Extraction method of triptolide and application thereof

Abstract

This application relates to the field of natural product chemistry, specifically disclosing a method for extracting triptolide and its application. The extraction method for triptolide includes the following steps: S1, pretreatment: After washing and drying the roots of Tripterygium wilfordii, it is subjected to ultrafine pulverization and plasma treatment to obtain Tripterygium wilfordii powder; S2, enzymatic hydrolysis: After enzymatic hydrolysis of the Tripterygium wilfordii powder, the enzyme is inactivated, centrifuged, concentrated, and dried to obtain crude triptolide; S3, purification: The crude triptolide is purified by high-speed countercurrent chromatography and vacuum dried to obtain refined triptolide. The above extraction method is simple and safe to operate, and the purity and extraction rate of the obtained triptolide are improved. The obtained triptolide can be mixed with total glucosides of paeony, glycyrrhizic acid, papain, poloxamer, xanthan gum, and propylene glycol to prepare a drug for treating rheumatoid arthritis, which has significant efficacy, high safety, and broad clinical application prospects.

Description

Technical Field

[0001] This application relates to the field of natural product chemistry, and more specifically, to a method for extracting triptolide and its application. Background Technology

[0002] Tripterygium ethyl acetate is a natural compound extracted from the plant Tripterygium wilfordii. It is a diterpenoid compound with a complex chemical structure, a white crystalline solid, practically insoluble in water at room temperature, but soluble in organic solvents. Tripterygium ethyl acetate possesses a variety of biological and pharmacological activities. On the one hand, it exhibits potent anti-inflammatory and immunomodulatory effects, making it suitable for treating autoimmune and inflammatory diseases. On the other hand, it also shows antitumor activity, inhibiting various cancer cells, including breast cancer, lung cancer, gastric cancer, and liver cancer. Therefore, triptolide has been extensively studied in scientific research to explore its potential pharmacological effects and clinical applications.

[0003] CN105601700A discloses a method for preparing triptolide from Tripterygium wilfordii. The method involves extracting Tripterygium wilfordii root by heating and reflux with an ethanol-water solution to obtain a total extract; dissolving the total extract several times with ethyl acetate until no further dissolution occurs to obtain the ethyl acetate fraction; sequentially chromatography the ethyl acetate fraction using a neutral alumina column, an MCIGEL column, and a silica gel column to obtain crude triptolide; and finally recrystallizing with ethyl acetate to obtain pure triptolide. However, this method is relatively complex, and the extraction rate and purity of the obtained triptolide need improvement, failing to meet current medicinal needs. Therefore, there is an urgent need to propose a new extraction method for triptolide to lay the foundation for its wider application. Summary of the Invention

[0004] To address the issues of low extraction rate and purity of existing triptolide extraction methods, this application provides a method for extracting triptolide and its application.

[0005] A method for extracting triptolide, employing the following technical solution:

[0006] A method for extracting triptolide includes the following steps:

[0007] S1. Pretreatment: After washing and drying, the roots of Tripterygium wilfordii are subjected to ultra-fine grinding and plasma treatment to obtain Tripterygium wilfordii powder.

[0008] S2. Enzymatic hydrolysis: The Tripterygium wilfordii powder obtained in step S1 is enzymatically hydrolyzed, the enzyme is inactivated, centrifuged, concentrated, and dried to obtain crude Tripterygium wilfordii B.

[0009] S3. Purification: The crude triptolide obtained in step S2 is purified by high-speed countercurrent chromatography and vacuum dried to obtain the refined triptolide.

[0010] The enzymatic hydrolysis includes three steps: the first step uses cellulase and hemicellulase; the second step uses a complex enzyme of protease, amylase, xylanase and pectinase; and the third step uses β-glucanase.

[0011] By adopting the above technical solution, in the process of extracting triptolide, the roots of Tripterygium wilfordii are pulverized and then subjected to plasma treatment, resulting in numerous tiny pores on the surface of the obtained Tripterygium wilfordii powder. This increases the contact area between the powder and the enzyme during subsequent enzymatic hydrolysis, thereby improving the hydrolysis efficiency and accelerating the release of triptolide. The three-step enzymatic hydrolysis significantly improves the extraction rate of triptolide. Finally, the crude product undergoes high-speed countercurrent purification to further improve the purity of triptolide and greatly reduce its toxicity. The triptolide extraction method of this application is simple in steps, has a short process, is suitable for industrial production, and yields triptolide with high purity, possessing broad market prospects.

