Method for purifying and separating 10-DAB III from taxus chinensis

Abstract

A method for purifying and separating 10-DAB III from taxus chinensis relates to the technical field of natural product extraction, and comprises the following steps: 1) raw material treatment: taking taxus chinensis branches and leaves, drying, crushing with a crusher, and sieving to obtain taxus chinensis powder; 2) aqueous two-phase extraction and purification: taking the taxus chinensis powder, adding polyethylene glycol and ammonium sulfate, then adding water, stirring, standing, forming an aqueous two-phase, and extracting to obtain an upper-phase extracting solution; 3) ultrafiltration centrifugal purification: diluting the upper-phase extraction liquid obtained in the step 2), and further purifying by using an ultrafiltration centrifugal method to obtain a 10-DAB III solution; and 4) freeze-drying: freeze-drying the 10-DAB III solution obtained in the step 3), and removing moisture to obtain the 10-DAB III freeze-dried powder. The method has the advantages of simple process route, avoidance of recrystallization and other processes in which a large amount of toxic organic solvents are used, greenness and environmental protection, the purity of the obtained sample can reach more than 90%, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly to a method for purifying and separating 10-DABⅢ from Taxus chinensis. Background Art

[0002] Paclitaxel is regarded as an effective anti-cancer drug due to its special mechanism of inhibiting cancer cell division. The main ways to obtain paclitaxel include extraction from bark, semi-synthesis, total chemical synthesis, etc. Extracting paclitaxel from bark will cause the death of Taxus chinensis trees, which is not conducive to sustainable development. The total chemical synthesis has cumbersome steps, low yield and high cost. Therefore, paclitaxel drugs in the domestic market are generally synthesized by semi-chemical synthesis. The process of semi-chemically synthesizing paclitaxel from 10-DABⅢ has been relatively mature. Therefore, it is crucial to find a green and economical method for extracting 10-DABⅢ in large quantities.

[0003] 10-DABⅢ, also known as 10-deacetylbaccatin, is a taxane derivative and mainly exists in the leaves, bark, roots and other parts of Taxus chinensis trees. Extracting 10-DABⅢ from Taxus chinensis branches and leaves is a sustainable and renewable extraction method. At present, there are various ways to extract 10-DABⅢ from Taxus chinensis branches and leaves. Chinese Patent (CN201710444797.8) discloses a method for extracting 10-DABⅢ from Taxus chinensis, including steps such as initial pulverization, enzymatic hydrolysis, aqueous two-phase extraction, macroporous resin enrichment, elution, crystallization and purification, etc. The selected ethanol / salt aqueous two-phase system has no strong selectivity for 10-DABⅢ. In the upper phase, not only 10-DABⅢ exists, but also a large amount of other taxane derivatives enzymatically hydrolyzed exist. It is necessary to enrich with macroporous resin and then crystallize and purify. The steps are cumbersome, and a large amount of organic solvents are used in recrystallization, resulting in problems such as high toxicity.

[0004] Therefore, there is a need for a method for extracting 10-DABⅢ from Taxus chinensis with a simple process route, green economy and low solvent toxicity. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems in the prior art and provide a method for purifying and separating 10-DABⅢ from Taxus chinensis.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for purifying and separating 10-DABⅢ from Taxus chinensis, comprising the following steps:

[0008] 1) Raw material treatment: Take Taxus chinensis branches and leaves, dry them, pulverize them with a pulverizer and then sieve to obtain Taxus chinensis powder;

[0009] 2) Aqueous two-phase extraction and purification: Take Taxus chinensis powder, add polyethylene glycol and ammonium sulfate, then add water, stir and let stand until an aqueous two-phase system is formed. After extraction, the upper-phase extract is obtained.

[0010] 3) Ultrafiltration and centrifugation purification: Dilute the upper-phase extract obtained in step 2), and further purify it by ultrafiltration and centrifugation to obtain a 10-DABⅢ solution.

[0011] 4) Freeze-drying: Freeze-dry the 10-DABⅢ solution obtained in step 3) to remove water and obtain 10-DABⅢ freeze-dried powder.

[0012] In step 1), the particle size of the Taxus chinensis powder is 40 - 100 mesh.

[0013] In step 2), the average relative molecular mass of the polyethylene glycol is 4000 - 20000.

[0014] In step 2), in the aqueous two-phase system formed by polyethylene glycol, ammonium sulfate and water, the mass concentration of polyethylene glycol is 15% - 25%, and the mass concentration of ammonium sulfate is 10% - 14%.

[0015] In step 2), the mass ratio of the Taxus chinensis powder to the aqueous two-phase system formed by polyethylene glycol, ammonium sulfate and water is 1:10 - 20.

