CN112402471A - Preparation method of total asiatic acid, total asiatic acid prepared by preparation method and application of total asiatic acid - Google Patents

Abstract

The invention belongs to the field of pharmacy, and particularly relates to a preparation method of total asiatic acid, the total asiatic acid prepared by the preparation method and application of the total asiatic acid. A preparation method of total asiatic acid comprises the following steps: (1) dissolving the asiaticoside alcohol solvent, adding a poor solvent, and recrystallizing to obtain refined asiaticoside; (2) hydrolyzing the refined asiaticoside product with alkaline matter to obtain coarse asiaticoside product; (3) adding an ethanol solution into the crude product of the total acid of the asiatic centella, dropwise adding an alkaline solution until the pH value of a liquid phase is 9-11, then adding an acidic solution until the pH value of the liquid phase is 6-9, carrying out first solid-liquid separation to obtain a first solid phase, dissolving the first solid phase with methanol, adding an acidic solution until the pH value of the liquid phase is 4-5, concentrating, adding water, carrying out second solid-liquid separation to obtain a second solid phase, namely the total acid of the asiatic centella. The total content of asiatic acid, madecassic acid and asiatic acid B in the total asiatic acid obtained by the preparation method is more than or equal to 95%, and the preparation method is suitable for industrialization.

Description

Preparation method of total asiatic acid, total asiatic acid prepared by preparation method and application of total asiatic acid

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a preparation method of total asiatic acid, the total asiatic acid prepared by the preparation method and application of the total asiatic acid.

Background

Pulmonary fibrosis is the terminal change of a large group of lung diseases characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammatory injury and tissue structure destruction, namely structural abnormality (scar formation) caused by abnormal repair after normal alveolar tissues are damaged. Pulmonary fibrosis seriously affects the respiratory function of the human body, manifested as dry cough and progressive dyspnea (insufficient subjective qi), and the respiratory function of the patient is continuously worsened with the aggravation of the disease condition and the lung injury. The incidence and mortality of idiopathic pulmonary fibrosis increases year by year, with an average survival period of only 2.8 years after diagnosis, with mortality rates higher than that of most tumors, known as a "neoplastic-like disease".

In recent years, the research on the application of Chinese herbal medicines to the treatment of pulmonary fibrosis is more, and certain progress is made. Centella asiatica (L.) Urban is a plant of Centella asiatica of the family umbelliferae, and its leaves resemble horseshoe-shaped or half-copper coin, so it is also called as Centella asiatica, glechoma longituba, etc., and it is also called as huh mint, liverwort, etc. in folk. Centella has a long history of application in the traditional medicine field of many countries and regions, and the traditional Chinese medicine of China has two thousand years of history of internal and external application of centella, and is originally collected in one book of Shen nong Ben Cao Jing.

The chemical components of centella asiatica mainly comprise triterpenes, flavonoids, polyacetylenes, volatile oils and the like. Triterpenes mainly include triterpenoid saponins such as asiaticoside, madecassoside, asiaticoside B, etc., and triterpenic acids such as asiatic acid, madecassic acid, asiatic acid B, etc. Triterpenic acid substances in the chemical components of centella asiatica are a large group of components which are researched most early and have the most deep biological activity. Centella asiatica triterpenic acids mainly comprise asiatic acid, madecassic acid and asiatic acid B, and have antioxidant, anti-fibrosis, anti-tumor and analgesic effects. Jihe warship and the like (the protective effect of asiaticoside on bleomycin-induced pulmonary fibrosis of rats, modern medicine 2014, 11 th volume 42 and 1309 th page) research the action mechanism of asiaticoside having the protective effect on pulmonary fibrosis of rats; the Shanghai pharmaceutical industry research institute (patent application Nos. 200710037474.3, 200610117236.9, 03116105.7) also conducted systematic studies on the effect of asiaticoside against pulmonary fibrosis. The patent application No. 200610118041.6 reports that asiatic acid also has anti-pulmonary fibrosis effect.

At present, the active ingredients of asiatic acid, madecassic acid and asiatic acid B in asiatic total acid are separately separated and researched by column chromatography or preparation of liquid phase after asiaticoside is hydrolyzed. The method has large solvent consumption and high three-waste treatment cost, and is not suitable for industrial production. Meanwhile, according to literature reports and experimental results, the asiatic acid, the madecassic acid and the asiatic acid B are all effective on liver pulmonary fibrosis, have curative effects on chronic progressive fibrotic diseases and provide hopes for delaying organ failure.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of total asiatic acid, the total asiatic acid prepared by the preparation method and application of the total asiatic acid. Through the asiaticoside crystallization, the asiaticoside hydrolysis and the asiaticoside crystallization, white or white-like solid asiaticoside with the total acid content of 95-98% can be obtained, and the proportion of the main active ingredients of the asiaticoside, madecassic acid and asiatic acid B is consistent with the proportion of the asiaticoside. The asiaticoside hydrolyzing process is optimized to obtain white or quasi-white solid asiatic acid with total acid content not less than 95%.