[0012] Preferably, the conditions for ultrafine pulverization are: a rotation speed of 4000-6000 rpm and a time of 20-30 min.

[0013] Preferably, the plasma treatment conditions are: vacuum degree of 70-90 Pa, power of 600-800 W, treatment time of 1-2 min, and introduction of a mixed gas composed of nitrogen and oxygen in a volume ratio of 2:1.

[0014] By adopting the above technical solution, this application uses plasma treatment of Tripterygium wilfordii, which on the one hand helps to destroy cell walls and cell membranes, change surface properties, and make triptolide in Tripterygium wilfordii roots easier to dissolve or release; on the other hand, the high-energy particles and active species generated by plasma can destroy microorganisms such as bacteria, fungi and viruses, remove microbial contaminants in Tripterygium wilfordii roots, thereby improving the purity and quality of triptolide; this application strictly controls the technical parameters of plasma treatment, effectively improves the extraction rate, shortens the extraction time, and further improves the extraction rate and purity of triptolide.

[0015] Preferably, the mass ratio of cellulase to hemicellulase is 2:1-2.

[0016] Preferably, the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0017] Preferably, the total mass of the cellulase and hemicellulase is 1-2% of the Tripterygium wilfordii powder; the mass of the complex enzyme is 2-4% of the Tripterygium wilfordii powder; and the mass of the β-glucanase is 0.5-1% of the Tripterygium wilfordii powder.

[0018] Preferably, the first enzymatic hydrolysis step is performed at a temperature of 50-55℃ for 3-6 hours and at a pH of 4-5.5; the second enzymatic hydrolysis step is performed at a temperature of 45-50℃ for 8-10 hours and at a pH of 4-6; and the third enzymatic hydrolysis step is performed at a temperature of 48-60℃ for 4-8 hours and at a pH of 4.8-5.5.

[0019] By adopting the above technical solution, the enzymatic hydrolysis of Tripterygium wilfordii powder involves a three-step process. The first step utilizes the combined action of cellulase and hemicellulase to effectively degrade cellulose and hemicellulose, thus accelerating the efficiency of the second step. The second step employs a complex enzyme system consisting of protease, amylase, xylanase, and pectinase, with the mass ratio of each enzyme strictly controlled within a specific range. This allows for synergistic effects among the enzymes, effectively disrupting the cell wall structure, causing localized collapse, dissolution, or loosening of the cell wall, accelerating the rate of triptolide dissolution from cells, increasing the extraction rate of triptolide, and shortening the extraction time. The third step uses β-glucanase to hydrolyze the glucosinolate bonds in Tripterygium wilfordii, reducing toxic components or inhibiting the release of active components, which helps to reduce the toxicity of crude Tripterygium wilfordii. Simultaneously, this application controls the enzyme dosage, hydrolysis temperature, time, and pH value within a specific range, significantly improving the extraction rate of triptolide.

[0020] Preferably, the solvent system of the high-speed countercurrent chromatograph is a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water, with a volume ratio of 0.05:5-7:8-10:3-4:5, wherein the lower phase is the mobile phase and the upper phase is the stationary phase.

[0021] By adopting the above technical solution, this application utilizes high-speed countercurrent chromatography with a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol, and water as the solvent system, and strictly controls the volume ratio of each solvent. After 1-ethyl-3-methylimidazolium acetate dissolves in the organic solvent, it forms an ionic liquid mixed organic solvent. 1-ethyl-3-methylimidazolium acetate readily forms strong hydrogen bonds, van der Waals forces, and electrostatic interactions with triptolide molecules, thereby increasing the distribution ratio of triptolide molecules in the solvent, increasing the extraction efficiency, obtaining high purity triptolide, and producing a product of excellent quality, thus giving triptolide a broad market prospect.

