic acid: acetonitrile: methanol: 2mmol/Lβ-CD formic acid water (pH=3～4)=35:15:50 isocratic elution

Under the conditions of this preparation process, in further purification and separation (HPLC semi-preparative column preparation cosmosil C18 flow rate 5mL/min mobile phase is acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76) , the asiatic acid obtained reaches more than 98.5%.

3. Synthesis of asiatic acid choline salt

The synthesis process of asiatic acid choline salt is as follows:

Mix 1g (1.98mmol) Asiatic acid with absolute ethanol at room temperature, heat to 60°C until completely dissolved, add 0.24g (1.98mmol) choline to the solution, and stir at this temperature for 0.5h to obtain a clear solution , the stirring speed was 50rpm, and the solvent was evaporated at normal temperature, and the product was put into a vacuum oven at 50°C for drying to obtain 1.3g of asiatic acid choline salt, whose structural formula is as shown in the following formula I.

The purity of the obtained asiatic acid choline salt was 97%. The identification data of gained Asiatic acid choline salt are as follows:

¹ H-NMR: δ5.05(2H), δ4.2(1H), δ3.8(3H), δ3.4(4H), δ3.1(12H), δ2.21(1H), δ1.25 (2H), δ0.98(3H), δ0.93(3H), δ0.74(3H), δ0.54(3H), δ0.92(3H), δ0.82(3H).

GC-MS(EI,m/z): EI-MS m/z: Asiatic acid choline salt EI-MS m/z: [488+206+H] ⁺ choline EI-MS m/z 207.18[2M- 2H ₂ O+H] ⁺ Asiatic acid: EI-MS m/z 487.47 [MH] ⁻ .

Example 2

Mix 1g (0.52mmol) Asiatic acid with absolute ethanol at room temperature, heat to 70°C until completely dissolved, add 0.51g (0.52mmol) choline to the solution, and stir at this temperature for 2 hours to obtain a clear solution, The stirring speed was 100 rpm, the solvent was evaporated at normal temperature, and the product was put into a vacuum drying oven at 50° C. for drying to obtain 1.3 g of asiatic acid choline salt, and the purity of the obtained asiatic acid choline salt was 97%.

Example 3

Mix 1g (4.98mmol) asiatic acid with anhydrous methanol at room temperature, heat to 60°C until completely dissolved, add 0.532g (46%) choline solution to the solution, and stir at this temperature for 1 hour to obtain a clear solution , the stirring speed is 100rpm, and the solvent is evaporated at normal temperature, and the product is put into a 50°C vacuum oven for drying to obtain 1.15g of asiatic acid choline salt, and the purity of the gained asiatic acid choline salt is 97%.

Example 4

Mix 18g (1.98mmol) asiatic acid with absolute ethanol at room temperature, heat to 75°C until completely dissolved, add 9.42g (46%) choline solution to the solution, and stir at this temperature for 1.5 hours to obtain a clear solution , stirring speed is 100rpm, evaporates and removes solvent at normal temperature, puts this product into 50 ℃ of vacuum ovens and dries, obtains asiatic acid choline salt 18.02g, the purity of gained asiatic acid choline salt is 97%.

Example 5

Mix 32g (0.98mmol) asiatic acid with absolute ethanol at room temperature, heat to 75°C until completely dissolved, add 17.2g (46%) choline solution to the solution, and stir at this temperature for 2 hours to obtain a clear solution , the stirring speed was 100rpm, and the solvent was evaporated at normal temperature, and the product was put into a vacuum oven at 50°C for drying to obtain 29.87g of asiatic acid choline salt, and the purity of the gained asiatic acid choline salt was 97%.

Example 6

Mix 0.5g (3.01mmol) asiatic acid with absolute ethanol at room temperature, heat to 55°C until completely dissolved, add 0.158g (46%) choline solution to the solution, and stir at this temperature for 0.5 hours to obtain clarification solution, the stirring speed is 100rpm, and the solvent is evaporated at normal temperature, and the product is put into a vacuum oven at 50° C. for drying to obtain 0.31 g of asiatic acid choline salt, and the purity of the obtained asiatic acid choline salt is 97%. .

Example 7 Asiatic acid choline salt solubility detection

Conducted according to GB/T 21845-2008 Chemical Water Solubility Test Method;

The solubility of asiatic acid choline salt was measured to be greater than 350 mg/ml.

And the solubility of asiatic acid measured under the same situation<5mg/ml, the solubility of asiatic acid diethylamine salt<5mg/ml.

The results show that the solubility of asiatic acid choline salt is significantly improved compared with that of asiatic acid and other existing types of asiatic acid salts.

Embodiment 8 prepares asiatic acid choline salt injection

Preparation method: add 3 mg of asiatic acid choline salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Autoclaved, that is.

Embodiment 9 prepares asiatic acid choline salt injection

Preparation method: add 0.1 mg of asiatic acid choline salt to an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Autoclaved, that is.

Embodiment 10 prepares asiatic acid choline salt injection

Preparation method: add 5 mg of asiatic acid choline salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Sterilize by autoclaving, and the concentration of the obtained asiatic acid choline salt is 5 mg/ml.