The commercially available asiaticoside is obtained by extraction from centella asiatica, and the centella asiatica contains triterpenes, polyacetylenic olefins, flavonoids, alkaloids and polyphenol components, so that the content of the asiaticoside is low. The highest total content of asiaticoside, madecassoside and asiaticoside B in the total asiaticoside sold in the market at present is 80%, and the combination of experimental results shows that the total asiaticoside sold in the market is directly hydrolyzed to prepare the total asiatic acid, so that more impurities are generated, the total acid content is only 80-85%, and the total acid has poor appearance.

The first aspect of the invention provides a preparation method of total asiatic acid, which comprises the following steps:

(1) dissolving the asiaticoside alcohol solvent, adding a poor solvent, and recrystallizing to obtain refined asiaticoside;

(2) performing hydrolysis reaction on the refined asiaticoside product obtained in the step (1) and an alkaline substance to obtain a crude asiaticoside product;

(3) adding an ethanol solution into the crude asiatic centella total acid product obtained in the step (2), dropwise adding an alkaline solution until the pH value of a liquid phase is 9-11, then adding an acidic solution until the pH value of the liquid phase is 6-9, carrying out first solid-liquid separation to obtain a first solid phase, dissolving the first solid phase with methanol, adding an acidic solution until the pH value of the liquid phase is 4-5, concentrating, adding water, and carrying out second solid-liquid separation to obtain a second solid phase, namely asiatic centella total acid.

In some embodiments of the first aspect of the present invention, in the step (3), the ethanol solution is a mixed solution of water and absolute ethanol; preferably, the volume ratio of the water to the ethanol is 0.2-10: 1 (e.g., 10: 1, 5: 1, 2: 1, 1:1, 1: 2, 1: 5), more preferably 1: 1.

In some embodiments of the first aspect of the present invention, in the step (3), the crude asiatic total acid is mixed with the mixed solution of water and absolute ethyl alcohol in an amount relationship of: 4.5g of the crude product of the total acid of centella asiatica corresponds to 270.0ml of the mixed solution of water and absolute ethyl alcohol.

In certain embodiments of the first aspect of the present invention, in step (3), the alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution.

In certain embodiments of the first aspect of the present invention, in step (3), the acidic solution is glacial acetic acid.

In some embodiments of the first aspect of the present invention, in the step (3), before the first solid-liquid separation, the step (3) further comprises a temperature reduction step, wherein the temperature in the temperature reduction step is controlled to be 0-10 ℃ (such as 2 ℃, 5 ℃, 8 ℃) and the temperature is kept for crystallization for 30 minutes.

In some embodiments of the first aspect of the present invention, the method comprises one or more of the following 1) to 7):

1) in the step (1), the usage relationship of the asiaticoside and the methanol is as follows: 40.0g of total asiaticoside corresponds to 200.0ml of methanol;

2) in the step (1), the alcoholic solvent is selected from one or more of methanol, ethanol, isopropanol, n-propanol, n-butanol and tert-butanol;

3) in the step (1), the poor solvent is one or more selected from methyl tert-butyl ether, ethyl acetate, dichloromethane, acetonitrile, tetrahydrofuran, ethanol, isopropanol and n-butanol;

preferably, the dosage of the total asiaticoside and the poor solvent is in a relation of 40.0g of total asiaticoside to 100.0-500.0ml (such as 150.0ml, 200.0ml, 300.0ml, 400.0ml, 450.0ml) of the poor solvent, more preferably 200ml of the poor solvent;

4) in the step (1), after dissolving the asiaticoside alcohol solvent, decoloring by using medicinal carbon before adding a poor solvent;

5) in the step (1), the recrystallization is carried out at 15-25 ℃ (such as 18 ℃, 20 ℃ and 23 ℃) for 30 minutes of heat preservation crystallization;

6) in the step (2), the alkaline substance is selected from one or more of sodium hydroxide, lithium hydroxide, potassium hydroxide and calcium hydroxide;

preferably, the relationship between the usage amount of the refined asiaticoside and the alkaline substance is as follows: 10.0g of refined asiaticoside corresponding to 1.92-5.92g (such as 2.0g, 2.5g, 3.0g, 3.5g, 4.0g, 4.5g, 5.0g, 5.5g) of alkaline substance;

7) in step (2), the temperature of the hydrolysis reaction is 60-80 deg.C (such as 62 deg.C, 65 deg.C, 68 deg.C, 70 deg.C, 75 deg.C, 78 deg.C).