[0022] Preferably, the vacuum drying conditions are: vacuum degree of 2-3 Pa, temperature of 30-40℃, and drying until the surface moisture content is 0.7-0.9%.

[0023] Secondly, this application proposes an application of triptolide, employing the following technical solution:

[0024] An application of triptolide, specifically its use in medications for treating rheumatoid arthritis.

[0025] Preferably, the drug for treating rheumatoid arthritis comprises the following raw materials in parts by weight: 5-7 parts triptolide, 2-3 parts total glucosides of paeony, 1-4 parts glycyrrhizic acid, 1-2 parts papain, 0.1-0.3 parts poloxamer, 4-8 parts xanthan gum, and 5-10 parts propylene glycol.

[0026] Preferably, the drug for treating rheumatoid arthritis is prepared by the following method:

[0027] First, poloxamer, xanthan gum, and propylene glycol are mixed evenly. Then, triptolide, total paeoniflorin, glycyrrhizic acid, and papain are added. After homogenization, the mixture is freeze-dried and compressed into tablets to obtain triptolide polyglycoside tablets.

[0028] By adopting the above technical solution, the triptolide obtained in this application can be used as a drug for treating rheumatoid arthritis. The triptolide, together with total glucosides of paeony, glycyrrhizic acid, and papain, has a synergistic effect, which not only has a significant effect on treating rheumatoid arthritis, but also reduces toxicity and enhances efficacy, resulting in higher safety and better efficacy.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. The extraction method of triptolide in this application involves first washing and drying the roots of Tripterygium wilfordii, then subjecting them to ultrafine pulverization and plasma treatment to obtain Tripterygium wilfordii powder; subsequently, the powder undergoes three enzymatic hydrolysis processes, followed by enzyme inactivation, centrifugation, concentration, and drying to obtain crude triptolide; finally, it is purified by high-speed countercurrent chromatography and vacuum dried to obtain refined triptolide. The extraction process of this application is simple and safe to operate, effectively improving the extraction rate and purity of triptolide, and is suitable for industrial production.

[0031] 2. The triptolide of this application can be homogenized with poloxamer, xanthan gum, propylene glycol, total glucosides of paeony, glycyrrhizic acid, and papain, then freeze-dried and compressed into tablets to prepare a drug for treating rheumatoid arthritis. It has good therapeutic effects, significantly reduces the toxic side effects of triptolide, has high safety, and has broad clinical application prospects. Detailed Implementation

[0032] The present application will be further described in detail below with reference to the embodiments.

[0033] Examples 1-5 provide a method for extracting triptolide.

[0034] Example 1

[0035] A method for extracting triptolide includes the following steps:

[0036] S1, Preprocessing

[0037] After cleaning and drying, the roots of Tripterygium wilfordii are first subjected to ultra-fine pulverization at a speed of 4000 rpm for 20 minutes, and then subjected to plasma treatment at a vacuum of 70 Pa, a power of 600 W, and a treatment time of 1 minute. A mixed gas consisting of nitrogen and oxygen in a volume ratio of 2:1 is introduced to obtain Tripterygium wilfordii powder.

[0038] S2, Enzymatic hydrolysis

[0039] S21. Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 10 g of a mixture of cellulase and hemicellulase. Hydrolyze for 3 h at 50 °C and pH 4. Then, perform the second enzymatic hydrolysis by adding 20 g of a compound enzyme. Hydrolyze for 8 h at 45 °C and pH 4. Finally, perform the third enzymatic hydrolysis by adding 5 g of β-glucanase. Hydrolyze for 4 h at 48 °C and pH 4.8. Then, raise the temperature to 80 °C and inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0040] S22. First, concentrate the enzymatic hydrolysate obtained in step S21 under a vacuum of 0.04 MPa and a temperature of 50°C until the solid content is 80%; then dry it at 50°C to obtain crude triptolide.

[0041] The mass ratio of cellulase to hemicellulase is 2:1; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0042] S3, Purification

[0043] The crude triptolide obtained in step S2 was purified by high-speed countercurrent chromatography. The solvent system was a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water in a volume ratio of 0.05:5:8:3:5, with the lower phase being the mobile phase and the upper phase being the stationary phase. Subsequently, it was vacuum dried at a vacuum of 2 Pa and a temperature of 30 °C until the surface water content was 0.7%, yielding the refined triptolide.