Embodiment 11 prepares asiatic acid choline salt injection

Preparation method: Add 20mg of asiatic acid choline salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1ml, and encapsulate. Autoclaved, that is.

Embodiment 12 prepares asiatic acid choline salt injection

Preparation method: Add 200mg of asiatic acid choline salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1ml, and encapsulate. Autoclaved, that is.

Therapeutic effect of embodiment 13 asiatic acid choline salt on bleomycin-induced pulmonary fibrosis in rats

Male SD rats (purchased from Shanghai Slack Experimental Animal Co., Ltd.), weighing 250-300 g, were fasted overnight, anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and injected with bleomycin (5 U/kg) into the trachea. kg). The specific plan is as follows: cut the skin of the neck with minimal trauma, expose the trachea with the assistance of elbow ophthalmic forceps, use a micro-sampler to puncture the trachea, inject about 50 μl of bleomycin into the trachea, rotate rapidly and Stand upright for 5 minutes so that the bleomycin can enter the left and right lung lobes evenly. The whole operation is carried out on the operating table at about 60°C.

Drugs were administered to the experimental animals from the day after the modeling (wherein the day of modeling was the zeroth day, and the day after the modeling was the first day). And set asiatic acid choline salt treatment group, daily dosage is respectively 0.3mg/kg, 0.9mg/kg and 2.7mg/kg according to the body weight of mice, by intraperitoneal injection (i.p.) processing 28 days, dextran The metasone group was treated with 0.6 mg/kg po (oral) for 28 days, and the model group and blank group were given normal saline. For the preparation method of asiatic acid choline salt injection, please refer to the record in Example 7.

After the end of the test (on the twenty-ninth day), the rats in each group were tested for lung weight ratio (lung weight/body weight*100%), serological testing (tested by Hitachi 7080 biochemical detector) and serum hydroxyproline Acid content detection.

Wherein the detection method of the hydroxyproline content is slightly improved with reference to the method of Woessner Jr et al. The detection method of the hydroxyproline content is as follows.

Reagents: Hydroxyproline, Chloramine T Analytical grade Batch number: 20020510 Zhongjiang Pharmaceutical Group Shanghai Chemical Reagent Company

p-Dimethylaminobenzaldehyde AR batch number: 20010224 Shanghai Sanaisi Reagent Co., Ltd.

Ethylene glycol methyl ether AR batch number: 20030705 Sinopharm Chemical Reagent Co., Ltd.

Instrument: 721 Spectrophotometer UV755B Xiamen Analytical Instrument Factory

Hydroxyproline content detection method steps:

1) The sample is dried;

2) Take samples;

3) Put the sample in the hydrolysis tube, add 6N HCL (100 times the sample volume is 2ml);

4) sealing tube;

5) Hydrolysis at 138°C for 2 hours;

6) Adjust the pH value. Using methyl red as indicator;

7) Prepare hydroxyproline standard solution at the same time, dissolve in 0.001N HCL to make 0.1mg/ml solution, and then prepare 0.5, 1.0, 1.5, 2.0, 2.5ug/ml standard products;

8) Add 1ml of chloramine T solution to the diluted sample;

9) Take 2ml of sample and add 1ml of chloramine T solution;

Chloramine T solution preparation: 1.4g chloramine T was dissolved in 20ml water. Add ethylene glycol methyl ether and 50ml citrate buffer and mix;

10) Stand at room temperature at 25°C for 20 minutes;

11) Add 1ml of perchloric acid solution;

12) Place at room temperature for 5 minutes;

13) Add 1ml of p-dimethylaminobenzaldehyde solution (20g of p-dimethylaminobenzaldehyde is dissolved in ethylene glycol methyl ether to a total of 100ml, and cooled) and mix;

14) Heat at 60°C for 20 minutes, then cool in water;

15) Measure the absorbance at 557nm, Note: If the absorbance exceeds the range of the standard curve, dilute to within the range for testing;

16) Find out the amount of hydroxyproline contained in the sample according to the standard curve.

The specific test results are shown in the following Tables 1-4, wherein the number of grouped samples in Table 1 is obtained by counting on the 0th day, the total number of deaths is obtained by counting on the 29th day, and the results described in Table 2-4 are the results obtained by testing on the 29th day .

Table 1 Analysis of animal death in different samples after administration of bleomycin

Table 2 Lung index at 28 days after administration of bleomycin from the next day after modeling

*Compared with the model group, there is a significant difference at P<0.05**, and there is a very significant difference at P<0.01.

Table 3 Serological test results of the administration group on the next day of bleomycin modeling

*Compared with the model group, there is a significant difference at P<0.05**, and there is a very significant difference at P<0.01.

Table 4 Asiatic acid choline salt modeling next day administration group anti-pulmonary fibrosis hydroxyproline measured value

group Hydroxyproline blank control 19.10±2.14 Model 29.89±3.21 Dexamethasone 24.55±2.17 Asiatic acid choline salt i.p.0.3mg/kg 21.46±2.67 Asiatic acid choline salt i.p.0.9mg/kg 19.73±2.11 Asiatic acid choline salt i.p.2.7mg/kg 19.52±3.01 Asiatic acid potassium salt i.p.2.7mg/kg 31.17±2.58

Above-mentioned result shows: asiatic acid choline salt injection not only has significant difference with control group in respects such as lung weight, lung index, hydroxyproline content etc., and the death rate of subject is extremely low, and the obtained result shows that the present invention The curative effect of the asiatic acid choline salt provided on pulmonary fibrosis is very obvious.