A second aspect of the invention provides an asiatic total acid comprising asiatic acid, madecassic acid, asiatic acid B; the total content of asiatic acid, madecassic acid and asiatic acid B in the total asiatic acid is not less than 95% (such as 95%, 96%, 97%, 98%, 99%, 99.9%).

In certain embodiments, the total asiatic acid content of centella asiatica, madecassic acid and asiatic acid B is 95-98%.

In certain embodiments, the total asiatic acid is prepared from total asiaticoside.

In some embodiments, the total asiatic acid is prepared by the above preparation method.

The third aspect of the invention provides an application of the total asiatic acid in preparing a medicine for treating pulmonary fibrosis.

The fourth aspect of the present invention provides a solution preparation for inhalation of total asiatic acid, wherein the total content of asiatic acid, madecassic acid and asiatic acid B in the total asiatic acid is not less than 95%, and the solution preparation comprises:

(1) the effective component is a composition of three acids, wherein the concentration range of the asiatic acid B is as follows: 45-90ug/mL (e.g., 50ug/mL, 55ug/mL, 60ug/mL, 70ug/mL, 80ug/mL, 85 ug/mL); concentration range of madecassic acid: 150-350ug/mL (such as 170ug/mL, 200ug/mL, 240ug/mL, 280ug/mL, 300ug/mL, 320 ug/mL); the concentration range of asiatic acid is as follows: 250-550ug/mL (such as 270ug/mL, 300ug/mL, 350ug/mL, 400ug/mL, 450ug/mL, 500ug/mL, 520 ug/mL);

(2) an isotonicity adjusting agent;

(3) a solvent.

In the above solution preparation for inhalation, as a preferred embodiment, the solution preparation has an osmolality of 260-480mOsmol/kg (e.g., 280mOsmol/kg, 290mOsmol/kg, 300mOsmol/kg, 305mOsmol/kg, 310mOsmol/kg, 350mOsmol/kg, 400mOsmol/kg, 450 mOsmol/kg); preferably 280-320 mOmol/kg.

In the above solution formulation for inhalation, as a preferred embodiment, the pH of the solution formulation is 3.0 to 8.5 (e.g., 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5).

In the above solution formulation for inhalation, as a preferred embodiment, the solution formulation further comprises a pH adjuster; the pH regulator is preferably an inorganic base or a buffering agent; the inorganic base is preferably one or more of sodium hydroxide, sodium bicarbonate, disodium hydrogen phosphate, sodium citrate and sodium carbonate; the buffer is preferably citric acid-sodium citrate, citric acid-disodium hydrogen phosphate, potassium dihydrogen phosphate-disodium hydrogen phosphate or citric acid-sodium hydroxide. By controlling the pH value of the solution preparation, the irritation of the solution preparation on mucous membrane can be reduced, and the physical property of the solution preparation can be optimized, so that the aerosol forming capability of the solution preparation is improved.

In the above solution formulation for inhalation, as a preferred embodiment, the solvent is deionized water, distilled water, or water for injection.

In the invention, an ultrasonic atomizer or an air compression atomizer is adopted, the solution preparation of the effective components for inhalation is inhaled into a respiratory system in an atomization mode, can directly act on the focus part, and has quick response, safety and effectiveness. The effective components of centella can be directly delivered to the lung of a patient by an inhalation administration mode, thereby obviously relieving symptoms and shortening the course of disease. The medicine directly enters the respiratory tract, so that the toxic and side effects of the medicine are obviously reduced, which is particularly important for cancer patients.

The invention also provides a preparation method of the solution for inhalation, which comprises the following steps:

taking the total asiatic acid according to the prescription amount, and adding a solvent to obtain a mixture; mixing the mixture uniformly, adding an isotonic agent and a pH regulator, dissolving and clarifying, and fixing the volume to obtain the solution for inhalation; putting the solution for inhalation into an ampoule bottle to obtain the total acid solution preparation of the asiatic centella for inhalation.

Compared with the prior art, the invention has the following technical effects:

1. the invention provides an asiatic total acid, wherein the total content of asiatic acid, madecassic acid and asiatic acid B in the asiatic total acid is greater than or equal to 95%.

2. The preparation method can obtain white or white-like total asiatic acid solid, wherein the total content of asiatic acid, madecassic acid and asiatic acid B in the total asiatic acid is more than or equal to 95%, and the proportion of the main active ingredients of the total asiatic acid, including asiatic acid, madecassic acid and asiatic acid B is consistent with the proportion of total asiaticoside, so that the preparation method is suitable for industrialization.

