CN105017373B - Based on asiatic acid and the therapeutic agent of specific salts thereof - Google Patents

Abstract

The invention discloses the preparation method of a kind of asiatic acid, asiatic acid tromethane salt and preparation method thereof.The preparation method of asiatic acid includes: adds 8 × 4% NaOH/50% EtOH solution in asiaticoside powder and is heated to reflux, regulation pH to 45 sucking filtration, filter cake is successively with water, 30% EtOH solution washing, the backflow of 8 × dehydrated alcohol is added in gained filter cake, add activated carbon backflow for twice, filtrate is cooled to room temperature, and add water to obtain precipitation, is asiatic acid.The present invention produces asiatic acid and tromethane salt is the most convenient, and gained asiatic acid and the yield of asiatic acid tromethane salt and purity are the highest.Utilize asiatic acid tromethane salt disclosed by the invention to can be used for treatment of arthritis, psoriasis and other inflammation, it may also be used for the treatment fibrotic disease such as pulmonary fibrosis, diabetic nephropathy, and be obtained in that significant curative effect.

Description

Therapeutic agents based on asiatic acid and specific salts thereof

This application has an international filing date of January 08, 2009, an international application number of PCT/US2009/030458, a national application number of 200980108867.6, and a date of entering the Chinese national phase on September 9, 2010. The invention name is based on Asiatic acid A divisional application for an application for a therapeutic agent and a specific salt thereof.

priority claim

This application claims priority to the following US Provisional Application: Application No. 61/006,432, filed January 11, 2008, which is hereby incorporated by reference in its entirety into this application.

technical field

The invention relates to the field of chemistry, in particular to a preparation method of asiatic acid, a tromethamine asiatic acid salt and a preparation method thereof.

Background technique

Bontems, Bull.Sci.Pharmacol.49:186-91 (1941), reported for the first time that Asiatic acid and its trisaccharide saponins can be extracted from Centella asiatica. Centella asiatica is a small annual herbaceous plant of the genus Dill in the Umbelliferae family, which is produced in the Asia-Pacific Rim region. The chemical structures of asiatic acid and asiaticoside are as follows:

See: Polonsky, Compt. Rend. 232:1878-80 (1951); and Bull. Soc. Chim. 173-80 (1953).

Centella asiatica extract contains asiatic acid and asiaticoside, known to be useful in the treatment of skin scars and chronic ulcers. In a related aspect, the literature also discloses the use of centella asiatica extracts for the treatment of skin deformities associated with tuberculosis or leprosy. See, for example: Boiteau et al., Bull. Soc. Chim. 31:46-51 (1949).

The pharmacological mode of action for such wound healing is related to the induction of keratinization, see: May, Eur. J. Pharmacol. 4:331-39 (1968), in particular by promoting dermal fibroblast proliferation and including type I collagen synthesis of extracellular matrix. See, for example: Lu et al., Int'l J. Dermatol. 43:801-07 (2004); Skukla et al., J. Ethnopharmacol. 65:1-11 (1999). According to the report of Bonte et al., Bonte et al., Planta Med.60:133-35 (1994), by comparing asiaticoside and asiatic acid, it was found that the sugar moiety in the former molecule has the highest biological activity. Doesn't seem to be indispensable. And as discussed in Grimaldi et al., J. Ethnopharmacol. 28:235-41 (1990), following oral administration in humans, asiaticoside is completely converted to plasma asiatic acid.

Based on the recognition of these wound healing properties, proposals for dermatological preparations comprising asiatic acid or its derivatives were proposed in US Patent No. 5,834,437. In addition, see also U.S. Patent No. 6,417,349, in which, in an animal model of liver fibrosis, a water-soluble extract of madecassoside and madecassoside by intraperitoneal administration achieved a symptomatic effect, wherein The above-mentioned madecassoside is also a related compound produced by Centella asiatica. In addition to its wound-healing and anti-fibrotic functions, oral administration of asiaticoside has also been reported to reduce splenocytes and levels of inflammatory molecules, including COX , in a standard rodent model of (collagen-induced) arthritis. -2, PGE2, TNF-α, and IL-6, and thereby inhibited or reduced various arthritic parameters such as paw swelling, arthritis score, and synovial hyperplasia. See: Li et al., Yao Xue Xue Bao (Acta Pharma. Sinica) 42:698-703 (2007).

Although these and other disclosures suggest the therapeutic potential of asiatic acid and asiaticoside, the realization of this potential faces significant obstacles. The main problem is that the extraction of these two compounds is very difficult, and the yield and purity obtained are relatively low. Furthermore, little is known about the preparation of asiatic acid salts for use in therapeutic compositions. In US Patent No. 6,891,063, certain asiatic acid salts, mainly some ammonium salts, are reported for topical use.

Contents of the invention

In order to solve the above-mentioned technical problems that the extraction of asiatic acid is difficult and the yield and purity of the obtained product are relatively low, the present invention provides a method for preparing asiatic acid, a tromethamine asiatic acid salt and its Preparation.

Therefore, in order to solve the above-mentioned technical problems, one of the technical solutions adopted by the present invention is: a preparation method of asiatic acid, the preparation method comprising the following steps: adding 8× (V/W) 4%NaOH/50%EtOH solution, heat and reflux for 3-4 hours, add hydrochloric acid to the obtained hydrolyzate to adjust the pH to 4-5 and suction filter, discard the filtrate, and wash the obtained filter cake with water, 30% Wash with EtOH solution until the filtrate is nearly colorless, discard the filtrate, add 8 × (V/W) absolute ethanol to the obtained filter cake and reflux until the solid dissolves, add 2 × activated carbon, reflux for 30 minutes, suction filter while hot, discard For the used activated carbon, add 2× activated carbon to the filtrate and reflux for 30 minutes, suction filter while it is hot, discard the used activated carbon, cool the obtained filtrate to room temperature, add water until no new precipitate is formed, and the obtained precipitate is snow Oxalic acid, wherein the asiatic acid obtained has a purity of 98% (W/W).