Effect of Example 14 on rabbit ear hypertrophic scar model (prevention or treatment of scar hyperplasia)

Model establishment (references Li Xijun, Liu Dalie, Wang Jihui. Discussion on the method of establishment of rabbit ear hypertrophic scar model [J]. Chinese Cosmetic Medicine, 2006,15(5):499-500.):

In each rabbit (Shanghai Slack Experimental Animal Co., Ltd., weighing about 1.5 kg), two pieces of skin 1 cm×1 cm in size were excised from the ventral side of the left and right ears, with a distance of 3 cm, and were wrapped with sterile gauze. Grouping and administration: 21 rabbits in total, the left and right ears of each rabbit were used for the experiment (please refer to the literature for animal model establishment methods: Li Xijun, Liu Dalie, Wang Jihui. Discussion on the establishment method of rabbit ear hypertrophic scar model[J].China Aesthetic Medicine, 2006,15(5):499-500.).

Experimental animals were divided into normal skin group, model group, vitamin EC treatment group (with 0.5% sodium carboxymethyl cellulose suspension, 100 μg/ml), madecassoside treatment group (with 0.5% carboxymethyl cellulose Suspension made of sodium cellulose, 1 μg/ml), madecassic acid treatment group (suspension made with 0.5% sodium carboxymethyl cellulose, 200ng/ml), ethylenediamine salt of madecassic acid Group (suspension made with 0.5% sodium carboxymethylcellulose, 500 ng/ml), and group treated with choline madecassate (concentration of choline madecassate aqueous solution was 40 ng/ml). The administration method of each administration group is to smear 1ml of sample every day. On the 45th day after operation, the hypertrophic scar of rabbit ear was treated. In the 2nd, 4th, and 6th weeks after the administration, the SEQUIA512 color Doppler color ultrasonic diagnostic instrument 13Hz high-frequency probe was used to detect hypertrophic scars and skin thickness, and the hardness meter was used to measure hypertrophic scars and skin hardness (for experimental methods, please refer to the literature: Li Huiyuan, Liu Jianbo, Xia Wei, et al. Establishment and application of animal experimental models of hypertrophic scars [J]. Chinese Journal of Plastic Surgery, 2001,17(5):276-278.), and cut hypertrophic scar specimens from rabbit ears and skin samples for visual inspection, the experimental results are shown in Table 5 and Table 6.

Table 5 Thickness of hypertrophic scar (mm)

*p<0.05 compared with the model group; **p<0.01 compared with the model group

Table 6 Hypertrophic scar hardness (N/mm ² )

*p<0.05 compared with the model group; **p<0.01 compared with the model group

From the above experimental results, it can be known that the asiatic acid choline salt obtained in the present invention has a significant inhibitory effect on rabbit ear hypertrophic scars compared with other salts of asiaticoside and asiatic acid.

Preparation and identification of embodiment 15 asiatic acid glucosamine salt

1. Preparation of asiatic acid

Weigh 10 g of total asiaticosides, of which the content of asiaticoside is 35.5%, add 50% ethanol solution containing 4% NaOH, reflux extraction for 4 hours; add hydrochloric acid to adjust the pH value to 2-3, and let stand overnight. Centrifuge the sample, discard the filtrate, wash the solid with water and 30% ethanol in turn until the color of the filtrate is light to transparent; add absolute ethanol to dissolve the solid after washing, add granular activated carbon, and reflux for 1 hour for decolorization; filter while it is hot, and add Deionized water, let it stand overnight; Centrifuge to get Centella asiatic acid (containing asiatic acid, brahmic acid, isobrahmic acid and betulinic acid) tulinic acid) and other triterpene acids), i.e. intermediate I, wherein the asiatic acid content measured by HPLC method is 55%; an appropriate amount of intermediate I is taken and separated by silica gel column chromatography, and the eluent is petroleum ether: ethyl acetate Gradient elution (the volume ratio of petroleum ether and ethyl acetate in the eluent is 10:1 ~ 1:10), to obtain the crude product I of asiatic acid, wherein the content of asiatic acid is 83%; through HPLC semi-preparative column (cosmosil C18 Flow rate 5mL/min mobile phase is acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76) prepares the pure product of asiatic acid, wherein the content of asiatic acid is 98.2%, and the yield is 49% .

2. HPLC analysis of asiatic acid

HPLC analysis conditions: Chromatographic column: Agela Promosil C18 4.6×250mm, 5μm, detection wavelength: 205nm; column temperature: 30°C; flow rate: 1ml/min;

Mobile phase: A, asiaticoside: acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76 isocratic elution;

B. Asiatic acid: acetonitrile: methanol: 2mmol/Lβ-CD formic acid water (pH=3～4)=35:15:50 isocratic elution

Under the conditions of this preparation process, in further purification and separation (HPLC semi-preparative column preparation cosmosilC18 flow rate 5mL/min mobile phase is acetonitrile: 2mmol/Lβ-CD formic acid water (pH=3～4)=24:76), The asiatic acid obtained reaches above 98.5%.