3. The asiatic centella total acid is initiatively prepared into the atomization inhalation preparation, the bioavailability of the medicament can be greatly improved through pulmonary administration, the first pass effect of the medicament is reduced, the toxicity of liver and kidney is reduced, the treatment effect is improved, and a new treatment idea is provided for the treatment of fibrosis.

4. The preparation method of the total asiatic acid solution preparation for inhalation has the advantages of few steps and easy operation, and is beneficial to industrial production.

Detailed Description

The processes for preparing the products and the preparations of the following examples are not limited to the words, and any method for preparing the drugs or preparations provided by the present invention is also within the scope of the present invention.

The chemicals used in the examples were those whose preparation methods were not clarified, the reagents or instruments used were those whose manufacturers were not aware of them, and they were all conventional products commercially available.

Example 1 recrystallization of Total glycosides of centella asiatica

Adding 40.0g of total asiaticoside (purchased from Siemens Mingze Biotechnology Co., Ltd.) and 200.0ml of methanol into a 500ml four-neck flask, stirring at room temperature until the total asiaticoside is dissolved completely, adding 8.0g of medicinal charcoal, stirring and heating to 70 ℃, keeping the temperature and stirring for 30 minutes, stirring and cooling to room temperature, filtering, and recovering filter residues.

Concentrating the filtrate at 50 deg.C under 0.10Mpa to 2 times volume, adding 200.0ml acetonitrile, cooling to 25 deg.C, keeping the temperature, crystallizing for 30 min, filtering, and drying the filter cake at 65 deg.C for 8 hr to obtain refined product 28.0g of total glycosides of herba Centellae.

Example 2 recrystallization of Total glycosides of centella asiatica

Adding 40.0g of total asiaticoside and 200.0ml of methanol into a 500ml four-mouth bottle, stirring at room temperature until the total asiaticoside and the methanol are completely dissolved, adding 8.0g of medicinal carbon, stirring, heating to 60 ℃, keeping the temperature, stirring for 30 minutes, stirring, cooling to 20 ℃, filtering, and recovering filter residues.

Concentrating the filtrate at 40 deg.C under 0.08Mpa to 1 times volume, adding 200.0ml ethyl acetate, cooling to 20 deg.C, keeping the temperature, crystallizing for 30 min, filtering, and drying the filter cake at 60 deg.C for 12 hr to obtain refined product of total glycosides of herba Centellae 29.0 g.

Example 3 recrystallization of Total glycosides of centella asiatica

Adding 40.0g of total asiaticoside and 200.0ml of methanol into a 500ml four-mouth bottle, stirring at room temperature until the total asiaticoside and the methanol are completely dissolved, adding 8.0g of medicinal carbon, stirring, heating to 65 ℃, keeping the temperature, stirring for 30 minutes, stirring, cooling to 25 ℃, filtering, and recovering filter residues.

Concentrating the filtrate at 45 deg.C under 0.09Mpa to 1 volume, adding 200.0ml methyl tert-butyl ether, cooling to 25 deg.C, keeping the temperature, crystallizing for 30 min, filtering, and drying the filter cake at 65 deg.C for 12 hr to obtain refined product of total glycosides of herba Centellae 35.0 g.

Example 4 preparation of crude Total acid of centella asiatica

80.0ml of a purified water/absolute ethyl alcohol mixed solvent (1/1 in volume ratio) and 10.0g of a refined asiaticoside (prepared according to the protocol of example 3) were added to a 250ml four-necked flask, and 3.2g of sodium hydroxide was added in portions while controlling the temperature in the flask at 20-30 ℃. And after the addition is finished, stirring until all the solution is clear, stirring and heating to 80 ℃ under the protection of nitrogen, keeping the temperature for reaction for 1 hour, and sampling for TLC.

When the raw material point disappears, the reaction is qualified, the reaction liquid is concentrated at 50 ℃ and 0.10Mpa until no fraction is produced, the concentration is stopped, the reaction liquid is cooled to 20 ℃, 100.0ml of drinking water is added into a reaction bottle, hydrochloric acid solution is dripped into the reaction bottle at the temperature of 20-30 ℃, the reaction liquid is stirred for 10 minutes, and then the pH value of the reaction liquid is detected to be 1-2.

And filtering the reaction solution, washing with water until the pH value is 6.5-7.0, and drying the filter cake at 60 ℃ for 12 hours to obtain 4.1g of the total asiatic acid crude product.

EXAMPLE 5 preparation of crude Total acid of centella asiatica

60.0ml of a purified water/absolute ethyl alcohol mixed solvent (1/1 in volume ratio) and 10.0g of a refined asiaticoside (prepared according to the protocol of example 3) were added to a 250ml four-necked flask, and 3.2g of sodium hydroxide was added in portions while controlling the temperature in the flask at 20-30 ℃. And after the addition is finished, stirring until all the solution is clear, stirring and heating to 65 ℃ under the protection of nitrogen, keeping the temperature for reaction for 1 hour, and sampling for TLC.