The preparation method of Asiatic acid of the present invention preferably comprises the following steps: 200g of asiaticoside with a purity of about 92% is dissolved in 8×(V/W) 4%NaOH/50%EtOH solution, and the resulting solution Heat and reflux in a water bath at 80°C for 3 to 4 hours, cool to room temperature, and adjust the pH of the solution to 4 to 5 by adding dilute hydrochloric acid in batches under stirring. Become viscous paste, this paste is suction-filtered with a Buchner funnel with a diameter of 200mm, discards the filtrate, and the precipitate is washed with water under reduced pressure for many times until the washing liquid is close to colorless, then washed with 30% ethanol, Discard the washing solution twice until the washing solution is nearly colorless, add 8 × (V/W) absolute ethanol to the above filter residue, heat the system to reflux in a water bath at 80°C until the solid is completely dissolved, and then add 2 × Activated carbon, continue to reflux for 30 minutes, use a Buchner funnel with a diameter of 200mm to filter while it is hot, remove the activated carbon, discard the used activated carbon, then add the same amount of activated carbon as the previous time to the filtrate, reflux for 30 minutes, use a diameter 200mm Buchner funnel for suction filtration while it is hot, cool the filtrate to room temperature, add water to it in batches, about 100-200ml each time, until no white precipitate occurs, the total volume of water required is about 4000ml, use a diameter of Suction filtration with a 200mm Buchner funnel, discard the filtrate, dry and crush the white solid in the funnel to obtain asiatic acid with a purity of 98% (W/W).

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 tromethamine salt, the preparation method comprising the following steps: mixing 2g Asiatic acid and 0.59g tromethamine Add it to 30ml of methanol, stir at room temperature for 24 hours, then reflux for 0.5 hours, concentrate the system, add a small amount of water, a precipitate is formed, filter, put the filter cake in an appropriate amount of methanol, heat to dissolve, filter, remove Concentrate the insoluble matter, add enough acetone to the concentrated solution to make the system uniform, cool, and a precipitate forms, and the solid obtained after filtration is dried at 50°C to obtain 1.5g of asiatic acid tromethamine salt as a beige solid.

In order to solve the above-mentioned technical problems, the third technical solution adopted by the present invention is: a kind of asiatic acid tromethamine salt, its structural formula is as follows:

The invention provides high-purity Asiatic acid. In one embodiment, the asiatic acid is pharmaceutical grade, preferably about 98% pure.

The present invention also provides salts prepared from high-purity Asiatic acid. The salts may be alkali metal salts, alkaline earth metal salts or optionally substituted ammonium salts. Specifically, it may be potassium salt, sodium salt or ammonium salt formed from tromethamine. The present invention also provides a solid preparation comprising any one or more salts mentioned above.

The present invention further provides derivatives of high-purity Asiatic acid. Said derivatives may be amides or esters. A preferred derivative is asiaticoside.

The present invention also provides a therapeutic composition, which is prepared by the following steps: preparing high-purity asiatic acid or asiaticoside and a pharmaceutically acceptable carrier to contain a therapeutically effective amount of asiatica Formulations of oxalic acid or asiaticoside.

The present invention further provides a method for treating or preventing fibrotic diseases. The method comprises administering the therapeutic composition described above to a subject who has or is at risk of having a disease. Typical fibrotic diseases involved in the present invention are radiation pneumonitis and fibrosis, idiopathic pulmonary fibrosis, diabetes, kidney disease and chronic obstructive pulmonary disease (COPD).

The present invention also provides a method for treating or preventing an inflammatory disease, comprising administering the above-mentioned therapeutic composition to a subject who has the disease or is at risk of the disease. These inflammatory disease types may be arthritis, inflammatory bowel disease, and psoriasis.

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: using the preparation method of asiatic acid and asiatic acid tromethamine salt of the present invention, it is relatively easy to extract asiatic acid, and the obtained products asiatic acid and asiatic acid tromethamine The yield and purity of the salt are high. The pharmaceutical composition prepared by using the asiatic acid prepared by the invention can be used to treat arthritis, psoriasis and other inflammations, and can also be used to treat fibrotic diseases such as pulmonary fibrosis and diabetic nephropathy, and can obtain significant curative effect.

Description of drawings

Figure 1 is an overlay spectrum of asiatic acid and its salts.

Fig. 2 is the ¹ H-NMR (300 MHz, DMSO-d ₆ ) spectrum of asiatic acid (Lot071109) and its sodium salt (AJF09, 99b).

Fig. 3 is the ¹³ C-NMR (300 MHz, DMSO-d ₆ ) spectrum of asiatic acid (Lot071109) and its sodium salt (AJF09, 99b).

Fig. 4 is a ¹³ C-NMR (300 MHz, DMSO-d ₆ ) spectrum of asiatic acid (Lot071109) and its sodium salt (AJF09,99b) at a chemical shift of 11-41 ppm.

Figure 4a is the complete ¹ H-NMR spectrum of asiatic acid in DMSO-d ₆ .

Fig. 4b is an enlarged view of the ¹ H-NMR spectrum of asiatic acid.

Figure 4c is the complete ¹ H-NMR spectrum of asiatic acid sodium salt (AJF09,99b).

Fig. 4d is an enlarged view of the ¹ H-NMR spectrum of asiatic acid.

Figure 4e is the complete ¹³ C-NMR spectrum of asiatic acid in DMSO-d ₆ .

Fig. 4f is an enlarged view of the ¹³ C-NMR spectrum of Asiatic acid.

Fig. 4g is the complete ¹³ C-NMR spectrum of asiatic acid sodium salt in DMSO-d ₆ .

Fig. 4h is an enlarged view of the ¹³ C-NMR spectrum of asiatic acid sodium salt.

Figure 5a is the PXRD spectrum of asiatic acid.

Figure 5b is the PXRD spectrum of asiatic acid sodium salt.

Fig. 6 is the FTIR spectrogram of asiatic acid.

Fig. 7 is the FTIR spectrum of asiatic acid sodium salt (AJF09,82).

Figure 8 is the FTIR spectrum of asiatic acid sodium salt (AJF09,99b).

Fig. 9 is the FTIR spectrum of asiatic acid sodium salt (AJF09,99a).

Fig. 10 is a thermogravimetric analysis (TGA) diagram of asiatic acid.

Fig. 11 is a thermogravimetric analysis (TGA) diagram of asiatic acid sodium salt (AJF09,82).

Fig. 12 is a thermogravimetric analysis (TGA) diagram of asiatic acid sodium salt (AJF09,99b).

Figure 13 is a standard linear curve.

Figure 14 is a schematic representation of the process for preparing asiaticoside.

Figure 15 is a schematic representation of the preparation process of asiatic acid of pharmaceutical grade purity.

Fig. 16a is the ¹ H-NMR spectrum of potassium salt of asiatic acid.

Fig. 16b is the ¹ H-NMR spectrum of the salt formed by asiatic acid and trometamol.

Fig. 16c is the ¹ H-NMR spectrum of the sodium salt of asiatic acid phosphate.

