A method for purifying madecassoside

Abstract

The present invention belongs to the field of medicine, and specifically relates to a method for purifying madecassoside. Specifically, it comprises the following steps: (1) using methanol as a solvent to dissolve a crude madecassoside product; (2) concentrating and distilling off the solvent; (3) adding seed crystals, cooling and crystallizing; (4) filtering and drying. The present invention effectively controls the crystal nucleation and crystal growth rate of madecassoside by selecting key influencing factors such as pulping solvent, pulping solvent volume, purification method, dissolution volume, concentration crystallization volume, crystallization temperature, drying method and temperature, drying method and temperature, thereby ensuring the crystal quality of the product. Using the purification method of the present invention, a white or off-white crystalline powder can be obtained, wherein the methanol solvent residue is less than 0.3%, and the content of madecassoside is higher than 90%.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a method for purifying madecassoside. Background Art

[0002] Madecassoside is also known as Madecassoside, with a CAS number of 34540-22-2 and a molecular formula of C 48 H 78 O 20 , molecular weight: 975.13, structure as shown in formula Ⅰ:

[0003]

[0004] Modern pharmacological research reports that madecassoside has the effects of removing scars and promoting wound healing, and can be used to treat skin ulcers, such as stubborn trauma, skin tuberculosis, leprosy, etc. At present, madecassoside is mainly extracted from Centella asiatica, which contains triterpenes, polyacetylenes, flavonoids, alkaloids and polyphenol components (including n-heptacosane, 2,4,6-tri-tert-butylbenzene, β-sitosterol, lauric acid, kaempferol, p-hydroxybenzoic acid, stigmasterol-3-O-β-D-glucose, asiatic acid, madecassoside, terminal oleic acid, marigold, quercetin, hexacosanol octanoate, carotene, vanillic acid, succinic acid, asiaticobioside, etc.). The above components are quite different in structure from madecassoside, and the above impurities can be effectively removed by crystallization. However, the structures of the two components, Centella asiatica (Formula II) and Centella asiatica B (Formula III), are relatively close to those of hydroxymadecassoside and are difficult to remove using conventional crystallization purification methods alone. They are the main impurities in the preparation process of hydroxymadecassoside. Therefore, how to effectively remove asiaticaside B and Centella asiatica is particularly important for the quality control of hydroxymadecassoside.

[0005]

[0006] Currently, the purification processes for hydroxymadecassoside reportedly include: separation by preparative high performance liquid chromatography analysis using cyclodextrin as a mobile phase additive. The disadvantages are: a large amount of solvent is used, the production cost is high, and it is not suitable for future industrial production. Summary of the invention

[0007] The object of the present invention is to solve the above problems and provide a method for purifying madecassoside.

[0008] The method for purifying madecassoside of the present invention uses a crude madecassoside prepared by an existing method as a raw material, and undergoes a refining and crystallization step to obtain a madecassoside product with a relatively better crystal form, less dissolved residue and high content of related substances.

[0009] Wherein, madecassoside can be prepared by the following method:

[0010] CN102477063A discloses a method for purifying madecassoside and asiaticaoside B, wherein high-purity madecassoside is obtained by separation and purification through preparative high performance liquid chromatography.

[0011] CN107903296A discloses a method for purifying madecassoside, wherein high-purity madecassoside is obtained by separation and purification through a macroporous resin column.

[0012] CN101983966B discloses a process for simultaneously preparing asiaticoside and madecassoside from total glycosides with low asiaticoside content. The total glycosides with low asiaticoside content are dissolved by heating with methanol at 50-65°C, and cooled and crystallized to obtain high-purity madecassoside and asiaticoside, respectively. However, the purity and yield of madecassoside obtained by this process are relatively low.

[0013] The method for preparing madecassoside is not limited to the above method, and may be other methods for preparing crude madecassoside in the prior art.

[0014] The method for purifying madecassoside of the present invention comprises the following steps:

[0015] (1) using methanol as a solvent to dissolve the crude madecassoside;

[0016] (2) Concentrating and removing the solvent;

[0017] (3) adding seed crystals, cooling and crystallizing;

[0018] (4) Filter and dry to obtain the product.

[0019] in,

[0020] Step (1) is preferably heated to dissolve, and the dissolving temperature is 60-65°C;

[0021] In step (1), the volume of methanol is 10-20 times the weight of the crude madecassoside, i.e., 10-20V; preferably, the volume of methanol is 15V; specifically, 300mL of methanol is added to 20.00g of the crude madecassoside;

[0022] In step (2), the concentration is continued at 40-50°C and P≤-0.08MPa until the residual volume of the system is 7-9V, and then the concentration is stopped;

[0023] In step (3), 0.01 times the amount of madecassoside seed crystals of the crude madecassoside is added, the temperature is naturally lowered to 20-30° C., and the temperature is kept for crystallization for 0.5-1.0 hour;

[0024] In step (4), the filter cake is vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain a refined madecassoside.