When the raw material point disappears, the reaction is qualified, the reaction liquid is concentrated at 50 ℃ and 0.10Mpa until no fraction is produced, the concentration is stopped, the reaction liquid is cooled to 25 ℃, 100.0ml of drinking water is added into a reaction bottle, hydrochloric acid solution is dripped into the reaction bottle at the temperature of 20-30 ℃, the reaction liquid is stirred for 10 minutes, and then the pH value of the reaction liquid is detected to be 1-2.

And filtering the reaction solution, washing with water until the pH value is 6.5-7.0, and drying the filter cake at 65 ℃ for 8 hours to obtain 4.5g of the total asiatic acid crude product.

Example 6 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol (the volume ratio of the purified water to the absolute ethyl alcohol is 1/5) and 4.5g of crude asiatic centella total acid (prepared according to the scheme of the embodiment 5) into a 500ml four-mouth bottle, dropwise adding a sodium hydroxide solution into the reaction bottle at the temperature of 20-30 ℃ until the pH value of a reaction solution is 9-10, stirring until the sodium hydroxide solution is completely dissolved, dropwise adding glacial acetic acid into the reaction bottle at the temperature of 20-30 ℃ until the pH value of the reaction solution is 7-8, precipitating a large amount of solid in a reaction system, stirring and cooling to 0 ℃, preserving heat and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, controlling the temperature in the reaction bottle to be 20-30 ℃, dropwise adding glacial acetic acid until the pH value of a reaction solution is 4-5, concentrating the reaction solution to 2 times of volume under the condition of 40 ℃ and 0.08Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying for 12 hours at 60 ℃ to obtain 1.0g of the total asiatic acid finished product.

Example 7 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol (the volume ratio of the purified water to the absolute ethyl alcohol is 5/1) and 4.5g of crude asiatic centella total acid (prepared according to the scheme of the embodiment 5) into a 250ml four-mouth bottle, dropwise adding a sodium hydroxide solution into the reaction bottle at the temperature of 20-30 ℃ until the pH value of a reaction solution is 9-10, stirring until the sodium hydroxide solution is completely dissolved, dropwise adding glacial acetic acid into the reaction bottle at the temperature of 20-30 ℃ until the pH value of the reaction solution is 7-8, precipitating a large amount of solid in a reaction system, stirring and cooling to 5 ℃, preserving heat and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, controlling the temperature in the reaction bottle to be 20-30 ℃, dropwise adding glacial acetic acid until the pH value of a reaction solution is 4-5, concentrating the reaction solution to 1 time of volume under the condition of 45 ℃ and 0.09Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying for 12 hours at 65 ℃ to obtain 3.0g of the total asiatic acid finished product.

Example 8 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol mixed solvent (the volume ratio of purified water to absolute ethyl alcohol is 1/1) and 4.5g of crude asiatic acid (prepared according to the scheme of example 5) into a 250ml four-mouth bottle, dropwise adding a sodium hydroxide solution into the bottle at 20-30 ℃ until the pH value of the reaction solution is 9-10, stirring until the sodium hydroxide solution is completely dissolved, dropwise adding glacial acetic acid into the bottle at 20-30 ℃ until the pH value of the reaction solution is 7-8, separating out a large amount of solids from the reaction system, stirring and cooling to 8 ℃, keeping the temperature and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, controlling the temperature in the reaction bottle to be 20-30 ℃, dropwise adding glacial acetic acid until the pH value of a reaction solution is 4-5, concentrating the reaction solution to be 1.5 times of the volume of the reaction solution at 45 ℃ and 0.10Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying for 10 hours at 65 ℃ to obtain 2.0g of the total asiatic acid finished product.

Example 9 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol mixed solvent (the volume ratio of purified water to absolute ethyl alcohol is 2/1) and 4.5g of crude asiatic acid (prepared according to the scheme of example 5) into a 250ml four-mouth bottle, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding a sodium hydroxide solution until the pH value of a reaction solution is between 9 and 11, stirring until the sodium hydroxide solution is completely dissolved, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding glacial acetic acid until the pH value of the reaction solution is between 8 and 9, separating out a large amount of solids from a reaction system, stirring and cooling to 10 ℃, preserving heat and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, dropwise adding glacial acetic acid at the temperature of 20-30 ℃ in the reaction bottle until the pH value of the reaction solution is 4-5, concentrating the reaction solution to 2 times of the volume at-50 ℃ and 0.08-Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying at 65 ℃ for 12 hours to obtain 2.3g of the total asiatic acid finished product.