Figure 16d is the ¹ H-NMR spectrum of the amide derivative of asiatic acid triacetate.

Figure 17a is the mass spectrum of Asiatic acid potassium salt.

Figure 17b is the mass spectrum of the salt formed by asiatic acid and tromethamine.

Figure 17c is the mass spectrum of the sodium salt of asiatic acid phosphate.

Figure 17d is the mass spectrum of the amide derivative of asiatic acid triacetate.

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.

Based on the above problems, the present inventors provide an easy-to-implement method for obtaining high-purity asiaticoside and asiatic acid suitable for pharmacological research. In addition, they also prepared and identified asiatic acid salt. The physical properties of these salts can all meet the requirements of formulations and meet the requirements for treating inflammation and fibrosis diseases.

Embodiment 1 prepares asiatic acid and asiatic acid salt

Ⅰ. Preparation of asiatic acid with pharmaceutical grade purity

A. Preparation of asiaticoside with a purity of 92%

Crude asiaticoside powder can be purchased from the market, for example, from Guangxi Changzhou Natural Products Development Co., Ltd. (www.changzhou-centella.com).

First add 4×60% (V/W) EtOH to the crude asiaticoside powder, then heat the mixture to 70-90°C (eg, 80°C) and keep the system under reflux until the solid dissolves. After the solution was cooled, it was placed in a refrigerator at 5°C for 16 hours to allow it to crystallize. Amorphous precipitates were formed in the system, filtered, and the recovered precipitates were washed with 60% ethanol until the color turned white. The precipitate is then dried at 55˜70° C. (eg, 60° C.) and a pressure of −0.09˜−0.11 MPa (eg, −0.10 MPa).

Add 30×80% (V/W) acetone aqueous solution to the dry solid obtained above, and heat to reflux for 1 hour until the solid dissolves. Filter, and place the filtrate in a refrigerator at 10°C for 24 hours to crystallize. Amorphous precipitates were generated in the system, filtered, and the recovered precipitates were washed with 80% acetone until the color turned white. The precipitate is dried and crushed to obtain asiaticoside powder. Fig. 14 is a schematic diagram of this operation process.

B. Preparation of asiatic acid with a purity of 98%

200g of asiaticoside (purity about 92%) prepared by the above method was dissolved in 8×(V/W) 4%NaOH/50%EtOH solution (1600ml), and the resulting solution was heated to reflux in a water bath at 80°C for 3 ~4 hours, cool to room temperature or near room temperature.

Then, adjust the pH of the solution to 4-5 by adding dilute hydrochloric acid (pre-diluted 1-2 times) in small amounts (for example, about 10 ml each time) under stirring. During this process, precipitation was continuously formed, and finally, the whole system became a viscous paste.

The paste was suction-filtered with a Buchner funnel with a diameter of 200 mm, the filtrate was discarded, and the precipitate was washed with water under reduced pressure for several times until the washing liquid was nearly colorless. Then wash with 30% ethanol until the washing solution is almost colorless. Both washes were discarded.

Add 8×(V/W) absolute ethanol (about 1600ml) to the above filter residue, and heat the system to reflux in a water bath at 80° C. until the solid is completely dissolved. Then add 2×activated carbon (about 400g) and continue to reflux for 30 minutes. Use a Buchner funnel with a diameter of 200mm to filter while hot to remove activated carbon. Discard the used activated carbon, add the same amount of activated carbon as before to the filtrate, reflux for 30 minutes, and suction filter while it is hot with a Buchner funnel with a diameter of 200 mm.

The filtrate was cooled to room temperature, and water was added therein in batches, about 100-200ml each time, until no white precipitate was produced, and the total volume of water required was about 4000ml. Suction filtration with a Buchner funnel with a diameter of 200 mm, discard the filtrate, dry and crush the white solid in the funnel to obtain asiatic acid with a purity of 98% (W/W). Fig. 15 is a schematic diagram of this operation process.

Ⅱ. Pharmacological activity of asiatic acid

Treatment of rats with bleomycin is the preferred method for establishing an animal model of fibrosis. As described in more detail below, the asiatic acid and asiaticoside compositions of the present invention significantly increased the survival rate of bleomycin-treated rats, regardless of whether the treatment was one day or seven days after bleomycin administration start. This result was unexpected because dexamethasone, a drug traditionally used to treat pulmonary fibrosis, did not significantly improve the survival rate of rats under the same experimental conditions.

Since dexamethasone mainly acts on inflammatory pathways, the experimental results of the present invention show that asiatic acid and asiaticoside, in addition to having an inhibitory effect on inflammatory responses, also play a role in other pathways related to or directly related to fibrosis. effect. For example, asiatic acid reduced bleomycin-induced pulmonary fibrosis as measured by lung histopathology. Treatment with asiatic acid starting seven days after bleomycin injury was more effective than treatment with dexamethasone, suggesting that asiatic acid not only targets the inflammatory response but acts more directly on fibrosis than dexamethasone.

Much better than dexamethasone, both asiatic acid and asiaticoside significantly reduced bleomycin-induced pulmonary fibrosis, whether treatment was initiated one day or seven days after bleomycin injury weight-to-weight ratio.

Asiatic acid and asiaticoside also significantly reduced bleomycin-induced fibrosis as measured by serum levels of hydroxyproline (collagen precursor) and type III collagen. Asiatic acid was as effective as or slightly superior to dexamethasone, regardless of whether treatment was initiated one day or seven days after bleomycin injury.

Asiatic acid significantly reduced bleomycin-induced fibrosis-mediating cytokines transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-β), regardless of whether treatment was initiated one day or seven days after bleomycin injury. -α), which is comparable to dexamethasone.

Ⅲ. Treatment indications and dosage forms

The experimental results fully show that the pharmaceutical-grade asiatic acid and high-purity asiaticoside prepared according to the method of the present invention are effective in treating inflammation-related diseases and diseases related to accumulation of fibroblasts and cell matrix ("fibrotic diseases") ) in validity. These inflammatory diseases are, for example, psoriasis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), and arthritis (including rheumatoid arthritis, osteoarthritis and psoriatic arthritis). These disorders are characterized by abnormal activity of the body's immune system and elevated levels of inflammatory molecules such as COX-2, certain cytokines, and prostaglandins. Such fibrotic diseases are, for example, radiation pneumonitis and fibrosis, idiopathic pulmonary fibrosis and diabetic nephropathy (characterized by renal fibrosis).