[0044] Example 2

[0045] A method for extracting triptolide includes the following steps:

[0046] S1. Pretreatment: After washing and drying the roots of Tripterygium wilfordii, they are first subjected to ultra-fine pulverization at a speed of 4500 rpm for 22 min. Then, they are subjected to plasma treatment at a vacuum of 75 Pa and a power of 650 W for 1.2 min. A mixed gas consisting of nitrogen and oxygen in a volume ratio of 2:1 is introduced to obtain Tripterygium wilfordii powder.

[0047] S2, Enzymatic hydrolysis:

[0048] S21. Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 12 g of a mixture of cellulase and hemicellulase. Hydrolyze for 3.5 h at 51 °C and pH 4.2. Then, perform the second enzymatic hydrolysis by adding 25 g of a compound enzyme. Hydrolyze for 8.5 h at 47 °C and pH 4.5. Finally, perform the third enzymatic hydrolysis by adding 6 g of β-glucanase. Hydrolyze for 5 h at 50 °C and pH 5. Then, heat to 80 °C and inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0049] S22. First, concentrate the enzymatic hydrolysate obtained in step S21 under a vacuum of 0.04 MPa and a temperature of 50°C until the solid content is 80%; then dry it at 50°C to obtain crude triptolide.

[0050] The mass ratio of cellulase to hemicellulase is 2:1.2; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0051] S3. Purification: The crude triptolide obtained in step S2 was purified by high-speed countercurrent chromatography. The solvent system was a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water in a volume ratio of 0.05:5:8.5:3.3:5. The lower phase was the mobile phase and the upper phase was the stationary phase. Subsequently, vacuum drying was carried out at a vacuum degree of 2.3 Pa and a temperature of 33 °C until the surface water content was 0.75%, thus obtaining the purified triptolide.

[0052] Example 3

[0053] A method for extracting triptolide includes the following steps:

[0054] S1. Pretreatment: After washing and drying the roots of Tripterygium wilfordii, they are first subjected to ultra-fine pulverization at a speed of 5000 rpm for 25 minutes. Then, they are subjected to plasma treatment at a vacuum of 80 Pa, a power of 700 W, and a treatment time of 1.5 minutes. A mixed gas consisting of nitrogen and oxygen in a volume ratio of 2:1 is introduced to obtain Tripterygium wilfordii powder.

[0055] S2, Enzymatic hydrolysis:

[0056] S21. Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 15 g of a mixture of cellulase and hemicellulase. Hydrolyze at 53℃ and pH 5 for 4 h. Then, perform the second enzymatic hydrolysis by adding 30 g of a compound enzyme. Hydrolyze at 48℃ and pH 5 for 9 h. Finally, perform the third enzymatic hydrolysis by adding 8 g of β-glucanase. Hydrolyze at 54℃ and pH 5.2 for 6 h. Then, raise the temperature to 80℃ and inactivate the enzyme for 10 min. Centrifuge at 4000 rpm for 10 min to remove the precipitate to obtain the enzymatic hydrolysate.

[0057] S22. First, concentrate the enzymatic hydrolysate obtained in step S21 under a vacuum of 0.04 MPa and a temperature of 50°C until the solid content is 80%; then dry it at 50°C to obtain crude triptolide.

[0058] The mass ratio of cellulase to hemicellulase is 2:1.5; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0059] S3. Purification: The crude triptolide obtained in step S2 was purified by high-speed countercurrent chromatography. The solvent system was a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water in a volume ratio of 0.05:6.5:9:3.5:5. The lower phase was the mobile phase and the upper phase was the stationary phase. Subsequently, vacuum drying was carried out at a vacuum degree of 2.5 Pa and a temperature of 35 °C until the surface water content was 0.8%, thus obtaining the purified triptolide.