Example 10 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol mixed solvent (the volume ratio of purified water to absolute ethyl alcohol is 1/1) and 4.5g of crude asiatic acid (prepared according to the scheme of example 5) into a 250ml four-mouth bottle, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding a sodium hydroxide solution until the pH value of a reaction solution is between 9 and 11, stirring until the sodium hydroxide solution is completely dissolved, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding glacial acetic acid until the pH value of the reaction solution is between 8 and 9, separating out a large amount of solids from a reaction system, stirring and cooling to 10 ℃, preserving heat and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, dropwise adding glacial acetic acid at the temperature of 20-30 ℃ in the reaction bottle until the pH value of the reaction solution is 4-5, concentrating the reaction solution to 2 times of the volume at 50 ℃ and 0.08Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying at 65 ℃ for 12 hours to obtain 1.3g of the total asiatic acid finished product.

Example 11 Total acid recrystallization of centella asiatica

Adding 270.0ml of purified water/absolute ethyl alcohol mixed solvent (the volume ratio of purified water to absolute ethyl alcohol is 1/1) and 4.5g of crude asiatic acid (prepared according to the scheme of example 5) into a 250ml four-mouth bottle, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding a sodium hydroxide solution until the pH value of a reaction solution is between 9 and 11, stirring until the sodium hydroxide solution is completely dissolved, controlling the temperature in the reaction bottle to be between 20 and 30 ℃, dropwise adding glacial acetic acid until the pH value of the reaction solution is between 6 and 7, separating out a large amount of solids from a reaction system, stirring and cooling to 10 ℃, preserving heat and crystallizing for 30 minutes, and filtering.

Adding 135.0ml of methanol and a filter cake into a 250ml four-mouth bottle, stirring until the methanol and the filter cake are completely dissolved, dropwise adding glacial acetic acid at the temperature of 20-30 ℃ in the reaction bottle until the pH value of the reaction solution is 4-5, concentrating the reaction solution to 2 times of the volume at 50 ℃ and 0.08Mpa, adding 90.0ml of purified water, stirring, mixing uniformly, filtering, washing with water until the pH value is 6.5-7.0, and drying at 60-65 ℃ for 12 hours to obtain 1.2g of the total asiatic acid finished product.

The content of the total asiatic acid prepared in examples 6 to 11 was measured by the following method:

as determined by HPLC, mobile phase: β -cyclodextrin (4mmol/L, pH 4.0 adjusted with acetic acid) -methanol (40: 60), column: InfinityLab Poroshell 120 EC-C184.6 x 150mm 4 μm, flow rate: 0.8ml/min, column temperature: 30 ℃, wavelength: 204nm, the results are shown in Table 1.

TABLE 1

	Asiatic acid B	Madecassic acid	Asiatic acid
				Example 6	t＝20.278min,area％＝5.69％	t＝25.873min,area％＝29.8％	t＝47.461min,area％＝60.11％
Example 7	t＝20.290min,area％＝13.68％	t＝25.886min,area％＝31.25％	t＝47.411min,area％＝46.3％
				Example 8	t＝21.204min,area％＝9.63％	t＝25.862min,area％＝34.33％	t＝47.338min,area％＝52.81％
Example 9	t＝21.748min,area％＝11.4％	t＝26.692min,area％＝32.93％	t＝49.082min,area％＝49.71％
				Example 10	t＝21.210min,area％＝14.19％	t＝25.860min,area％＝35.57％	t＝47.351min,area％＝45.73％
Example 11	t＝21.776min,area％＝11.36％	t＝26.737min,area％＝37.33％	t＝49.145min,area％＝48.37％

Note: t represents the time of peak and area% represents the percentage of integrated area, i.e., the percentage of the substance.

Example 12 Total acid centella asiatica inhalation solution

Prescription:

active ingredients: total asiatic acid 500 mg;

isotonic adjusting agent: sodium chloride (in appropriate amount, adjusted to osmotic pressure (osmolality) 300mOsmol/kg)

pH regulator: 2.0g/ml aqueous disodium hydrogen phosphate solution

Water for injection: 1000ml

The preparation method comprises the following steps: taking a proper amount of snow-accumulated total acid, dispersing the snow-accumulated total acid by 80 percent of formula amount of water, controlling the temperature to be 30-40 ℃, adding the formula amount of sodium chloride for dissolving, adjusting the pH to 7.5 by using 2.0g/ml of disodium hydrogen phosphate aqueous solution, clarifying, fixing the volume and filling into an ampoule bottle to obtain the snow-accumulated total acid.