Therefore, one of the objects of the present invention is to provide a therapeutic composition, which is a medicament prepared from high-purity asiatic acid or asiaticoside and other commonly used ingredients, especially suitable for internal use. The therapeutic composition of the present invention may include one or more pharmaceutically acceptable carriers, excipients or stabilizers, specifically see Remington-The Science and Practice of Pharmacy21 ^st ed. (2005), as long as snow The antifiber effect of oxalic acid or asiaticoside is not adversely affected by other ingredients in the composition.

Depending on the desired mode of administration, the therapeutic composition of the present invention may be an injection for parenteral administration. For this purpose, the therapeutic compositions of the present invention can be prepared in aqueous solutions, containing physiologically compatible buffered solutions, such as Hank's solution, Ringer's solution or physiological saline.

The present invention further provides a solid preparation, which can be prepared by mixing a salt of asiatica (see the next part) and a pharmaceutically acceptable solid base, suitable for oral, buccal, sublingual, rectal or Vaginal administration. High-purity asiatic acid, asiaticoside or asiaticoside may be mixed with one or more solid excipients, optionally grinding the resulting mixture, sometimes after addition of suitable excipients, before processing This mixture is thus made into tablets or dragees of the therapeutic composition of the present invention. Suitable excipients are fillers such as sugars (lactose, sucrose, mannitol, sorbitol, etc.) and celluloses such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, formazan, etc. cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC) and/or polyvinylpyrrolidone (PVP: povidone). Disintegrants such as cross-linked polyvinylpyrrolidone, agar, alginic acid or alginate (eg sodium alginate) may also be added.

Dragees with suitable coatings may employ concentrated sugar solutions, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbomer gel, polyethylene glycol (PEG), and/or titanium dioxide, Lacquer solution, and appropriate organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragees for distinction or to identify combinations of different doses of active ingredient.

Among the therapeutic compositions involved in the present invention, those suitable for oral administration include: hard capsules made of gelatin and sealed soft capsules made of gelatin and plasticizers (such as glycerin or sorbitol), loaded with high-purity product A solid preparation made of notacid or asiaticoside. The hard capsules described above may contain active ingredients and fillers (such as lactose), binders (such as starch) and/or lubricants (such as talcum powder or magnesium stearate), and optionally add one or more a stabilizer. In soft capsules, the active ingredient may be present as a solution or as a suspension in a suitable liquid, which may be fatty oil, liquid paraffin, or liquid polyethylene glycol (PEG), and optionally added one or more stabilizers.

Another administration method of the therapeutic composition involved in the present invention is inhalation or insufflation, and direct administration to the respiratory tract. See, eg, US Patent No. 5,607,915 and published PCT applications WO 97/39745 and WO 99/47196. The present invention therefore intends to provide a liquid formulation suitable for administration by a nebulizer, a liquid spray device, or an electrospray (EHD) spray device.

In one embodiment, the composition may contain a pharmaceutically acceptable liquid carrier, such as alcohol, water, polyethylene glycol or perfluorocarbon. Other materials may optionally be added to modify the spray characteristics of the active ingredient solutions or suspensions. Said other raw materials may be liquids such as ethanol, ethylene glycol, polyethylene glycol or fatty acids. Other methods may also be used to formulate solutions or suspensions suitable for use with spray devices according to the invention. See: U.S. Patent Nos. 5,112,598 and 5,556,611.

The active inhalation formulation of the present invention can adopt a dry powder carrier suitable for a dry powder inhaler. Described carrier can be monosaccharide (such as fructose, mannitol, Arabidan, xylitol and glucose) and hydrate thereof, disaccharide (such as lactose, maltose, and sucrose) and polysaccharide (such as starch, dextrin and dextran sugar). For example, the Asiatic acid salt of the present invention can be made into a dry powder in an ultrafine pulverizer as described in US Patent No. 5,376,386 and one or more of the above-mentioned carriers.

Ⅳ. Preparation and Characterization of Medicinal Asiatica Salt

Considering that asiatic acid can be better dissolved in methanol, the present invention plans to prepare asiatic acid salt, for example, by dissolving asiatic acid in methanol solutions of sodium hydroxide, sodium carbonate and ammonium hydroxide respectively. The preparation and characterization of Asiatic acid salt will be described in detail below.

A. Screening of Asiatic acid salt

Since asiatic acid is readily soluble in methanol (see below), the corresponding salts can be prepared by dissolving asiatic acid in methanolic solutions of sodium hydroxide, sodium carbonate, and ammonium hydroxide, respectively.

1. Reaction of asiatic acid with sodium carbonate (AJF09,82)

0.992g of asiatic acid was dissolved in 27.5ml of methanol, 2.0043g of anhydrous sodium carbonate was added to the solution and shaken vigorously. The methanol-asiatic acid solution was then transferred to a round-bottomed flask and evaporated to dryness to produce a white fine powder of asiatic acid sodium salt, which was vacuum-dried at 100° C. for 2 hours to obtain about 1 g of a sample.

2. Action of asiatic acid and methanol solution of ammonium hydroxide (AJF09,99a)

Add about 6ml of ammonium hydroxide in methanol dropwise to 0.4923g of asiatic acid, most of the asiatic acid dissolves, and new flakes begin to appear in the system, then add 1ml of ammonium hydroxide in methanol and stir overnight. Precipitation was formed in the system, and 0.0741 g of asiatic acid ammonium salt was obtained after filtration.

3. Action of asiatic acid and methanol solution of sodium hydroxide (AJF09,99b)

Add 0.4961g of asiatic acid to 7.0ml of 0.2M methanol solution of sodium hydroxide. After all the solids are dissolved, add saturated sodium chloride solution drop by drop to the system until the system is cloudy and precipitates form, then add 7.0ml of deionized water Dissolve the precipitated sodium chloride. After filtering, the filter residue was vacuum-dried at 100°C for 2 hours to obtain 0.4938 g of asiatic acid sodium salt.

4. Potassium asiatic acid

As shown in Formula 4, 4g (8.196mmol) of asiatic acid was added to 30ml of methanol and heated to dissolve all the solids. Add a solution made of 0.56g (8.187mmol) potassium hydroxide and 20ml methanol into the system until the pH of the system becomes 8-9. The solution was decolorized with charcoal and concentrated. Then add enough acetone to the concentrate to make the system uniform. After cooling, a precipitate formed, and after filtration, 3.5 g of potassium salt of asiatic acid was obtained as off-white crystals. Figure 16a shows the ¹ H-NMR spectrum of the product. Figure 17a shows the mass spectrum of this product.