[0025] Furthermore, after filtering in step (4), a sample can be taken for HPLC detection. If the relevant content is less than 90%, steps (1) to (3) are repeated and the detection is continued.

[0026] Preferably, the purification method is:

[0027] To a 500ml reaction bottle, add 15V (300mL) of anhydrous methanol and madecassoside (20.00g) in sequence, stir and mix for 10-15 minutes, stir and heat to 60-65°C, and after madecassoside is completely dissolved, concentrate the crystallization system at 40-50°C, P≤-0.08MPa until the system reaches 7-9V and then stop concentrating; add 0.20g madecassoside to the crystallization system, cool naturally to 20-30°C, and then keep warm for crystallization for 0.5-1.0 hour, filter the reaction system, and take a sample of the filter cake for HPLC detection.

[0028] If the relevant content is less than 90%, repeat the crystallization operation and continue to test; when the relevant content is greater than 90%, the filter cake is vacuum dried at 55-60°C, P≤-0.08MPa for 8-10 hours to obtain refined hydroxy-madecassoside.

[0029] The invention uses the crude madecassoside prepared by the existing method as a raw material, and through the refining and crystallization steps, obtains a madecassoside product with better relative crystal form, less dissolution residue and high content of related substances. By selecting key influencing factors such as pulping solvent, pulping solvent volume, purification method, dissolution volume, concentrated crystallization volume, crystallization temperature, drying method and temperature, the nucleation and crystal growth rate of madecassoside crystals are effectively controlled, thereby ensuring the crystal quality of the product. Using the purification method of the invention, a white or off-white crystalline powder can be obtained, the methanol solvent residue of which is less than 0.3%, and the madecassoside content is higher than 90%.

[0030] The optimization process of the purification method of the present invention is further illustrated by test examples below:

[0031] 1.1 Pulping solvent

[0032] Table 1 Madecassoside beating solvent

[0033]

[0034]

[0035] Isopropanol, methanol and acetonitrile were selected to investigate the pulping effect of madecassoside. According to the test results in Table 1, methanol as a pulping solvent significantly increased the relevant content of madecassoside, while other solvents had poor pulping effects, so methanol was finally determined as the pulping solvent.

[0036] 1.2 Volume of pulping solvent

[0037] Table 2 Madecassoside beating solvent volume

[0038]

[0039] Although methanol as a pulping solvent can obtain a higher content of madecassoside, the yield of methanol as a pulping solvent is low, so the amount of methanol used was investigated (5V, 10V, 20V). According to the test results in Table 2, with the increase in the amount of methanol used, the content of madecassoside is improved to a certain extent, but the yield is significantly reduced. It is speculated that other impurities may be closer in structure to madecassoside, and their solubility in methanol is somewhat different. Subsequently, the purification method was screened to investigate whether the content of madecassoside and the crystallization yield can be effectively increased by recrystallization.

[0040] 1.3 Purification method

[0041] Table 3 Purification methods of madecassoside

[0042]

[0043] Since the yield of methanol pulping is low, the purification method was investigated. According to the test results in Table 3, the content and yield of madecassoside obtained by dissolving with the same volume of methanol and concentrating and crystallizing are significantly improved, and the product properties are good. Therefore, recrystallization was selected to purify and increase the content of madecassoside, and different crystallization conditions were screened.

[0044] 1.4 Dissolution volume

[0045] Table 4 Madecassoside dissolution volume

[0046]

[0047] The present invention investigates different dissolution volumes. According to the test results in Table 4, as the volume of methanol decreases, the dissolution rate of madecassoside significantly increases and decreases. When the volume of methanol is less than 10.0V, part of madecassoside cannot be dissolved. For the madecassoside described in the present invention, insufficient dissolution during the recrystallization process may cause partial impurities to be wrapped, thereby causing the crystallization purification effect to deteriorate. Therefore, the dissolution volume is determined to be 15.0V, which reduces the amount of solvent used, facilitates production operations, and can ensure the rapid dissolution of madecassoside.

[0048] 1.5 Concentrated crystallization volume

[0049] Table 5 Madecassoside Concentration Crystallization Volume

[0050]

[0051] The present invention investigates different recrystallization concentration and crystallization volumes. According to the test results in Table 5, as the concentration residual volume decreases, the madecassoside content changes slightly, but the yield changes significantly. When the concentration and crystallization volume is 7.0-9.0V, the madecassoside content and yield are both higher.