[0060] Example 4

[0061] A method for extracting triptolide includes the following steps:

[0062] S1. Pretreatment: After washing and drying the roots of Tripterygium wilfordii, they are first subjected to ultra-fine pulverization at a speed of 5500 rpm for 28 minutes. Then, they are subjected to plasma treatment at a vacuum of 85 Pa, a power of 750 W, and a treatment time of 1.8 minutes. A mixed gas consisting of nitrogen and oxygen in a volume ratio of 2:1 is introduced to obtain Tripterygium wilfordii powder.

[0063] S2, Enzymatic hydrolysis:

[0064] S21. Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 18 g of a mixture of cellulase and hemicellulase. Hydrolyze for 5.5 h at 54℃ and pH 5.4. Then, perform the second enzymatic hydrolysis by adding 35 g of a compound enzyme. Hydrolyze for 9.5 h at 48℃ and pH 5.5. Finally, perform the third enzymatic hydrolysis by adding 9 g of β-glucanase. Hydrolyze for 7.5 h at 57℃ and pH 5.2. Then, heat to 80℃ to inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0065] S22. First, concentrate the enzymatic hydrolysate obtained in step S21 under a vacuum of 0.04 MPa and a temperature of 50°C until the solid content is 80%; then dry it at 50°C to obtain crude triptolide.

[0066] The mass ratio of cellulase to hemicellulase is 2:1.8; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0067] S3. Purification: The crude triptolide obtained in step S2 was purified by high-speed countercurrent chromatography. The solvent system was a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water in a volume ratio of 0.05:6.5:9.5:3.8:5. The lower phase was the mobile phase and the upper phase was the stationary phase. Subsequently, vacuum drying was carried out at a vacuum degree of 2.8 Pa and a temperature of 38 °C until the surface water content was 0.85%, thus obtaining the purified triptolide.

[0068] Example 5

[0069] A method for extracting triptolide includes the following steps:

[0070] S1. Pretreatment: After washing and drying the roots of Tripterygium wilfordii, they are first subjected to ultra-fine pulverization at a speed of 6000 rpm for 30 minutes. Then, they are subjected to plasma treatment at a vacuum of 90 Pa, a power of 800 W, and a treatment time of 2 minutes. A mixed gas consisting of nitrogen and oxygen in a volume ratio of 2:1 is introduced to obtain Tripterygium wilfordii powder.

[0071] S2, Enzymatic hydrolysis:

[0072] S21. Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 20 g of a mixture of cellulase and hemicellulase. Hydrolyze for 6 h at 55℃ and pH 5.5. Then, perform the second enzymatic hydrolysis by adding 40 g of a compound enzyme. Hydrolyze for 10 h at 50℃ and pH 6. Finally, perform the third enzymatic hydrolysis by adding 10 g of β-glucanase. Hydrolyze for 8 h at 60℃ and pH 5.5. Then, raise the temperature to 80℃ and inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0073] S22. First, concentrate the enzymatic hydrolysate obtained in step S21 under a vacuum of 0.04 MPa and a temperature of 50°C until the solid content is 80%; then dry it at 50°C to obtain crude triptolide.

[0074] The mass ratio of cellulase to hemicellulase is 1:1; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0075] S3. Purification: The crude triptolide obtained in step S2 was purified by high-speed countercurrent chromatography. The solvent system was a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water in a volume ratio of 0.05:7:10:4:5. The lower phase was the mobile phase and the upper phase was the stationary phase. Subsequently, it was vacuum dried at a vacuum degree of 3 Pa and a temperature of 40 °C until the surface water content was 0.9%, thus obtaining the purified triptolide.

[0076] To verify the extraction method of triptolide provided in this application, and the purity and extraction rate of the obtained triptolide, the applicant set up comparative examples 1-10, in which:

[0077] Comparative Example 1

[0078] Comparative Example 1 is the same as Example 1, except that plasma treatment is not performed in step S1. The specific operation of step S1 is as follows:

[0079] After washing and drying the roots of Tripterygium wilfordii, they were subjected to ultrafine grinding at 400 rpm for 20 minutes to obtain Tripterygium wilfordii powder.