5. Asiatic acid tromethamine salt

As shown in formula 5, 2g (4.09mmol) asiatic acid and 0.59g (4.623mmol) trometamol were added to 30ml of methanol, stirred at room temperature for 24 hours, and then refluxed for 0.5 hours. The system was concentrated, and a small amount of water was added. Precipitation is formed, filter, put the filter cake in an appropriate amount of methanol, heat to dissolve, filter, remove insoluble matter, and concentrate. Add enough acetone to the concentrate to make the system uniform. After cooling, a precipitate formed, and the obtained solid was filtered and dried at 50° C. to obtain 1.5 g of asiatic acid tromethamine salt as a beige solid. Figure 16b shows the ¹ H-NMR spectrum of the product. Figure 17b shows the mass spectrum of this product.

6. Sodium salt of asiatic acid phosphate

As shown in the first step reaction in Formula 6, 12ml of pyridine was cooled to -10°C in an ice-salt bath, and 2.2g (0.01435mol) of POH was added dropwise thereto. Two minutes later, a solution made of 2 g (0.004098 mol) asiatic acid and 6 ml pyridine was added dropwise thereto, and the reaction was stirred for 1.5 hours. Slowly add 40ml of ice water to the system and stir overnight at room temperature. Most of the solvent was evaporated to give a solid which was dried in vacuo. The solid was added to 20 ml of 4N hydrochloric acid to obtain a colloidal precipitate, which was filtered and the filter cake was washed with water to obtain a crude product. Dissolve it in methanol, decolorize it with activated carbon, concentrate, and then add enough acetone to the concentrated solution to make the system uniform. Precipitation was formed in the system, and after filtration, a colloidal phosphate was obtained.

As shown in the last reaction step in Formula 6, the crude product of the previous step reaction was dissolved in methanol, and 1N sodium hydroxide methanol solution was added until the pH of the system became 8-9. Decolorize with activated carbon, concentrate, and then add enough acetone to the concentrate to make the system uniform. After cooling, precipitation appeared in the system. After filtration, the resulting solid was dried at 50°C to obtain 2.2 g of a beige solid, which was the sodium salt of asiatic acid phosphate. Figure 16c shows the ¹ H-NMR spectrum of this compound. Figure 17c shows the mass spectrum of this compound.

7. Amide derivatives of asiatic acid triacetate

As shown in the first step reaction in Formula 7, under stirring, dissolve 5 g of Asiatic acid in 10 ml of pyridine, cool to 10°C, add 15 ml of acetic anhydride, and stir at room temperature for 20 hours. Add 200ml of ice water to the system, and a precipitate forms. Filter, wash with water, and dry the obtained solid at 50°C. 6.28 g of asiatic acid triacetate white powder was obtained.

As shown in the second step reaction in Formula 7, 6.5g (0.01058mol) asiatic acid triacetate was added to 100ml of dichloromethane, cooled in an ice-water bath, 1.2g (0.01186mol) triethylamine, 1.55g (0.0111mol) of glycine ethyl ester hydrochloride and 0.2g (0.00164mol) of 4-dimethylaminopyridine, and the reaction mixture was reacted at 0-5°C for 0.5 hours. Add 2.37g (0.0115mol) of DCC solution in 50ml of dichloromethane dropwise to the system, control the rate of addition, and complete the addition in 0.5 hours. After the addition, the reaction was stirred at 0-5°C for 2 hours, and then stirred at room temperature for 24 hours. After filtration, the filtrate was washed successively with 1N hydrochloric acid, saturated sodium bicarbonate solution and water to neutral pH. The solvent was distilled off under reduced pressure to obtain 9 g of an amide derivative of asiatic acid triacetate in the form of a gum.

The above colloidal crude product was placed on a silica gel column, and column chromatography was performed with dichloromethane/acetone (20:1) as eluent, and the desired components were collected and concentrated. The solid was removed by filtration, and the filtrate was vacuum-dried to obtain 2.82 g of white crystals. Figure 16d shows the 1H-NMR spectrum of this compound. Figure 17d shows the mass spectrum of this compound.

B. HPLC Analysis of Asiatic Acid

Certain asiatic acid salts prepared as described above were subjected to HPLC analysis.

·Preparation of standard samples

Add about 5 mg of Asiatic acid to a 25 ml volumetric flask, and then add 1 ml of methanol to dissolve all the solids. Then dilute to the mark with mobile phase and mix well.

· Chromatographic parameters

Column: Agilent, Zorbax Eclipse XDBC8, 250mm×4.6mm mobile phase: KH ₂ PO ₄ (3g/L)pH2.5/acetonitrile (55:45) Flow rate: 1.0mL/min wavelength: 204nm Injection volume: 10 μL Column temperature: 30℃ testing time: 20 minutes

· System adaptability

For each test, a standard solution (200 μg/mL asiatic acid in methanol) was used to inject 6 times to demonstrate the repeatability of the system. And calculate the percent relative standard deviation (%RSD) of replicate injections. After the first injection of the standard solution, calculate the tailing factor and the number of theoretical plates for asiatic acid to calculate the RSD.

System Qualified Criteria

· Among the test results of six repeated injections, the percentage of relative standard deviation (%RSD) corresponding to the area of the first peak is not more than 3.0%.

· The number of theoretical plates is not less than 10000.

• The tailing factor should not be greater than 1.5.

·The resolution is not less than 2.0.

Test Results

Asiatic acid Relative Standard Deviation (%RSD) 0.5 Number of theoretical plates 15322 Tailing factor 1.0 resolution 2.7

Calculate the recovery of asiatic acid and its salts (Table 1). The retention times of asiatic acid and its various salts are identical, confirming that the salts do not undergo major structural changes relative to asiatic acid (Figure 1).

Table 1. Recovery of asiatic acid and its salts

Solubility in water and methanol

The experimental results of solubility were obtained from the solubility of asiatic acid and a sodium salt (AJF09,99b) in water and methanol. See Table 2 for specific data. Asiatic acid is only slightly soluble in water (0.03mg/ml), while its sodium salt is 228 times more soluble in water. This is strong evidence for salt formation and greatly increases drug utility for dosage form preparation.

Sample Preparation

Separately filter the methanol and water saturated solutions of Asiatic acid and its salts, and dilute the others with mobile phase except the aqueous solution of Asiatic acid. Except the methanol solution of sodium salt prepared 2 samples, the other samples of the same kind were prepared 3 times.