[0052] 1.6 Crystallization temperature

[0053] Table 6 Madecassoside crystallization temperature

[0054]

[0055] The present invention investigates different crystallization temperatures (20-30°C, 10-20°C, -10-0°C). According to the test results in Table 6, as the crystallization temperature decreases, the relevant content of madecassoside changes slightly, but the yield decreases significantly. Analysis shows that this is because the interfacial energy of madecassoside in methanol is relatively small, making it difficult to form crystals, and the induction period of madecassoside crystals under different temperature conditions is different. Therefore, the optimal crystallization temperature is determined to be 20-30°C after comprehensive consideration.

[0056] 1.7 Drying method and temperature

[0057] Table 7 Madecassoside Drying Method

[0058]

[0059] Table 8 Madecassoside Drying Temperature

[0060]

[0061] According to literature reports, the thermal stability of madecassoside is poor, so the present invention conducted a study on the stability of madecassoside in methanol solution. According to the test results in Table 7, madecassoside was observed to be degraded when stirred at 50-60°C for 2 hours in methanol solution, and the content of madecassoside was significantly reduced. Therefore, it is preferred to investigate whether the low-temperature drying conditions can obtain qualified madecassoside.

[0062] According to the test results in Table 8, when the drying temperature is 40-45°C, the methanol residual content of madecassoside is relatively high, and the methanol residual content of madecassoside obtained when the drying temperature is increased to 55-60°C is 0.030%, which meets the medium limit requirements of ICHQ3C; at the same time, compared with vacuum drying, the content of madecassoside obtained by forced air drying is slightly lower. Therefore, it is determined that the drying method is vacuum drying at 55-60°C for 8-10 hours, and madecassoside with methanol solvent residue meeting the limit can be obtained.

[0063] 2. Sample testing

[0064] 2.1 Analysis methods

[0065] 2.1.1 Gas chromatography

[0066] Analytical method: Chinese Pharmacopoeia 2015 edition Part IV General Principles 0861 Method 2

[0067] Chromatographic conditions: a capillary column with 6% cyanopropyl and 94% polydimethylsiloxane as the stationary phase; the starting temperature was 40°C, maintained for 10 minutes, raised to 230°C at a rate of 20°C per minute, and maintained for 10 minutes; the injection port temperature was 200°C, the detector temperature was 250°C; the headspace bottle equilibrium temperature was 85°C, and the equilibrium time was 30 minutes.

[0068] Preparation of the test solution: Take about 0.1 g of the sample of Example 1 of the present invention, accurately weigh it, put it in an empty bottle, accurately add 2 ml of N,N-dimethylformamide to dissolve it, and seal it to use as the test solution.

[0069] Preparation of reference solution: Accurately weigh an appropriate amount of methanol, place it in a volumetric flask, quantitatively dilute it with N,N-dimethylformamide to make a solution containing approximately 300 μg of methanol per 1 ml, accurately measure 2 ml, place it in an empty bottle, and seal it as the reference solution.

[0070] Determination method: Take headspace samples of the test solution and the reference solution respectively, record the chromatogram, and calculate the peak area according to the external standard method. The residual methanol content should meet the requirements.

[0071] 2.1.2 Liquid chromatography

[0072] Reference: Chinese Pharmacopoeia 2015 Edition Part 1 Total glycosides of Centella asiatica

[0073] Analytical method: Determined by high performance liquid chromatography (General Rule 0512)

[0074] Chromatographic conditions: octadecylsilane bonded silica gel as filler (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile: water (2 mmol / L beta-cyclodextrin) = 24:76 as mobile phase; detection wavelength 205 nm, column temperature 30°C.

[0075] Preparation of the test solution: Take an appropriate amount of the sample of Example 1 of the present invention, dissolve it in 50% methanol and dilute it to make a solution containing about 1 mg of the product per 1 ml.

[0076] Determination method: Accurately measure 10 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0077] 2.2 Test results

[0078]

[0079] Compared with the prior art, the present invention has the following technical effects:

[0080] The purity of commercially available madecassoside is 70-85%, which is relatively low and is not suitable for pharmaceutical research. The invention has a simple process, low production cost, less pollution, high product yield and purity, is suitable for industrial production, and does not involve highly toxic solvents, is environmentally friendly, and has the advantages of low requirements for instruments and equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Methanol 300ppm standard limit gas phase spectrum

[0082] Figure 2 Gas phase spectrum of methanol solvent residue in the sample of the present invention

[0083] Figure 3 Content determination blank control liquid phase spectrum

[0084] Figure 4 Liquid phase spectrum of sample content determination of the present invention DETAILED DESCRIPTION

[0085] The following examples are provided for a better understanding of the present invention, but they are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the materials, reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0086] Example 1

[0087] 300 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred and mixed for 10-15 minutes, stirred and heated to 60° C., and after madecassoside was completely dissolved, the crystallization system was concentrated at 50° C., P≤-0.08 MPa until the system reached 8V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 25° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was sampled for HPLC detection. When the relevant content was greater than 90%, the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 16.2 g of madecassoside refined product with a purity of 91.58%.