[0080] Comparative Example 2

[0081] Comparative Example 2 is the same as Example 1, except that only one enzymatic hydrolysis is performed in step S21. The specific operation of step S21 is as follows:

[0082] Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. Add 6.7 g cellulase, 3.3 g hemicellulase, 4.4 g protease, 8.8 g amylase, 4.4 g xylanase, 2.2 g pectinase and 5 g β-glucanase for enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 50℃ and pH 4 for 10 h. Then, the temperature is raised to 80℃ and the enzyme is inactivated for 10 min. The precipitate is removed by centrifugation at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0083] Comparative Example 3

[0084] Comparative Example 3 is the same as Example 1, except that only two enzymatic hydrolysis operations are performed in step S21. The specific operation of step S21 is as follows:

[0085] Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, perform the first enzymatic hydrolysis by adding 10-20 g of a mixture of cellulase and hemicellulase. Hydrolyze for 3 h at 50 °C and pH 4. Then, perform the second enzymatic hydrolysis by adding 20 g of a compound enzyme and 5 g of β-glucanase. Hydrolyze for 8 h at 45 °C and pH 4. Subsequently, raise the temperature to 80 °C and inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0086] The mass ratio of cellulase to hemicellulase is 2:1; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0087] Comparative Example 4

[0088] Comparative Example 4 is the same as Example 1, except that in step S21, the second enzymatic hydrolysis is performed first, followed by the first enzymatic hydrolysis. The specific operation of step S21 is as follows:

[0089] Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, add 20 g of compound enzyme and hydrolyze for 8 h at 45 °C and pH 4. Then, add 10 g of a mixture of cellulase and hemicellulase and hydrolyze for 3 h at 50 °C and pH 4. Finally, add 5 g of β-glucanase and hydrolyze for 4 h at 48 °C and pH 4.8. Then, raise the temperature to 80 °C and inactivate the enzyme for 10 min. Centrifuge at 4000 rpm for 10 min to remove the precipitate to obtain the enzymatic hydrolysate.

[0090] The mass ratio of cellulase to hemicellulase is 2:1; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0091] Comparative Example 5

[0092] Comparative Example 5 is the same as Example 1, except that in step S21, the third enzymatic hydrolysis is performed first, followed by the second enzymatic hydrolysis. The specific operation of step S21 is as follows:

[0093] Take 1 kg of Tripterygium wilfordii powder obtained in step S1 and add it to 2 L of water. First, add 10 g of a mixture of cellulase and hemicellulase and hydrolyze for 3 h at 50 °C and pH 4. Then add 5 g of β-glucanase and hydrolyze for 4 h at 48 °C and pH 4.8. Finally, add 20 g of a complex enzyme and hydrolyze for 8 h at 45 °C and pH 4. Subsequently, raise the temperature to 80 °C and inactivate the enzyme for 10 min. Centrifuge to remove the precipitate at 4000 rpm for 10 min to obtain the enzymatic hydrolysate.

[0094] The mass ratio of cellulase to hemicellulase is 2:1; the complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.5.

[0095] Comparative Example 6

[0096] Comparative Example 6 is the same as Example 1, except that the complex enzyme is obtained by mixing protease, amylase and xylanase in a mass ratio of 1:2:1.

[0097] Comparative Example 7

[0098] Comparative Example 7 is the same as Example 1, except that the complex enzyme is obtained by mixing protease, amylase and pectinase in a mass ratio of 1:2:0.5.

[0099] Comparative Example 8

[0100] Comparative Example 8 is the same as Example 1, except that the complex enzyme is obtained by mixing protease, xylanase and pectinase in a mass ratio of 1:1:0.5.

[0101] Comparative Example 9

[0102] Comparative Example 9 is the same as Example 1, except that the complex enzyme is obtained by mixing amylase, xylanase and pectinase in a mass ratio of 2:1:0.5.

[0103] Comparative Example 10

[0104] Comparative Example 10 is the same as Example 1, except that the solvent system is a mixed solution of n-hexane, chloroform, methanol and water in a volume ratio of 5:8:3:5.