Table 2. Results of solubility experiments

C. Nuclear Magnetic Resonance (NMR) Spectrum

The preliminary determination of ¹ H-NMR and ¹³ C-NMR spectra of asiatic acid and its sodium salt (AJF09,99b) was carried out on a Varian 300MHz nuclear magnetic resonance instrument in deuterated DMSO solution. Overall, the chemical shifts and splitting patterns (multiplicity) of the protons in the NMR spectra obtained were consistent with the structure of Asiatic acid (see Figure 2). By comparing the spectra of asiatic acid and its salts, it can be found that the chemical shifts of some peaks have changed, especially between 4.0 and 4.6 ppm, which is consistent with the result that the formation of salts causes electrons to change.

The chemical shift of carbon in the ¹³ C-NMR spectrum is also consistent with the structure of Asiatic acid. Figure 3 and Figure 4 are the ¹³ C-NMR spectra of asiatic acid and its sodium salt, respectively. In the ¹³ C-NMR spectrum of Asiatic acid, the signals with carbon chemical shifts at 36, 24 and 17 ppm changed in the corresponding ¹³ C-NMR spectrum of the sodium salt, which also indicated the formation of the salt.

Further NMR analysis tests were carried out on a 400MHz nuclear magnetic resonance instrument in order to obtain more accurate details and to confirm the results of the preliminary tests. Through careful analysis of the obtained ¹ H-NMR and ¹³ C-NMR spectra (see Figure 4a~h), it can be seen that, except for the chemical shift change caused by the formation of the sodium carboxylic acid salt, the structure of the sodium salt is relatively For acid, there is no change. The most notable changes in chemical shifts that support salt formation are:

a. In the ¹ H-NMR spectrum of asiatic acid, there is a hydrogen signal at the position of 11.9 ppm, which usually corresponds to the hydrogen in the carboxyl group. However, there is no such signal in the spectrum of its sodium salt.

b. In the ¹³ C-NMR spectrum, the signal of the carbon atom in the carboxyl group also changes, which can be explained by the formation of salt.

D. X-ray powder diffraction

The instrument used in the X-ray diffraction experiment is Shimadzu Lab X, XRD-6000, and the test standard is referring to: United States Pharmacopoeia (USP) <941>. Fig. 5a is a diffraction pattern of asiatic acid, and the broad peak in the figure is a characteristic peak of amorphous. According to the method described in IV.A.3 above, the preparation scale of the sodium salt was enlarged to 2 g, and analyzed by HPLC, the results as in the aforementioned IV.B were obtained, and the X-ray powder diffraction spectrum of the sodium salt Consistent with the characteristics of crystals (see Figure 5b).

E. Fourier Transform Infrared Spectrum

Asiatic acid, asiatic acid sodium salt (AJF09,82 and AJF09,99b) and asiatic acid ammonium salt (AJF09,99a) were tested by Fourier transform infrared spectroscopy. Figure 6 is the infrared spectrum of the obtained asiatic acid, in which the peak near 1697cm ^-1 is a typical characteristic peak of carboxylic acid.

The IR spectra of the sodium salts AJF09,82 (Fig. 7) and AJF09,99b (Fig. 8) clearly show the weakening of the intensity of the characteristic peaks of carboxylic acids around 1697 cm ^-1 and the appearance of peaks around 1545 cm ^-1 and 1390 cm ^-1 . The reason for this change is the presence of carboxylate groups as opposed to carboxylic acid groups. This is spectroscopic evidence supporting the formation of asiatica. Ammonium salt (AJF09,99a) has a characteristic peak near 1390cm ^-1 in the infrared spectrum (Fig. 9), which also confirms the existence of carboxylate, and there is also a characteristic peak of carboxylic acid near 1690cm ^-1 in this figure, which shows that It is caused by incomplete conversion from acid to salt.

F. Thermogravimetric Analysis (TGA)

The TGA in the present invention is carried out on a Q5000 thermal analyzer. Calibration of the instrument is carried out with reference to the United States Pharmacopoeia (USP) <891> with nickel standard. Asiatic acid and two sodium salts AJF09,82 and AJF09,99b were subjected to thermogravimetric analysis (see Figures 10-12 for the experimental results). It can be seen from the figure that asiatic acid begins to decompose at 400°C, while the two sodium salts show better heat resistance, which is a common phenomenon in the change of salt formation.

G. Determination of sodium content

The instrument used to determine the sodium content is a Perkin Elmer AAnalyst 300 atomic absorption spectrophotometer, which is equipped with a HGA 850 graphite furnace, and uses an oxidizing air-acetylene flame to detect at a wavelength of 589nm. The standard solution of sodium was prepared by 1000mg/L sodium chloride solution. When the sodium content is determined by this method, it is usually carried out in the presence of an alkali metal (such as cesium) in order to control the degree of ionization. Therefore, experiments should first be used to determine the amount of cesium (in the form of cesium chloride), which is very necessary to improve the sensitivity of the instrument to sodium. It is determined through experiments that 0.3% cesium chloride is used for all samples and standard items of the present invention.

Standard sodium chloride solution 1 (1000ppm Na)

Place accurately weighed 2.53g of sodium chloride in a 1.0L volumetric flask, add an appropriate amount of deionized water to dissolve it completely, and then dilute to the mark with deionized water to obtain the required sodium chloride standard solution.

3% cesium chloride solution

Place accurately weighed 3g of cesium chloride in a 100mL volumetric flask, add an appropriate amount of deionized water to dissolve it completely, and then dilute to the mark with deionized water to obtain the required cesium chloride solution.

0.3% cesium chloride solution (dilute solution)

Place accurately weighed 6g of cesium chloride in a 2.0L volumetric flask, add an appropriate amount of deionized water to dissolve it completely, and then dilute to the mark with deionized water to obtain a cesium chloride solution of the required concentration.

Diluent 1 (0.3% cesium chloride solution containing 100ppm Na)

Transfer 10.0ml of standard sodium chloride solution 1 (1000ppm Na) and 10.0ml of 3% cesium chloride solution to a 100ml volumetric flask, and then dilute to the mark with deionized water.

Diluent 2 (0.3% cesium chloride solution containing 10ppm Na)

Transfer 10.0ml of diluent 1 (0.3% cesium chloride solution containing 100ppm Na) to a 100ml volumetric flask, and dilute to the mark with 0.3% cesium chloride solution (dilute solution).