[0088] Example 2

[0089] 360 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred for 10-15 minutes, heated to 65° C. with stirring, and after madecassoside was completely dissolved, the crystallization system was concentrated at 50° C., P≤-0.08 MPa until the system reached 7V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 25° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 17.0 g of madecassoside refined product with a purity of 91.00%.

[0090] Example 3

[0091] 200 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred for 10-15 minutes, heated to 60° C. with stirring, and after madecassoside was completely dissolved, the crystallization system was concentrated at 45° C., P≤-0.08 MPa until the system reached 8V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 20° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 16.1 g of madecassoside refined product with a purity of 90.42%.

[0092] Example 4

[0093] 400 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred for 10-15 minutes, heated to 65° C. with stirring, and after madecassoside was completely dissolved, the crystallization system was concentrated at 50° C., P≤-0.08 MPa until the system reached 9 V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 30° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 15.7 g of madecassoside refined product with a purity of 92.01%.

[0094] Example 5

[0095] 360 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred for 10-15 minutes, heated to 60° C. with stirring, and after the madecassoside was completely dissolved, the crystallization system was concentrated at 45° C., P≤-0.08 MPa until the system reached 8V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 30° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 16.6 g of madecassoside refined product with a purity of 91.53%.

[0096] Example 6

[0097] 260 mL of anhydrous methanol and 20.00 g of madecassoside were added to a 500 ml reaction bottle in sequence, stirred for 10-15 minutes, heated to 65° C. with stirring, and after madecassoside was completely dissolved, the crystallization system was concentrated at 40° C., P≤-0.08 MPa until the system reached 7V, and then the concentration was stopped. 0.20 g of madecassoside was added to the crystallization system, and the temperature was naturally lowered to 20° C. and then kept warm for crystallization for 0.5-1.0 hours. The reaction system was filtered, and the filter cake was vacuum dried at 55-60° C., P≤-0.08 MPa for 8-10 hours to obtain 17.01 g of madecassoside refined product with a purity of 91.05%.

Claims

1. A method for purifying madecassoside, comprising the following steps: (1) using methanol as a solvent to dissolve the crude madecassoside; (2) Concentrating and removing the solvent; (3) adding seed crystals, cooling and crystallizing, the crystallization temperature is 20-30°C; (4) Filter and dry to obtain; In the step (1), the volume of methanol is 10-20 times the weight of the crude madecassoside, i.e., 10-20V, where the unit of V is mL / g; In the step (2), concentration is stopped after the residual volume of the system is 7-9V, where the unit of V is mL / g.

2. The purification method according to claim 1, characterized in that In the step (1), methanol is heated to dissolve, and the dissolution temperature is 60-65°C.

3. The purification method according to claim 1, characterized in that In the step (1), the volume of methanol is 15V, and the unit of V is mL / g.

4. The purification method according to claim 1, characterized in that In the step (2), the concentration is stopped after the system residual volume is 7-9V at 40-50°C and P≤-0.08MPa, where the unit of V is mL / g.

5. The purification method according to claim 1, characterized in that In the step (3), 0.01 times the amount of madecassoside crude product madecassoside seed crystals are added, the temperature is naturally lowered to 20-30° C., and the temperature is kept for crystallization for 0.5-1.0 hour.

6. The purification method according to claim 1, characterized in that In the step (4), the filter cake is vacuum dried at 55-60° C. and P≤-0.08 MPa for 8-10 hours to obtain a refined madecassoside.

7. The purification method according to claim 1, characterized in that After filtration in step (4), a sample may be taken for HPLC detection. If the relevant content is less than 90%, steps (1) to (3) are repeated and detection is continued.

8. The purification method according to claim 1, characterized in that To a 500 ml reaction bottle, 300 ml of anhydrous methanol and 20.00 g of madecassoside were added in sequence, the mixture was stirred for 10-15 minutes, the mixture was heated to 60-65° C. while stirring, and after the madecassoside was completely dissolved, the crystallization system was concentrated at 40-50° C., P≤-0.08 MPa until the system reached 7.0-9.0 V, and then the concentration was stopped, wherein the unit of V was mL / g; 0.20 g of madecassoside was added to the crystallization system, the mixture was naturally cooled to 20-30° C., and then the mixture was kept warm for crystallization for 0.5-1.0 hour, the reaction system was filtered, and the filter cake was sampled for HPLC detection.

Images