[0105] The extraction methods, extraction rates, and purity of triptolide in Examples 1-5 and Comparative Examples 1-10 were tested respectively, and the results are shown in Table 1:

[0106] Extraction rate of triptolide: The extraction rate of triptolide is obtained by the ratio of the mass of the obtained high-quality triptolide to the mass of triptolide powder;

[0107] Purity of triptolide: The purity of the refined triptolide was determined using high performance liquid chromatography.

[0108] Table 1:

[0109] Extraction rate / % purity / % Example 1 3.65 99.5 Example 2 3.76 99.7 Example 3 3.95 99.9 Example 4 3.88 99.9 Example 5 3.83 99.8 Comparative Example 1 3.12 98.1 Comparative Example 2 1.86 91.6 Comparative Example 3 2.24 93.5 Comparative Example 4 2.89 94.2 Comparative Example 5 2.95 95.3 Comparative Example 6 3.12 97.2 Comparative Example 7 3.03 96.9 Comparative Example 8 3.26 97.8 Comparative Example 9 3.18 97.5 Comparative Example 10 3.49 94.2

[0110] As shown in Table 1 above, the extraction rate and purity of triptolide obtained by the extraction methods of triptolide in Examples 1-5 of this application are much higher than those in Comparative Examples 1-10, which fully demonstrates that the extraction method of this application can extract triptolide from Tripterygium wilfordii to a great extent and has broad application prospects.

[0111] As can be seen from Example 1 and Comparative Example 1, Example 1 not only underwent ultrafine grinding in step S1, but also plasma treatment. Compared with Comparative Example 1, which only underwent ultrafine grinding, Example 1 had a higher extraction rate and higher purity of triptolide.

[0112] As can be seen from Example 1 and Comparative Examples 2 and 3, the Tripterygium wilfordii powder in Example 1 underwent three-step enzymatic hydrolysis, compared to only one-step enzymatic hydrolysis in Comparative Example 2 and two-step enzymatic hydrolysis in Comparative Example 3. The extraction rate and purity of Tripterygium wilfordii ethyl obtained in Example 1 were significantly higher than those in Comparative Examples 2 and 3.

[0113] As can be seen from Example 1 and Comparative Examples 4 and 5, in the enzymatic hydrolysis process of Example 1, the first step of enzymatic hydrolysis uses cellulase and hemicellulase; the second step of enzymatic hydrolysis uses a complex enzyme of protease, amylase, xylanase and pectinase; and the third step of enzymatic hydrolysis uses β-glucanase. Compared with Comparative Examples 4 and 5, the three-step enzymatic hydrolysis sequence in Example 1 is more conducive to the extraction of triptolide.

[0114] As can be seen from Example 1 and Comparative Examples 6-9, the compound enzyme in Example 1 was obtained by mixing protease, amylase, xylanase and pectinase. Compared with Examples 6-9, the extraction rate of triptolide in Example 1 was significantly improved.

[0115] As can be seen from Example 1 and Comparative Example 10, the solvent system of the high-speed countercurrent chromatograph in Example 1 is a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water. Compared with Comparative Example 10, the extraction process of Example 1 helps to improve the purity of triptolide.

[0116] An application example provides an application of triptolide.

[0117] The application of triptolide in the treatment of rheumatoid arthritis, the treatment of rheumatoid arthritis includes the following raw materials: 6g triptolide, 2g total glucosides of paeony, 2g glycyrrhizic acid, 1.5g papain, 0.2g poloxamer, 6g xanthan gum, 10g propylene glycol, and triptolide is prepared by Example 1;

[0118] Drugs for treating rheumatoid arthritis are prepared by the following methods:

[0119] First, poloxamer, xanthan gum, and propylene glycol are mixed and stirred at 500 rpm for 40 minutes at 50°C. Then, triptolide, total glucosides of paeony, glycyrrhizic acid, and papain are added and homogenized at 60 MPa for 40 minutes. Finally, the mixture is freeze-dried at 40°C for 2 hours and then compressed into tablets to obtain a drug for treating rheumatoid arthritis.

[0120] In this application, the properties of the rheumatoid arthritis treatment drug prepared in this application and the commercially available diclofenac sodium enteric-coated tablets (Beijing Novartis Pharmaceutical Co., Ltd.) were tested by establishing an adjuvant-induced arthritis (AA) rat model. The results are shown in Table 2.