Example 13 Total acid centella asiatica inhalation solution

Prescription:

active ingredients: 800mg of centella total acid;

isotonic adjusting agent: sodium chloride (in appropriate amount, adjusted to osmotic pressure (osmolality) 300mOsmol/kg)

pH regulator: 1.0g/ml sodium citrate solution

Water for injection: 1000ml

The preparation method comprises the following steps: taking a proper amount of snow-accumulated total acid, dispersing the snow-accumulated total acid by 80 percent of formula amount of water, controlling the temperature to be 20-30 ℃, adding the formula amount of sodium chloride for dissolving, using 1.0g/ml sodium citrate solution to adjust the pH value to 8.0, clarifying, fixing the volume and filling into an ampoule bottle to obtain the snow-accumulated total acid.

EXAMPLE 14 Total acid centella asiatica inhalation solution

Prescription:

active ingredients: 100mg of centella total acid;

isotonic adjusting agent: sodium chloride (in appropriate amount, adjusted to osmotic pressure (osmolality) 300mOsmol/kg)

pH regulator: 0.1M sodium hydroxide solution

Water for injection: 1000ml

The preparation method comprises the following steps: taking a proper amount of snow-accumulated total acid, dispersing the snow-accumulated total acid by 80 percent of formula amount of water, controlling the temperature to be 30-40 ℃, adding the formula amount of sodium chloride for dissolving, adjusting the pH to 8.5 by using 0.1M of sodium hydroxide, clarifying, fixing the volume and filling into an ampoule bottle to obtain the snow-accumulated total acid.

Example 15 Total acid centella asiatica inhalation solution

Prescription:

active ingredients: 100mg of centella total acid;

isotonic adjusting agent: sodium chloride (in appropriate amount, adjusted to osmotic pressure (osmolality) 300mOsmol/kg)

pH regulator: 0.5g/ml sodium bicarbonate solution

Water for injection: 1000ml

The preparation method comprises the following steps: taking a proper amount of snow-accumulated total acid, dispersing the snow-accumulated total acid by 80 percent of formula amount of water, controlling the temperature to be 30-40 ℃, adding the formula amount of sodium chloride for dissolving, adjusting the pH to 8.3 by using 0.5g/ml sodium bicarbonate solution, clarifying, fixing the volume and filling into an ampoule bottle to obtain the snow-accumulated total acid.

Stability test of inhalation solution

The method for measuring the total acid content comprises the following steps:

a chromatographic column: octadecylsilane chemically bonded silica gel column (Innoval ODS-2C18, 4.6X 150mm, 5 μm).

Mobile phase: 0.2mmol aqueous beta-cyclodextrin solution: methanol (15: 85);

column temperature: 35 ℃; detection wavelength: 210 nm; flow rate: 1.0 ml/min;

sample introduction volume: 40 mu l of the solution;

diluent agent: water-methanol (15: 85).

The inhalation solution obtained in example 1-3 was placed in a constant temperature and humidity cabinet at 40 + -2 deg.C and RH 25% + -5% relative humidity, and sampled at the end of 3 months and 6 months respectively to determine its properties, pH and content. The results are shown in table 2:

TABLE 2 test results of 3-month and 6-month accelerated imbibition of solutions

It can be seen from table 1 that the inhalation formulation of the present invention has stable pH and content through accelerated experiments.

In addition to the above solution quality criteria, examples 12-15 were also performed to examine the fine particle dose, delivered dose, and delivered rate of the samples, and the results showed that the fine particle dose, delivered dose, and delivered rate of the samples examined in examples 12-15 were all within the specifications.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.

Claims (18)

1. A preparation method of total asiatic acid is characterized by comprising the following steps:

(1) dissolving the asiaticoside alcohol solvent, adding a poor solvent, and recrystallizing to obtain refined asiaticoside;

(2) performing hydrolysis reaction on the refined asiaticoside product obtained in the step (1) and an alkaline substance to obtain a crude asiaticoside product;

(3) adding an ethanol solution into the crude asiatic centella total acid product obtained in the step (2), dropwise adding an alkaline solution until the pH value of a liquid phase is 9-11, then adding an acidic solution until the pH value of the liquid phase is 6-9, carrying out first solid-liquid separation to obtain a first solid phase, dissolving the first solid phase with methanol, adding an acidic solution until the pH value of the liquid phase is 4-5, concentrating, adding water, and carrying out second solid-liquid separation to obtain a second solid phase, namely asiatic centella total acid.

2. The method for preparing total asiatic acid according to claim 1, wherein said ethanol solution is a mixed solution of water and absolute ethanol in said step (3);

preferably, the volume ratio of water to ethanol is 0.2-10: 1, more preferably 1: 1.

3. The method for preparing total asiatic acid according to claim 1, wherein in said step (3), said crude asiatic acid is mixed with said water and absolute ethanol solution in the amount relationship of: 4.5g of the crude product of the total acid of centella asiatica corresponds to 270.0ml of the mixed solution of water and absolute ethyl alcohol.