3. Synthesis of asiatic acid glucosamine salt

The synthesis process of asiatic acid glucosamine salt is as follows:

Mix 18g of asiatic acid with absolute ethanol at room temperature, heat to 60°C until completely dissolved, add 7.19g of N-methyl-D-glucosamine to the solution, and stir at this temperature for 0.5h to obtain a clear solution. The stirring speed was 50 rpm, the solvent was evaporated at room temperature, and the product was dried in a vacuum oven at 50°C to obtain 25.6 g of glucosamine asiatic acid white powder, whose structural formula is shown in the following formula IV.

The purity of the obtained asiatic acid glucosamine salt was 97%. The identification data of gained asiatic acid glucosamine salt are as follows:

GC-MS (EI, m/z): asiatic acid glucosamine salt 682.51[488+195-H] ^- N-methyl-D-glucosamine EI-MS m/z 196.15[M+H]+, Asiatic acid: EI-MS m/z 487.47 [MH] ^- .

Example 16

Mix 1g (1.98mmol) asiatic acid with absolute ethanol at room temperature, heat to 70°C until completely dissolved, add 0.3g (2.48mmol) glucosamine to the solution, and stir at this temperature for 2 hours to obtain a clear solution , the stirring speed is 100rpm, and the solvent is evaporated at normal temperature, and the product is put into a vacuum drying oven at 50°C for drying to obtain 1.4g of asiatic acid glucosamine salt, and the purity of the obtained asiatic acid glucosamine salt is 97 %.

Example 17

Mix 18g Asiatic acid with absolute ethanol at room temperature, heat to 70°C until completely dissolved, add 7.19g N-methyl-D-glucosamine to the solution, and stir at this temperature for 1.5 hours to obtain a clear solution. The solvent was evaporated at normal temperature at 100 rpm, and the product was dried in a vacuum oven at 50° C. to obtain 25.6 g of asiatic acid glucosamine salt, and the purity of the obtained asiatic acid glucosamine salt was 97%.

Example 18

Mix 1g of asiatic acid with absolute ethanol at room temperature, heat to 55°C until completely dissolved, add 0.81g of N-methyl-D-glucosamine to the solution, stir at this temperature for 0.5 hours to obtain a clear solution, stir The speed was 100 rpm, and the solvent was evaporated at room temperature, and the product was dried in a vacuum oven at 55° C. to obtain 1.32 g of asiatic acid glucosamine salt, and the purity of the obtained asiatic acid glucosamine salt was 97%.

Example 19

Mix 10g (1.1mmol) asiatic acid with absolute ethanol at room temperature, heat to 60°C until completely dissolved, add 1.77g (0.52mmol) glucosamine to the solution, and stir at this temperature for 1.5 hours to obtain a clear solution , the stirring speed was 100rpm, and the solvent was evaporated at normal temperature, and the product was put into a vacuum drying oven at 60°C for drying to obtain 6.10g of asiatic acid glucosamine salt, and the purity of the obtained asiatic acid glucosamine salt was 97 %.

Example 20

Mix 30g (2.5mmol) asiatic acid with absolute ethanol at room temperature, heat to 75°C until completely dissolved, add 18.2g (5mmol) glucosamine to the solution, and stir at this temperature for 2 hours to obtain a clear solution. The stirring speed is 100rpm, the solvent is evaporated at normal temperature, the product is put into a vacuum drying oven at 55°C for drying, and 40.2g of asiatic acid glucosamine salt is obtained, and the purity of the obtained asiatic acid glucosamine salt is 97% .

Embodiment 21 Asiatic acid glucosamine salt solubility detection

Conducted according to GB/T 21845-2008 Chemical Water Solubility Test Method;

The solubility of asiatic acid glucosamine salt was measured to be greater than 250 mg/ml.

In solutions exceeding 30 mg/ml, asiatic acid glucosamine salt forms a gel after 1 hour.

And the solubility of asiatic acid measured under the same situation<5mg/ml, the solubility of asiatic acid diethylamine salt<5mg/ml.

The results show that the solubility of asiatic acid glucosamine salt is significantly improved compared with that of asiatic acid and other existing types of asiatic acid salts.

Example 22 Preparation of asiatic acid glucosamine salt injection

Preparation method: add 3 mg of asiatic acid glucosamine salt to an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Autoclaved, that is.

Example 23 Preparation of asiatic acid glucosamine salt injection

Preparation method: Add 0.1 mg of asiatic acid glucosamine salt to an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Autoclaved, that is.

Example 24 Preparation of asiatic acid glucosamine salt injection

Preparation method: add 5 mg of asiatic acid glucosamine salt to an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1 ml, and encapsulate. Sterilize by autoclaving, and the concentration of the obtained asiatic acid glucosamine salt is 5 mg/ml.

Example 25 Preparation of asiatic acid glucosamine salt injection

Preparation method: Add 20mg of asiatic acid glucosamine salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1ml, and encapsulate. Autoclaved, that is.