[0121] (1) Establishment of rat AA model

[0122] Wistar rats were randomly divided into four groups of five rats each: a control group, a model group, a group receiving a drug for treating rheumatoid arthritis prepared according to the guidelines, and a group receiving diclofenac sodium enteric-coated tablet (10 mg / kg, once daily). Except for the control group, all other groups received a subcutaneous injection of 0.1 mL Freund's complete adjuvant into the right hind paw. The control group received an equal volume of physiological saline injected into the bottom of its right hind paw using the same method.

[0123] (2) Dosage and administration method

[0124] The control group and model group received no treatment. Starting on day 1 after modeling, the rats were administered 2 mL of a drug for treating rheumatoid arthritis and diclofenac sodium enteric-coated tablets (10 mg / kg, once daily) via gavage for 28 days. The length of the right hind paw (mm) was measured using calipers 1 hour before administration and on days 1, 7, 14, 21, and 28 after administration, and the average value was recorded. The comparison of paw swelling in each group is shown in Table 2.

[0125] Table 2:

[0126] No. 0d 1d 7th day 14th 21st 28th Application examples 6.83 8.45 7.71 7.42 7.38 7.35 Diclofenac sodium enteric-coated tablets 6.84 8.37 7.83 7.67 7.66 7.64 Model group 6.51 8.82 8.67 8.59 8.52 8.43 Blank group 6.83 6.78 7.13 7.27 7.28 7.30

[0127] As shown in Table 2, the drug prepared in this application has a significant effect on improving the symptoms of arthritis in rats, and its therapeutic effect is better than that of diclofenac sodium enteric-coated tablets. It can improve the clinical symptoms and signs and joint function of patients, reduce pain, and regulate the level of related rheumatoid factors, and has broad clinical application prospects.

[0128] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for extracting triptolide, characterized in that, Includes the following steps: S1. Pretreatment: After washing and drying, the roots of Tripterygium wilfordii are subjected to ultra-fine grinding and plasma treatment to obtain Tripterygium wilfordii powder. S2. Enzymatic hydrolysis: The Tripterygium wilfordii powder obtained in step S1 is enzymatically hydrolyzed, the enzyme is inactivated, centrifuged, concentrated, and dried to obtain crude Tripterygium wilfordii B. S3. Purification: The crude triptolide obtained in step S2 is purified by high-speed countercurrent chromatography and vacuum dried to obtain the refined triptolide. The enzymatic hydrolysis includes three steps: the first step uses cellulase and hemicellulase; the second step uses a complex enzyme of protease, amylase, xylanase and pectinase; and the third step uses β-glucanase. The complex enzyme is obtained by mixing protease, amylase, xylanase and pectinase in a mass ratio of 1:2:1:0.

5. The plasma treatment conditions are: vacuum degree of 70-90 Pa, power of 600-800 W, treatment time of 1-2 min, and introduction of a mixed gas composed of nitrogen and oxygen in a volume ratio of 2:

1. The first step of enzymatic hydrolysis is performed at a temperature of 50-55℃ for 3-6 hours, with a pH of 4-5.5; the second step is performed at a temperature of 45-50℃ for 8-10 hours, with a pH of 4-6; and the third step is performed at a temperature of 48-60℃ for 4-8 hours, with a pH of 4.8-5.

5. The solvent system of the high-speed countercurrent chromatograph is a mixed solution of 1-ethyl-3-methylimidazolium acetate, n-hexane, chloroform, methanol and water, with a volume ratio of 0.05:5-7:8-10:3-4:

5. The lower phase is the mobile phase and the upper phase is the stationary phase.

2. The method for extracting triptolide according to claim 1, characterized in that, The mass ratio of cellulase to hemicellulase is 2:1-2.

3. The method for extracting triptolide according to claim 1, characterized in that, The total mass of the cellulase and hemicellulase is 1-2% of the Tripterygium wilfordii powder; the mass of the compound enzyme is 2-4% of the Tripterygium wilfordii powder; and the mass of the β-glucanase is 0.5-1% of the Tripterygium wilfordii powder.