[0016] In step 2), the stirring time is 2 - 3 min.

[0017] In step 2), the extraction time is 20 - 30 min.

[0018] In step 3), the upper-phase extract is diluted 6 - 8 times.

[0019] In step 3), the molecular weight cut-off of the ultrafiltration and centrifugation tube selected is 3 kD.

[0020] In step 3), the ultrafiltration and centrifugation speed is 6000 - 10000 g, and the centrifugation time is 10 - 20 min.

[0021] In the following examples of the present invention, the Taxus chinensis selected is Taxus chinensis var. mairei, and a large amount of 10-DABⅢ is contained in the branches and leaves of Taxus chinensis var. mairei, which is easier to extract.

[0022] Compared with the prior art, the beneficial effects achieved by the technical solution of the present invention are:

[0023] 1. The polyethylene glycol / ammonium sulfate aqueous two-phase system selected in the present invention has strong selectivity for 10-DABⅢ. During the aqueous two-phase extraction, 10-DABⅢ is highly enriched in the upper phase, and there are fewer impurities with similar properties, which is convenient for subsequent treatment.

[0024] 2. The process route of the present invention is simple, avoiding processes that use a large amount of toxic organic solvents such as recrystallization, being green and environmentally friendly. The purity of the obtained sample reaches more than 90%, reducing the production cost.

[0025] 3. The present invention combines aqueous two-phase extraction with ultrafiltration centrifugation, significantly reducing the purification time, solving the problem that in the existing process, 10-DABⅢ needs to be further purified by macroporous resin, crystallization, etc. to meet the same purity requirements, and improving the production efficiency.

[0026] 4. In the present invention, the particle size of the Taxus chinensis powder is 40-100 mesh, which is more conducive to the progress of aqueous two-phase extraction.

[0027] 5. The present invention uses polyethylene glycol with an average relative molecular mass of 4000-20000, which is more conducive to the formation of the aqueous two-phase system and enriches 10-DABⅢ into the upper phase.

[0028] 6. The present invention adopts specific aqueous two-phase concentrations and the mass ratio of Taxus chinensis powder to the aqueous two-phase system, which is conducive to the distribution of 10-DABⅢ in the aqueous two-phase system and the progress of aqueous two-phase extraction.

[0029] 7. The ultrafiltration centrifugal tube selected in the present invention has a cut-off molecular weight of 3 kD, the ultrafiltration centrifugation speed is 6000-10000 g, and the centrifugation time is 10-20 min. Such specific ultrafiltration centrifugation conditions are conducive to the separation and purification of 10-DABⅢ and improve the purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the high-performance liquid chromatography diagram of the upper-phase sample after aqueous two-phase extraction in Example 1 of the present invention;

[0031] Figure 2 It is the high-performance liquid chromatography diagram of the sample after ultrafiltration centrifugation treatment in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the following further describes the present invention in detail with reference to the drawings and embodiments.

[0033] Example 1

[0034] Purifying and separating 10-DABⅢ from Taxus chinensis, including the following steps:

[0035] (1) Raw material treatment: Take the branches and leaves of Taxus chinensis var. mairei, dry them, crush them with a pulverizer and sieve them to obtain Taxus chinensis powder with a mesh size of 60.

[0036] (2)Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus chinensis powder obtained in step (1), add 3.00 g of polyethylene glycol (average relative molecular mass is 10,000), 1.50 g of ammonium sulfate and 10.50 g of water, stir for 2 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 9 ml of the upper-phase extraction solution; As Figure 1 shown, 10-DABⅢ peaks at about 5 min, and polyethylene glycol, ammonium sulfate and other substances peak at about 1 - 3 min;

[0037] (3)Ultrafiltration and centrifugation purification: Dilute the upper-phase extraction solution rich in 10-DABⅢ obtained in step (2) by 8 times, select a 3 kd ultrafiltration and centrifugation tube, and further purify it by ultrafiltration and centrifugation method. The centrifugation speed is 8000 g and the centrifugation time is 20 min to obtain 23 ml of 10-DABⅢ solution with higher purity; As Figure 2 shown, most of the impurities are removed by ultrafiltration and centrifugation to obtain a 10-DABⅢ solution with higher purity;

[0038] (4)Freeze-drying: Freeze-dry the 10-DABⅢ solution obtained in step (3) at a vacuum degree of 20 Pa and a temperature of -40 °C for 48 h to remove water, and obtain 6.3838 mg of 10-DABⅢ freeze-dried powder with a purity of 88.10%.