Example 26 Preparation of asiatic acid glucosamine salt injection

Preparation method: Add 200mg of asiatic acid glucosamine salt into an appropriate amount of water for injection, adjust the pH value to 7 with hydrochloric acid/sodium hydroxide, add water for injection to 1ml, and encapsulate. Autoclaved, that is.

Example 27 Therapeutic effect of asiatic acid glucosamine salt on bleomycin-induced pulmonary fibrosis in rats

Male SD rats (purchased from Shanghai Slack Experimental Animal Co., Ltd.), weighing 250-300 g, were fasted overnight, anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and injected with bleomycin (5 U/kg) into the trachea. kg). The specific plan is as follows: cut the skin of the neck with minimal trauma, expose the trachea with the assistance of elbow ophthalmic forceps, use a micro-sampler to puncture the trachea, inject about 50 μl of bleomycin into the trachea, rotate rapidly and Stand upright for 5 minutes so that the bleomycin can enter the left and right lung lobes evenly. The whole operation is carried out on the operating table at about 60°C.

Drugs were administered to the experimental animals from the day after the modeling (wherein the day of modeling was the zeroth day, and the day after the modeling was the first day). And establish asiatic acid glucosamine salt treatment group, the daily dose is respectively 0.3mg/kg, 0.9mg/kg and 2.7mg/kg according to the body weight of mice, by intraperitoneal injection (i.p.) processing 28 days, ground The dexamethasone group was treated with 0.6mg/kg po (oral) for 28 days, and the model group and the blank group were given normal saline. For the preparation method of asiatic acid glucosamine salt injection, please refer to the description in Example 21.

After the end of the test (on the twenty-ninth day), the rats in each group were tested for lung weight ratio (lung weight/body weight*100%), serological testing (tested by Hitachi 7080 biochemical detector) and serum hydroxyproline Acid content detection.

Wherein the detection method of the hydroxyproline content is slightly improved with reference to the method of Woessner Jr et al. The detection method of the hydroxyproline content is as follows.

Reagents: Hydroxyproline, Chloramine T Analytical grade Batch number: 20020510 Zhongjiang Pharmaceutical Group Shanghai Chemical Reagent Company

p-Dimethylaminobenzaldehyde AR batch number: 20010224 Shanghai Sanaisi Reagent Co., Ltd.

Ethylene glycol methyl ether AR batch number: 20030705 Sinopharm Chemical Reagent Co., Ltd.

Instrument: 721 Spectrophotometer UV755B Xiamen Analytical Instrument Factory

The detection method operating steps of hydroxyproline content comprises:

1) The sample is dried;

2) Take samples;

3) Put the sample in the hydrolysis tube, add 6N HCL (100 times the sample volume is 2ml);

4) sealing tube;

5) Hydrolysis at 138°C for 2 hours;

6) Adjust the pH value. Using methyl red as indicator;

7) Prepare hydroxyproline standard solution at the same time, dissolve in 0.001N HCL to make 0.1mg/ml solution, and then prepare 0.5, 1.0, 1.5, 2.0, 2.5μg/ml standard products;

8) Add 1ml of chloramine T solution to the diluted sample;

9) Take 2ml of sample and add 1ml of chloramine T solution;

Chloramine T solution preparation: 1.4g chloramine T was dissolved in 20ml water. Add ethylene glycol methyl ether and 50ml citrate buffer and mix;

10) Stand at room temperature at 25°C for 20 minutes;

11) Add 1ml of perchloric acid solution;

12) Place at room temperature for 5 minutes;

13) Add 1ml of p-dimethylaminobenzaldehyde solution (20g of p-dimethylaminobenzaldehyde is dissolved in ethylene glycol methyl ether to a total of 100ml, and cooled) and mix;

14) Heat at 60°C for 20 minutes, then cool in water;

15) Measure the absorbance at 557nm, Note: If the absorbance exceeds the range of the standard curve, dilute to within the range for testing;

16) Find out the amount of hydroxyproline contained in the sample according to the standard curve.

The specific test results are shown in Table 7-10 below, wherein the number of grouped samples in Table 7 is obtained by counting on the 0th day, the total number of deaths is obtained by counting on the 29th day, and the results described in Table 8-10 are the results obtained by testing on the 29th day .

Table 7 Analysis of death of animals administered with different samples after bleomycin modeling

Table 8 Lung index of 28 days after administration of bleomycin from the next day after modeling

*Compared with the model group, there is a significant difference at P<0.05**, and there is a very significant difference at P<0.01.

Table 9 Serological test results of bleomycin modeling

*Compared with the model group, there is a significant difference at P<0.05**, and there is a very significant difference at P<0.01.

Table 10 Asiatic acid glucosamine salt modeling next day administration group anti-pulmonary fibrosis hydroxyproline measured value

The above results show that: Asiatic acid glucosamine salt injection not only has significant differences with the control group in terms of lung weight, lung index, hydroxyproline content, etc., but also has a very low mortality rate of the subjects. The curative effect of the asiatic acid choline salt provided by the invention on pulmonary fibrosis is very obvious.