Linearity of System Response

The linearity of the test system refers to the characteristic that the system can give test results proportional to the concentration of the test sample within a certain range. In the present invention, the relationship between absorbance and sodium concentration was first measured. Before the test, prepare the sodium chloride standard solution earlier, and the preparation process is as follows: accurately transfer 2.0ml, 3.0ml, 4.0ml, 5.0ml and 6.0ml dilution 2 (0.3% cesium chloride solution containing 10ppm Na) respectively To five independent 50ml volumetric flasks, dilute to the mark with 0.3% cesium chloride solution to prepare standard solutions with concentrations of 0.4ppm, 0.6ppm, 0.8ppm, 1.0ppm and 1.2ppm respectively. In the test, the samples of each concentration were read 3 times, and the average value of the readings was used as the absorbance of the concentration. A linear curve can be obtained by plotting the absorbance of the sample against its concentration (Figure 13).

The test results of asiatic acid sodium salt AJF09,82 and AJF09,99b are shown in Table 3

Table 3. Sodium content in Asiatic acid salt

If it is assumed that there is one sodium ion per molecule of asiatic acid salt, then the sodium content of the sample should be 4.5%.

The animal experiment result of embodiment 2 products obtained therefrom

Effects on Rat Pulmonary Fibrosis Induced by Bleomycin

1. Purpose

In order to observe the effects of asiatic acid and asiaticoside on bleomycin-induced pulmonary fibrosis in rats, they were administered on the second day and the seventh day after the animal model of the disease was established.

2. Materials and Methods

Animals: SD rats (male), weighing 250-300 g, provided by Shanghai SLACC Co., Ltd.;

TFG-βKit, Human 96T ELISA Kit was purchased from Shanghai Jingmei;

TNF-αKit, Human 96T ELISA Kit was purchased from Shanghai Jingmei;

Asiaticoside (purity ≥ 92%);

Asiatic acid (purity ≥ 98%)

Bleomycin for injection, specification: 8mg/Ap, provided by Tianjin Taihe Pharmaceutical Co., Ltd.;

Dexamethasone, specification: 5mg/Ap, provided by Shanghai Xinyi Pharmaceutical.

3. Dosage and route of administration

4. Method

Male SD rats, weighing 250-300 g, were anesthetized with 3% cecobarbital, laid flat, and fixed on the operating table. The neck was sterilized with ethanol and cut open to expose the trachea. The injection needle was inserted into the space between the tracheal rings, and 5 mg/kg bleomycin was injected. Keep the rat upright and rotate to distribute the solution evenly in the lungs. Suture the incision.

After the rats regained consciousness, they were randomly divided into two groups: (i) receiving the drug administration on the second day after the establishment of the animal model; (ii) receiving the administration of the drug seven days after the establishment of the animal model. The two groups were further divided into the following groups: 27, 9 and 3 mg/kg p.o. × 28 days Asiatic acid group; 0.6 mg/kg p.o. × 28 days dexamethasone group; 36 mg/kg p.o. × 28 days asiaticoside group; Model group and blank group. Physiological saline was administered to the model group and the blank group. On the 28th day after the administration of asiatic acid and asiaticoside, the survival rate of the rats and the percentage of lung weight to body weight were calculated, and pathological and serum examinations were performed.

Table 4. Mortality of rats on days 7-28 of the test

Effects of asiatic acid treatment on lung mass index in rats with bleomycin-induced pulmonary fibrosis

Body and lung weights of rats were determined postmortem.

Lung weight ratio = (lung weight/body weight) x 100%

The results are shown in Table 5 and Table 6. In the table below, one star (*) means that the difference is obvious compared with the model group (P<0.05); two stars (**) means that the difference is very obvious (P<0.01)

Table 5. Lung mass index after asiatic acid administration for 28 days (administration started the next day after the model was established)

Table 6. Lung mass index after asiatic acid administration for 28 days (administration started on the 7th day after the model was established)

As shown in Table 5 and Table 6, the lung mass index of the rats in the high, medium and low doses of asiatic acid groups administered on the second or seventh day after modeling was significantly lower than that of the model group. The experimental results of the asiaticoside group were similar to those of the asiatic acid group. Lung mass index was relatively higher in the dexamethasone group, most likely due to dexamethasone-induced weight loss.

Serological effects of asiatic acid treatment on cytokines in rats with bleomycin-induced pulmonary fibrosis

Serum samples were prepared according to standard operating procedures (centrifugation at 3000 rpm for 10 minutes after sample collection) and stored at -20°C. The test was carried out according to the instructions on the kit, and the results are shown in Table 7.

Table 7. Serum test results for asiatic acid

Pathological Analysis of Asiatic Acid Effect on Bleomycin-Induced Pulmonary Fibrosis in Rats

The lung tissue was fixed in 10% formalin solution for one week, and the lower lobe was dehydrated and soaked in wax, then embedded in paraffin and sectioned with a thickness of 3-4 μm. Then routine hematoxylin-eosin (HE) staining was performed. Double special staining of collagen fibers and elastic fibers is performed in order to determine the degree of fibrosis. The staining status of the two groups of slices was observed, and the results are as follows.

Group 1 (dosing started the day after bleomycin modeling)

blank group

The morphology and structure of the lung tissue of the animal was complete, and there was no phenomenon of hemorrhage, hyperplasia or edema. However, there was an infiltration of inflammatory cells in the lung tissue of most animals. In some animals the condition was severe with localized hyperemia.

Double staining of collagen fibers and elastic fibers showed mild alveolar septum thickening and a few fibroblasts hyperplasia.

model group

Inflammatory cell infiltration (mainly lymphocytes) and alveolar septal hyperplasia existed in the lung tissue of experimental animals. In thickened lesions, there was moderate to severe focal thickening of alveolar septa and moderate hyperplasia of fibroblasts. In addition, there is severe alveolitis with possible fibroplasia in the periterminal bronchioles such that in some areas few alveoli are present. There was also mild alveolar atrophy and emphysema under the lung pleura.

Double staining of collagen fibers and elastic fibers showed that a large number of collagen fibers and elastic fibers proliferated in the lung tissue. In some areas there are few alveoli. Some tracheal walls also have proliferation of collagen fibers and elastic fibers. The lumen becomes smaller, and there is proliferation of a large number of collagen fibers and elastic fibers around the blood vessels.

Dexamethasone group

There was a slight infiltration of inflammatory cells (mainly lymphocytes) in the lung tissue. There is a small amount of inflammatory cell infiltration in the alveoli, and the alveolar septum is slightly hyperplastic. There was slight proliferation of fibroblasts, bulla formation, and mild subpleural atrophy and emphysema.

Staining of collagen fibers and elastic fibers showed that a small amount of collagen fibers and elastic fibers proliferated in the lung tissue.