[0039] Example 2

[0040] (1)Raw material treatment: Take the branches and leaves of Taxus chinensis var. mairei, dry them, crush them with a pulverizer and sieve them to obtain Taxus chinensis powder with a mesh size of 60;

[0041] (2)Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus chinensis powder obtained in step (1), add 3.75 g of polyethylene glycol (average relative molecular mass is 10,000), 1.50 g of ammonium sulfate and 9.75 g of water, stir for 5 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 10 ml of the upper-phase extraction solution;

[0042] (3)Ultrafiltration and centrifugation purification: Dilute the upper-phase extraction solution rich in 10-DABⅢ obtained in step (2) by 8 times, select a 3 kd ultrafiltration and centrifugation tube, and further purify it by ultrafiltration and centrifugation method. The centrifugation speed is 8000 g and the centrifugation time is 20 min to obtain 30 ml of 10-DABⅢ solution with higher purity;

[0043] (4)Freeze-drying: Freeze-dry the 10-DABⅢ solution obtained in step (3) at a vacuum degree of 20 Pa and a temperature of -40 °C for 48 h to remove water, and obtain 5.9476 mg of 10-DABⅢ freeze-dried powder with a purity of 90.39%.

[0044] Example 3

[0045] (1) Raw material treatment: Take Taxus chinensis var. mairei branches and leaves, dry them, pulverize them with a pulverizer and then sieve them to obtain Taxus powder with a mesh size of 60;

[0046] (2) Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus powder obtained in step (1), add 3.75 g of polyethylene glycol (average relative molecular mass is 4000), 1.50 g of ammonium sulfate and 9.75 g of water, stir for 5 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 9.4 ml of the upper-phase extract;

[0047] (3) Ultrafiltration and centrifugation purification: Dilute the upper-phase extract rich in 10-DABⅢ obtained in step (2) by 8 times, select a 1 kd ultrafiltration and centrifugation tube, and further purify it by ultrafiltration and centrifugation method. The centrifugation speed is 8000 g and the centrifugation time is 20 min to obtain 28 ml of 10-DABⅢ solution with higher purity;

[0048] (4) Freeze-drying: Freeze-dry the 10-DABⅢ solution obtained in step (3) at a vacuum degree of 20 Pa and a temperature of -40 °C for 48 h to remove water and obtain 5.0642 mg of 10-DABⅢ freeze-dried powder with a purity of 88.46%.

[0049] Example 4

[0050] (1) Raw material treatment: Take Taxus chinensis var. mairei branches and leaves, dry them, pulverize them with a pulverizer and then sieve them to obtain Taxus powder with a mesh size of 60;

[0051] (2) Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus powder obtained in step (1), add 3.75 g of polyethylene glycol (average relative molecular mass is 20000), 1.50 g of ammonium sulfate and 9.75 g of water, stir for 5 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 11.8 ml of the upper-phase extract;

[0052] (3) Ultrafiltration and centrifugation purification: Dilute the upper-phase extract rich in 10-DABⅢ obtained in step (2) by 8 times, select a 3 kd ultrafiltration and centrifugation tube, and further purify it by ultrafiltration and centrifugation method. The centrifugation speed is 8000 g and the centrifugation time is 20 min to obtain 33 ml of 10-DABⅢ solution with higher purity;

[0053] (4) Freeze-drying: Freeze-dry the 10-DABⅢ solution obtained in step (3) at a vacuum degree of 20 Pa and a temperature of -40 °C for 48 h to remove water and obtain 5.7663 mg of 10-DABⅢ freeze-dried powder with a purity of 87.84%.

[0054] Example 5

[0055] In step (2), the single-factor experiment method was used to investigate the effects of polyethylene glycol concentration, ammonium sulfate concentration, and the mass ratio of Taxus chinensis powder to the aqueous two-phase system on the extraction rate and yield of 10-DAB in the upper phase.

[0056] The average relative molecular mass of polyethylene glycol was fixed at 10,000, the mass ratio of Taxus chinensis powder to the aqueous two-phase system was 1:10, and the ammonium sulfate concentration was 8%. The effect of polyethylene glycol concentration on the extraction effect was investigated, and the results are shown in Table 1.

[0057] The average relative molecular mass of polyethylene glycol was fixed at 10,000, the mass ratio of Taxus chinensis powder to the aqueous two-phase system was 1:10, and the polyethylene glycol concentration was 20%. The effect of ammonium sulfate concentration on the extraction effect was investigated, and the results are shown in Table 2.

[0058] The average relative molecular mass of polyethylene glycol was fixed at 10,000, the ammonium sulfate concentration was 10%, and the polyethylene glycol concentration was 20%. The effect of the mass ratio of Taxus chinensis powder to the aqueous two-phase system on the extraction effect was investigated, and the results are shown in Table 3.