Effect of Example 28 on Rabbit Ear Hypertrophic Scar Model (Prevention or Treatment of Scar Hyperplasia)

Model establishment (references Li Xijun, Liu Dalie, Wang Jihui, Discussion on the establishment method of rabbit ear hypertrophic scar model [J]. Chinese Cosmetic Medicine, 2006,15(5):499-500.):

In each rabbit (Shanghai Slack Experimental Animal Co., Ltd., weighing about 1.5 kg), two pieces of skin 1 cm×1 cm in size were excised from the ventral side of the left and right ears, with a distance of 3 cm, and were wrapped with sterile gauze. Grouping and administration: There were 21 rabbits in total, and the left and right ears of each rabbit were used for the experiment. The experiment was divided into normal skin group, model group, vitamin EC treatment group (suspension made with 0.5% CMCNa, 100 μg/ml), asiaticoside treatment group (suspension made with 0.5% CMCNa, 1 μg/ml ), asiatic acid treatment group (suspension made with 0.5% CMCNa, 200ng/ml), ethylenediamine asiatic acid treatment group (suspension made with 0.5% CMCNa, 500ng/ml), asiatic acid Glucosamine oxalate salt treatment group (the concentration of glucosamine asiatic acid glucosamine salt solution is 40 ng/ml). The administration method of each administration group is to smear 1ml of sample every day. On the 45th day after operation, the hypertrophic scar of rabbit ear was treated. At 2, 4, and 6 weeks after administration, the hypertrophic scars and skin thickness were detected with a SEQUIA512 color Doppler color ultrasonic diagnostic instrument with a 13Hz high-frequency probe, and the hypertrophic scars and skin hardness were measured with a hardness tester [Li Huiyuan, Liu Jianbo, Xia Wei, et al. Establishment and application of animal experimental models of hypertrophic scars [J]. Chinese Journal of Plastic Surgery, 2001,17(5):276-278.]. The rabbit ear hypertrophic scar specimens and skin specimens were cut for visual inspection, and the results are shown in Tables 11 and 12:

Table 11 Hypertrophic scar thickness (mm)

*p<0.05 compared with the model group; **p<0.01 compared with the model group

From the results in Table 11, it can be seen that the asiatic acid glucosamine salt obtained in the present invention has an inhibitory effect on hypertrophic scars in rabbit ears.

Table 12 Hypertrophic scar hardness (N/mm ² )

*p<0.05 compared with the model group; **p<0.01 compared with the model group

It can be seen from the above results that the asiatic acid glucosamine salt obtained in the present invention has the effect of treating scar hyperplasia.

The effect of embodiment 29 on mouse subacute aging model

1. Model preparation and processing

1) Model preparation: 3-month-old clean-grade ICR female mice (purchased from Shanghai Slack Experimental Animal Center), weighing 28-32 g.

2) Process

First, the prepared subacute aging model mice were randomly divided into groups, including the control group and each treatment group. Treatment groups include D-galactose 1000mg/kg treatment group, vitamin EC treatment group (with 0.5% CMCNa to make suspension, 100 μ g/ml), asiaticoside treatment group (with 0.5% CMCNa to make suspension, 1 μg/ml), asiatic acid treatment group (suspension made with 0.5% CMCNa, 200ng/ml), ethylenediamine asiatic acid treatment group (suspension made with 0.5% CMCNa, 500ng/ml) and asiatic acid glucosamine salt treatment group (concentration of the aqueous solution of asiatic acid glucosamine salt is 40ng/ml), 10 rats in each group, and the back of each rat was shaved with an electric hair clipper.

The application process is as follows: the control group and the model group were smeared with 1ml of normal saline on the back skin every day; the D-galactose treatment group: daily subcutaneous injection of D-galactose 1000mg/kg on the back, and the injections were all performed aseptically; vitamin EC treatment Groups: daily back skin smear suspension 1ml; asiaticoside treatment group (concentration is 1 μg/ml), asiatic acid treatment group (concentration is 200ng/ml) and asiatic acid ethylenediamine salt treatment group (concentration 500ng/ml): the daily back skin administration site was treated with microneedles, and then smear 1ml of the suspension; asiatic acid glucosamine saline solution treatment group (40ng/ml): the daily back skin administration site was treated with microneedles After needle treatment, apply 1ml. After 42 days, all the mice were sacrificed by removing their eyes and bleeding, and immediately detected the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in the whole blood; Immediately put them into 10% formalin solution for fixation, and made tissue sections for histomorphological observation and quantitative analysis; the rest of the back skin was removed and quickly frozen for preservation, which was used for homogenization of skin tissue and determination of skin hydroxyproline.

Explanation of the experimental method used in the determination of the following results: the tissue section staining method refers to the method recorded in "Pathological Staining Technology" (2nd edition, edited by Liu Peihui, Beijing: People's Medical Publishing House, 2000.56-611), and elastic fiber staining adopts Weigert.s resorcinol fuchsin method; collagen fibers were stained by Mallory-Heidenhain.s modified method.