4. The method for preparing total asiatic acid according to claim 1, wherein said alkaline solution is sodium hydroxide solution or potassium hydroxide solution in said step (3).

5. The method for preparing total asiatic acid according to claim 1, wherein said acidic solution is glacial acetic acid in said step (3).

6. The method for preparing total asiatic acid according to claim 1, wherein said step (3) further comprises a temperature reduction step before said first solid-liquid separation;

preferably, the temperature in the temperature reduction step is controlled to be 0-10 ℃.

7. The method of preparing total asiatic acid according to claim 1, characterized by one or more of the following 1) to 7):

1) in the step (1), the usage relationship of the asiaticoside and the alcohol solvent is as follows: 40.0g of asiaticoside corresponds to 200.0ml of alcohol solvent;

2) in the step (1), the alcoholic solvent is selected from one or more of methanol, ethanol, isopropanol, n-propanol, n-butanol and tert-butanol;

3) the poor solvent is selected from one or more of methyl tert-butyl ether, ethyl acetate, dichloromethane, acetonitrile, tetrahydrofuran, ethanol, isopropanol and n-butanol;

preferably, the dosage relation of the asiaticoside and the poor solvent is that 40.0g of the asiaticoside corresponds to 100.0-500.0ml of the poor solvent, and more preferably 200ml of the poor solvent;

4) in the step (1), after dissolving the asiaticoside alcohol solvent, decoloring by using medicinal carbon before adding a poor solvent;

5) in the step (1), the recrystallization temperature is 15-25 ℃, and the heat preservation and crystallization are carried out for 30 minutes;

6) in the step (2), the alkaline substance is selected from one or more of sodium hydroxide, lithium hydroxide, potassium hydroxide and calcium hydroxide;

preferably, the relationship between the usage amount of the refined asiaticoside and the alkaline substance is as follows: 10.0g of refined asiaticoside product corresponds to 1.92-5.92g of alkaline substance;

7) in the step (2), the temperature of the hydrolysis reaction is 60-80 ℃.

8. An asiatic total acid, wherein said asiatic total acid comprises asiatic acid, madecassic acid, asiatic acid B; the total content of asiatic acid, madecassic acid and asiatic acid B in the total asiatic acid is not less than 95%.

9. The total asiatic acid according to claim 8, wherein the total asiatic acid has a combined content of 95-98% of asiatic acid, madecassic acid and asiatic acid B.

10. Total asiatic acid according to claim 8 or 9, wherein said total asiatic acid is prepared from total asiaticosides.

11. The total asiatic acid according to any one of claims 8 to 10, wherein said total asiatic acid is prepared by the method according to any one of claims 1 to 7.

12. Use of total asiatic acid according to any one of claims 9 to 11 for the manufacture of a medicament for the treatment of pulmonary fibrosis.

13. Solution formulation for inhalation of total asiatic acid according to any one of claims 9 to 11, characterized in that it has a total asiatic acid content of not less than 95%, madecassic acid and asiatic acid B, said solution formulation comprising:

(1) the effective component is a composition of three acids, wherein the concentration range of the asiatic acid B is as follows: 45-90 ug/mL; concentration range of madecassic acid: 150-; the concentration range of asiatic acid is as follows: 250-550 ug/mL;

(2) an isotonicity adjusting agent;

(3) a solvent.

14. The solution formulation for inhalation according to claim 13, wherein, as a preferred embodiment, said solution formulation has an osmolality of 260-480 mOsmol/kg; preferably 280-320 mOmol/kg.

15. The solution formulation for inhalation according to claim 13 or 14, wherein the pH of the solution formulation is 7.5-8.5.

16. The solution formulation for inhalation of any one of claims 13 to 15, further comprising a pH adjuster; the pH regulator is preferably an inorganic base or a buffering agent; the inorganic base is preferably one or more of sodium hydroxide, sodium bicarbonate, disodium hydrogen phosphate, sodium citrate and sodium carbonate; the buffer is preferably citric acid-sodium citrate, citric acid-disodium hydrogen phosphate, potassium dihydrogen phosphate-disodium hydrogen phosphate or citric acid-sodium hydroxide.

17. The solution formulation for inhalation of claims 13-16, wherein said solvent is deionized water, distilled water, or water for injection.

18. A method of preparing a solution formulation for inhalation according to any of claims 13 to 17, comprising the steps of:

taking the total asiatic acid according to the prescription amount, and adding a solvent to obtain a mixture; mixing the mixture uniformly, adding an isotonic agent and a pH regulator, dissolving and clarifying, and fixing the volume to obtain the solution for inhalation; putting the solution for inhalation into an ampoule bottle to obtain the total acid solution preparation of the asiatic centella for inhalation.