[0059] Table 1

[0060]

[0061] Table 2

[0062]

[0063] Table 3

[0064]

[0065] Comparative Example 1

[0066] (1) Raw material treatment: Take the branches and leaves of Taxus chinensis var. mairei, dry them, crush them with a pulverizer and sieve them to obtain Taxus chinensis powder with a mesh size of 60;

[0067] (2) Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus chinensis powder obtained in step (1), add 3.75 g of polyethylene glycol (average relative molecular mass of 10,000), 1.50 g of ammonium sulfate, and 9.75 g of water, stir for 5 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 10 ml of the upper-phase extraction solution;

[0068] (3) Freeze-drying: Freeze-dry the 10-DABⅢ upper-phase extraction solution obtained in step (2) under a vacuum of 20 Pa and a temperature of -40 °C for 48 h to remove water, obtaining 10-DABⅢ with a purity of 0.22% and 3.0218 g of polyethylene glycol freeze-dried powder.

[0069] Comparative Example 2

[0070] (1)Raw material treatment: Take the branches and leaves of Taxus chinensis var. mairei, dry them, crush them with a pulverizer and then sieve them to obtain Taxus powder with a mesh size of 60;

[0071] (2)Aqueous two-phase extraction and purification: Weigh 1.00 g of the Taxus powder obtained in step (1), add 3.75 g of anhydrous ethanol, 1.50 g of ammonium sulfate and 9.75 g of water, stir for 5 min, let it stand until the aqueous two-phase system forms, extract for 20 min, filter and then let it stand to obtain 11.5 ml of the upper-phase extraction solution;

[0072] Due to the use of ethanol, the ultrafiltration centrifugation method cannot be used to separate the target product 10-DABⅢ in the upper-phase extraction solution obtained in step (2); moreover, in the ethanol / ammonium sulfate aqueous two-phase system, 10-DABⅢ is not completely transferred to the upper phase, and the upper-phase extraction rate of 10-DABⅢ is 64.33%.

Claims

1. A method for purifying and separating 10-DABⅢ from Taxus chinensis, characterized in that: The following steps are involved: 1) Raw material processing: Take yew branches and leaves, dry them, crush them with a grinder and sieve them to obtain yew powder; 2) Two-phase aqueous extraction purification: Take yew powder, add polyethylene glycol and ammonium sulfate, then add water, stir and let stand until two-phase aqueous phase is formed, and extract to obtain the upper phase extract; 3) Ultrafiltration and centrifugation purification: After diluting the upper phase extract obtained in step 2), further purify it by ultrafiltration and centrifugation to obtain a 10-DABⅢ solution; 4) Freeze drying: freeze-dry the 10-DABⅢ solution obtained in step 3) to remove water and obtain 10-DABⅢ freeze-dried powder.

2. A method for purifying and isolating 10-DABⅢ from Taxus chinensis according to claim 1, characterized in that: In step 1), the particle size of the yew powder is 40-100 meshes.

3. A method for purifying and separating 10-DABⅢ from Taxus chinensis according to claim 1, characterized in that: In step 2), the average relative molecular mass of the polyethylene glycol is 4000 to 20000.

4. A method for purifying and isolating 10-DABⅢ from Taxus chinensis according to claim 1, characterized in that: In step 2), in the aqueous biphase formed by polyethylene glycol, ammonium sulfate and water, the mass concentration of polyethylene glycol is 15% to 25%, and the mass concentration of ammonium sulfate is 10% to 14%.

5. A method for purifying and separating 10-DABⅢ from Taxus chinensis according to claim 1, characterized in that: In step 2), the mass ratio of the aqueous two-phase formed by the yew powder, polyethylene glycol, ammonium sulfate and water is 1:10-20.

6. A method for purifying and isolating 10-DAB III from Taxus chinensis as claimed in claim 1, characterized in that: In step 2), the stirring time is 2 to 3 minutes.

7. A method for purifying and isolating 10-DAB III from Taxus chinensis as claimed in claim 1, characterized in that: In step 2), the extraction time is 20 to 30 minutes.

8. A method for purifying and isolating 10-DAB III from Taxus chinensis as claimed in claim 1, characterized in that: In step 3), the upper phase extract is diluted 6 to 8 times.

9. A method for purifying and separating 10-DABⅢ from Taxus chinensis according to claim 1, characterized in that: In step 3), the ultrafiltration centrifuge tube selected has a molecular weight cutoff of 3 kd.

10. A method for purifying and separating 10-DAB III from Taxus chinensis according to claim 1, characterized in that: In step 3), the ultrafiltration centrifugal speed is 6000~10000g, and the centrifugal time is 10~20min.

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