2. Determination of results

1) Preparation of 10% skin homogenate: take 0.5 g of depilated back skin tissue, rinse with pre-cooled physiological saline, remove subcutaneous fat and other connective tissue, dry with filter paper, weigh, and take 9 times the weight of the tissue block with a sterile needle Pre-cool normal saline, take 2/3 of the amount and pour it into the culture dish containing the tissue block, then use small ophthalmic scissors to cut the tissue block into rice grains and pour it into a homogenizer, and rinse the culture dish with the remaining 1/3 of normal saline. Pour into a homogenizer, use a high-speed homogenizer to make 10% tissue homogenate (in ice water), and freeze and thaw repeatedly 3 times to completely break the cells and free the contents in the liquid phase.

2) Determination of CAT and GSH-Px activity in whole blood: take 0.5ml of fresh 30% H ₂ O ₂ solution, add 50ml of distilled water and mix evenly, take 4ml out of it, add 26ml of 0.05mol/L phosphate buffer solution with pH 7.0, UV Spectrophotometer measures the A value of the 1cm light path at the wavelength of 230nm, adjusts the A value between 0.50 and 0.55, and uses it as the CAT substrate solution. Under the condition of 25°С, take 3ml of the substrate solution, quickly add 0.01ml of 1:50 hemolysis (purchased from Shanghai Lianke Biology), measure its A value immediately at 230nm wavelength, measure its A value after 1min, and measure the protein at the same time content. The activity of GSH-Px was determined according to the instructions of the GSH-Px kit of Nanjing Jiancheng Bioengineering Company.

3) Determination of skin biochemical indicators: refer to the method for measuring SOD activity recorded in the instructions of the superoxide dismutase (SOD) kit of Nanjing Jiancheng Bioengineering Company; determination of malondialdehyde (MDA) content in skin, determination of skin CAT activity, The determination of skin hydroxyproline content can be found in literature [Xu Shuyun, Chen Xiu, editor-in-chief. Pharmacological Experimental Methodology. 3rd Edition. Beijing: People's Health Publishing House, 2002.1464-1472]. Gained experimental result is shown in table 13 and table 14:

Table 13 CAT and GSH-Px activity detection results in mouse blood

*p<0.05 compared with normal group

Table 14 Detection results of mouse skin biochemical indicators

*p<0.05 compared with model group

From the above results, it can be seen that the asiatic acid glucosamine salt obtained in the present invention is similar to other groups, and has no significant impact on the activity of CAT and GSH-Px in mouse blood; it has no obvious impact on SOD activity, MDA content, and CAT in mouse skin. , but the content of hydroxyproline was higher than that of the model group and other groups, showing that it has a good anti-wrinkle effect.

Description of tissue slice results:

Control group: normal skin epidermis thickness, clear cell stratification, complete tissue structure, and obvious epidermal protrusions; the dermis is thicker, and the elastic fibers are arranged tightly and evenly.

Model group: the thickness of the epidermis becomes thinner, the cells are arranged in disorder, the dermis is significantly thinner, the number of cell layers is reduced, and the arrangement of collagen fibers in the dermis is loose; the total area of elastic fibers is significantly reduced, and thickened, denatured, and accumulated.

Asiaticoside treatment group, asiatic acid treatment group, Asiatic acid ethylenediamine salt treatment group: the thickness of the skin epidermis is close to normal, the cell layering is not clear, and the arrangement is relatively disordered; the dermis is thicker, and the elastic fibers are thickened, transsexual.

Vitamin EC treatment group and asiatic acid glucosamine salt treatment group: the thickness of the skin epidermis is close to normal, the cell layer is relatively clear, and the tissue structure is relatively complete; the thickness of the dermis is relatively normal, the elastic fibers are slightly thickened, arranged more closely, and distributed more evenly .

The above results indicate that the asiatic acid glucosamine salt has a more significant anti-wrinkle effect on the skin than other asiatic acid salt or compound treatment groups.

Effect of Example 30 on Mouse Skin Photoaging Model

1. Model preparation and processing

1) Model preparation: Take ICR female mice (purchased from Shanghai Slack Experimental Animal Center, weighing 20-25g), use an electric hair clipper to shave the back skin of the mice, about 2cm×2cm, bare skin, weighing 28-32g .

2) Process

The prepared skin photoaging model mice were randomly divided into groups and administered medicine, including control group and each treatment group. The skin of the control group was smeared with double distilled water once a day without any other treatment. The model group includes the photoaging model group. The skin is smeared with 1% 8-methoxypsoralen (8-MOP) once a day. After 1.5-2 hours, it is given 190μw of UVA irradiation, and the cumulative irradiation is 138 hours (cumulative intensity 95J/cm2) , take the skin within 24h after the irradiation; vitamin EC treatment group (suspension made with 0.5% CMCNa, 100 μg/ml); asiaticoside treatment group (suspension made with 0.5% CMCNa, 1 μg/ml) Asiatic acid treatment group (making suspension with 0.5% CMCNa, 200ng/ml); Asiatic acid ethylenediamine salt treatment group (making suspension with 0.5% CMCNa, 500ng/ml); Asiatic acid Glucosamine saline solution treatment group (40ng/ml, treated with microneedles at the administration site, applied 1% 8-MOP after the drug dries slightly, 1.5-2h later, given 190μw UVA irradiation, accumulatively irradiated for 138h, accumulative Intensity 95J/cm ² , the skin was taken within 24 hours after the end of the irradiation); after the experiment, the skin tissues were taken from the control group and each treatment group at the same time, fixed, stained with HE, and the hydroxyproline content was measured at the same time. The results are as follows: Table 15 shows.

2. Result analysis

1) Specimen preparation

The removed tissue pieces were fixed in 10% formaldehyde. After 12 hours, the fixed tissue blocks were taken out for embedding in wax blocks. After the wax block was sectioned, the sections were routinely stained with HE.

2) Determination of tissue hydroxyproline content

The determination method was operated according to the hydroxyproline detection kit (purchased from Lianke Biotechnology Co., Ltd.), and the tissue protein content was detected at the same time, and the t test was used to analyze the results.

Description of tissue slice results:

Control group: normal skin epidermis thickness, clear cell stratification, complete tissue structure, and obvious epidermal protrusions; the dermis is thicker, and the elastic fibers are arranged tightly and evenly.

Photoaging group: Compared with the normal control group, typical pathological changes of photoaging appeared: uneven thickness of the epidermis, frequent exfoliation, obvious thickening of the dermis, frequent infiltration of inflammatory cells, expansion of microvessels, reduction of hair follicles, and irregular sebaceous glands hyperplasia.

Asiaticoside treatment group, asiatic acid treatment group, asiatic acid ethylenediamine salt treatment group: the pathological conditions were similar to those of the photoaging group.

Compared with the photoaging group, the pathological changes of the vitamin EC group and the asiatic acid glucosamine salt group were significantly improved, the dermis was thinner than that of the photoaging group, and the infiltration of inflammatory cells was less.

Table 15 Tissue hydroxyproline content determination (hydroxyproline/total protein, )

*p<0.05 compared with photoaging group

From the above results, it can be seen that the hydroxyproline content in the tissue of the asiatic acid glucosamine salt treatment group was significantly increased compared with the photoaging group, and compared with the treatment groups of other compounds, the hydroxyproline content of the asiatic acid glucosamine salt treatment group The hydroxyproline content in the tissue increased the most, indicating that asiatic acid glucosamine salt had a significant protective effect on the photoaging skin of mice.

It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

1. a kind of asiatic acid choline salt, its structural formula is as shown in formula I: 2. a preparation method of asiatic acid choline salt, said preparation method comprising the following steps: in an organic solvent, asiatic acid and choline are carried out to the neutralization reaction as shown below to obtain 3. the preparation method as claimed in claim 2 is characterized in that, the temperature of described neutralization reaction is 55 ℃～75 ℃; The time of described neutralization reaction is 0.5～2 hours; Described organic solvent is Ethanol, methanol or isopropanol; in the neutralization reaction, the molar ratio of the asiatic acid to choline is 2:1 to 1:2; the molar concentration of the asiatic acid is 0.5 to 5 mol /L, the molar concentration of the choline is 0.5-5 mol/L. 4. A glucosamine asiatic acid salt, characterized in that the structural formula of the glucosamine asiatic acid salt is as shown in formula IV: 5. A preparation method of asiatic acid glucosamine salt, characterized in that the preparation method comprises the following steps: in an organic solvent, asiatic acid as shown in formula V and glucosamine as shown in formula VI Carry out the neutralization reaction shown below to get sugar amine 6. The preparation method according to claim 5, wherein the temperature of the neutralization reaction is 60°C to 70°C; the time of the neutralization reaction is 0.5 to 2 hours; the organic solvent is ethanol , methanol or isopropanol; in the neutralization reaction, the molar ratio of the asiatic acid to glucosamine is 2:1 to 1:2; the molar concentration of the asiatic acid is 0.5 to 5 mol /L; the molar concentration of the glucosamine is 0.5-5 mol/L. 7. Use of asiatic acid choline salt as claimed in claim 1 or asiatic acid glucosamine salt as claimed in claim 4 in the preparation of medicines for preventing or treating fibrosis. 8. The use according to claim 7, wherein the fibrosis is pulmonary fibrosis, and the pulmonary fibrosis is primary pulmonary fibrosis or secondary pulmonary fibrosis. 9. A pharmaceutical composition comprising asiatic acid choline salt as claimed in claim 1 or asiatic acid glucosamine salt as claimed in claim 4, characterized in that, the dosage form of said pharmaceutical composition is injection or solution. 10. Use of asiatic acid choline salt as claimed in claim 1 or asiatic acid glucosamine salt as claimed in claim 4 in the preparation of medicines or cosmetics for preventing or treating scar hyperplasia. 11. Use of asiatic acid glucosamine salt as claimed in claim 4 in the preparation of anti-aging medicine or cosmetics. 12. The use according to claim 11, characterized in that the aging is subacute aging or photoaging. 13. The use according to claim 11, wherein said aging is skin aging. 14. The use according to claim 13, characterized in that the skin aging includes skin tissue degeneration or skin physiological function degeneration. 15. purposes as claimed in claim 11, is characterized in that, the dosage form of described medicine is solution, suspension, coating agent, gel, ointment, transdermal absorption agent or biofilm agent; The dosage forms of cosmetics are solutions, suspensions, coatings, gels, ointments, transdermal absorption agents or biofilm agents.