Asiaticoside group

There was mild inflammation and alveolitis (mainly lymphocytes) in the lung tissue. The alveolar septa were slightly hyperplastic, and mild lung atrophy and emphysema symptoms were found under the alveolar septum. The degree and scope of the above pathological changes were similar to those in the dexamethasone group, but not as severe as in the model group.

The staining results of collagen fibers and elastic fibers were similar to those of the dexamethasone group.

Asiatic acid low dose group

Lung tissue with mild inflammation and alveolitis (mainly lymphocytes). Alveolar septum hyperplasia, and a little thickening, a small amount of fibroblast hyperplasia, slight lung atrophy and emphysema symptoms under the alveolar septum. The degree and scope of the above pathological changes were similar to those of the asiaticoside group.

The staining results of collagen fibers and elastic fibers were similar to those of the dexamethasone group.

Asiatic acid medium dose group

The lung tissue had a slight infiltration of inflammatory cells (mainly lymphocytes). Inflammation spread to the alveoli, with slight thickening and hyperplasia of the alveolar septum. A small amount of fibroblast hyperplasia, slight alveolar atrophy and emphysema under the alveolar septum.

Staining of collagen fibers and elastic fibers showed fibrous tissue hyperplasia, but it was significantly milder than that of the model group. The degree and range of pathological changes were similar to those of the low-dose group in most rats, but were milder than those of the low-dose group in a small number of rats in this group.

Asiatic acid high dose group

There was a slight infiltration of inflammatory cells (mainly lymphocytes) in the lung tissue. Inflammation spread to the alveoli, and the alveolar septa were slightly thickened. A small amount of fibroblast hyperplasia, mild alveolar atrophy and emphysema symptoms under the lung pleura.

Staining of collagen fibers and elastic fibers showed only a small amount of fibrous tissue proliferation, which was similar to the control group in some respects. The pathological changes in this group were milder than those in the low-dose group, middle-dose group and asiaticoside group.

Group 2 (dosing started seven days after bleomycin modeling)

The conditions of the blank group and the model group were similar to those of the same group in group 1 (dosing started on the second day after modeling).

Dexamethasone group

The lung tissue had a slight infiltration of inflammatory cells (mainly lymphocytes). There is a small amount of inflammatory cell infiltration in the alveoli, and the alveolar septum is slightly thickened. Fibroblasts were mildly hyperplastic, with numerous bullae formation, and mild subpleural atrophy and emphysema symptoms. The pathological changes were significantly more severe than those in the group receiving dexamethasone on the second day after modeling.

Collagen fiber and elastic fiber staining showed that the lung tissue had fibrous tissue hyperplasia, and the thickness of the alveolar wall and the degree of fibrosis were much more serious than those in the group receiving dexamethasone on the second day after modeling.

The asiaticoside and asiatic acid groups at different doses were similar to the group receiving dosing starting the day after bleomycin modeling.

5. Analysis

Bleomycin-induced pulmonary fibrosis is a "gold standard" model for studying the mechanisms of pulmonary fibrosis and for discovering anti-fibrotic, anti-inflammatory therapeutics. The biochemical and morphological changes in the lungs of many animals treated with bleomycin appear to be very similar to those in humans. Bleomycin treatment activates inflammatory and fibrotic pathways similar to those in inflammatory and fibrotic diseases such as diabetic nephropathy (kidney fibrosis). Furthermore, Gauldie et al. (Proc. Am. Thorac. Soc. 3:696-702, 2006) demonstrated that chronic obstructive pulmonary disease (COPD), including emphysema, relies on some of the same dysregulatory pathways. Accordingly, the present invention provides therapeutic compositions as hereinbefore described for use in the treatment of inflammatory and fibrotic diseases such as arthritis, inflammatory bowel disease, psoriasis, diabetic nephropathy and COPD.

Because the pneumonia caused by bleomycin and subsequent pulmonary fibrosis were very severe, 50-70% of the animals in the model group died. Animal death should be related to respiratory failure due to chronic inflammation and fibrosis. Dexamethasone did not protect rats from bleomycin-induced death, and surprisingly, asiaticoside and asiatic acid largely prevented bleomycin-induced death.

The results also showed that asiatic acid was able to reduce the levels of TNF-α and TGF-β, more effectively at the highest dose.

Conventional hematoxylin-eosin (HE) staining was used. Double staining of collagen fibers and elastic fibers was also performed to better observe the anti-fibrosis effect. The pathological results showed that asiatic acid effectively resisted bleomycin-induced pulmonary fibrosis, and its effect was dose-related. The effect of the high-dose asiatic acid group was better than that of the asiaticoside group, while the effect of the medium and low-dose asiatic acid group was similar to that of asiaticoside. Interestingly, there was a clear pathological difference between the two groups that started dexamethasone administration on the second day and the seventh day after modeling: the remission of pulmonary fibrosis was more pronounced when the drug was started on the second day. The reason may be due to the anti-fibrotic effect of dexamethasone by inhibiting inflammation. Dexamethasone is less effective once early inflammation occurs. Whereas asiatic acid and asiaticoside acted both in the initial inflammatory phase and in the later fibrotic phase, there was little variation in the effect between these groups administered on the second and seventh day after injury.

Thus, pure asiatic acid and asiaticoside showed adverse effects on bleomycin-induced inflammation and fibrosis.

Administration started seven days after bleomycin treatment, can affect the efficacy of dexamethasone, but can not significantly affect the effect of asiatic acid and asiaticoside, which shows that asiatic acid and asiaticoside are not only Acts by inhibiting inflammation that occurs shortly after bleomycin treatment. In contrast, experimental data suggest that both asiatic acid and asiaticoside have direct inhibitory effects on inflammatory and fibrotic processes.

Table 8. Determination value of hydroxyproline in asiatic acid anti-fibrosis experiment

Table 9. Content of type III collagen (asiatic acid administration started on the seventh day after modeling)

Claims (2)

1. the preparation method of an asiatic acid tromethane salt, it is characterised in that described preparation side Method comprises the following steps: join in 30ml methanol by 2g asiatic acid and 0.59g tromethane, After stirring 24 hours under room temperature, then refluxing 0.5 hour, system concentrated, add a small amount of water, there have to be heavy Forming sediment and generate, filter, be placed in proper amount of methanol by filter cake, heating is allowed to dissolve, and filters, and removes insoluble matter, Concentrate, in concentrated solution, add enough acetone make system uniform, cooling, there is precipitation to generate, institute after filtration Obtain solid to be dried at 50 DEG C, obtain 1.5g asiatic acid tromethane salt, for beige solid.

2. an asiatic acid tromethane salt, its structural formula is as follows: