A method for purifying paeoniflorin-6-O'-benzenesulfonate

Abstract

The invention provides a method for purifying paeoniflorin-6-O'-benzenesulfonate. The method can reduce the purification process of paeoniflorin-6-O'-benzenesulfonate, effectively remove impurities such as benzenesulfonyl chloride and its derivatives, iron, heavy metals, etc. in the preparation of a crude product of paeoniflorin-6-O'-benzenesulfonate, and obtain paeoniflorin-6-O'-benzenesulfonate with a content of more than 98%. In addition, while ensuring a high content, the method has low energy consumption, small solvent usage, and short time consumption, and is suitable for the industrial production of paeoniflorin-6-O'-benzenesulfonate.

Description

Technical Field

[0001] The invention belongs to the technical field of compound refining, and particularly relates to a method for purifying paeoniflorin-6-O'-benzenesulfonate. Background Art

[0002] Paeoniforin (Pae) ​​is derived from plants such as peony root and peony root. It is a major component of total peony glycosides. It has a wide range of pharmacological activities, including anti-inflammatory, immunomodulatory, antipyretic, analgesic, antihypertensive, antispasmodic and anti-tumor. It has low toxicity and few adverse reactions. It has been used in the clinical treatment of rheumatoid arthritis. Although Pae has significant pharmacological effects, its poor oral absorption and slow onset of action limit its use in large doses.

[0003] Therefore, Wei Wei et al. conducted a derivatization study on Pae and found that its derivative, paeoniflorin-6-O′-benzenesulfonate, had better drug absorption in animals, showing slower drug clearance, longer retention time and higher bioavailability.

[0004] The synthesis of peonyside-6-O'-benzenesulfonate is prepared by the reaction of peonyside and benzenesulfonyl chloride. Since there are multiple hydroxyl groups on peonyside, the reaction is not selective, so the reaction generates more impurities. In the prior art, even if peonyside with a content of at least 98% is used as a raw material to prepare peonyside-6-O'-benzenesulfonate, the product is purified by silica gel chromatography. On the one hand, only silica gel separation often leaves a small amount of unreacted benzenesulfonyl chloride and some side reaction products with polarity similar to peonyside-6-O'-benzenesulfonate; on the other hand, even if peonyside-6-O'-benzenesulfonate with a good content is obtained, the high content of the reaction raw materials leads to limited production of peonyside-6-O'-benzenesulfonate. For peonyside-6-O'-benzenesulfonate with a content of 95% to 98%, multiple column chromatography will not only consume a large amount of eluent and silica gel, but also lose a large amount of sample, reducing the yield of peonyside-6-O'-benzenesulfonate. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.

[0006] To this end, the present invention proposes a purification method for paeoniflorin-6-O′-benzenesulfonate, which can reduce the purification process of paeoniflorin-6-O′-benzenesulfonate, has low energy consumption, small amount of solvent, and short time consumption while ensuring high content, and is suitable for the industrial production of paeoniflorin-6-O′-benzenesulfonate.

[0007] There are multiple hydroxyl groups on paeoniflorin, and the reaction between paeoniflorin and benzenesulfonyl chloride is not selective. Figure 1As shown, after the reaction, in addition to the main component of paeoniflorin-6-O′-benzenesulfonate, up to 15 impurities are generated. Among them, impurity 7 is the reaction product of paeoniflorin lactone glycosides remaining in paeoniflorin and benzenesulfonyl chloride, and the other 14 impurities are all impurities generated during the reaction. It can be seen that after the reaction of paeoniflorin-6-O′-benzenesulfonate is completed, the components of the reaction system are relatively complex, and it is necessary to purify it to obtain a high-content paeoniflorin-6-O′-benzenesulfonate finished product.

[0008] Therefore, the present invention provides a method for purifying paeoniflorin-6-O′-benzenesulfonate, comprising the following steps:

[0009] S1: Extraction: adding water and alkyl halide to the solution containing crude peonyside-6-O′-benzenesulfonate for primary extraction, discarding the alkyl halide layer; adding carboxylate for extraction to obtain a carboxylate layer; washing the carboxylate layer with water to obtain crude peonyside-6-O′-benzenesulfonate A;

[0010] S2: separation: separating the crude product A of paeoniflorin-6-O′-benzenesulfonate by column chromatography to obtain a crude product B of paeoniflorin-6-O′-benzenesulfonate having a content of at least 95%;

[0011] S3: Recrystallization: Recrystallize the crude product B of paeoniflorin-6-O′-benzenesulfonate in a mixed solvent to obtain paeoniflorin-6-O′-benzenesulfonate with a content of at least 98%.

[0012] Generally speaking, in the preparation process of paeoniflorin-6-O′-benzenesulfonate, the reaction system of paeoniflorin and benzenesulfonyl chloride is an acetone system. According to the present invention, a halogenated alkane is added to the reaction solution after the reaction is completed. Then, the initial extract involves a three-phase system of acetone-water-halogenated alkane, in which acetone and water are miscible, acetone and halogenated alkane are miscible, and water and halogenated alkane are immiscible, resulting in stratification.

[0013] Therefore, the present invention first adopts this three-phase system, so that the impurities in the acetone system enter the halogenated alkane layer for preliminary impurity removal. The initial extraction can effectively remove impurities 1, impurity 2, impurity 3, impurity 5, and impurity 8 in the crude peonyside-6-O'-benzenesulfonate solution, and obtain a crude peonyside-6-O'-benzenesulfonate product A with a content of at least 90%; after the crude peonyside-6-O'-benzenesulfonate product A is separated by column chromatography, impurity 7 can be removed, and the contents of impurities 4, impurity 6, impurity 9, impurity 10, impurity 12, impurity 13 and impurity 14 can be further reduced; finally, by recrystallization, impurities 11 and impurity 15 can be significantly removed, and a finished peonyside-6-O'-benzenesulfonate product with a content of at least 98% can be obtained.

[0014] In some embodiments of the present invention, the content of paeoniflorin-6-O'-benzenesulfonate in the crude paeoniflorin-6-O'-benzenesulfonate in S1 is at least 70%. For example, at least 75%, preferably at least 80%, and more preferably at least 85%; preferably, the crude paeoniflorin-6-O'-benzenesulfonate can be prepared by reacting benzenesulfonyl chloride with paeoniflorin and / or benzenesulfonyl chloride with crude paeoniflorin; more preferably, the crude paeoniflorin-6-O'-benzenesulfonate can be prepared by any technology within the prior art or within the expected range of those skilled in the art.

[0015] The technical solution of the present invention provides a ratio range, and the amount (mass fraction) of acetone is controlled to be equivalent to 2 to 5 times of paeoniflorin.

[0016] In some preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O′-benzenesulfonate to the water in S1 is 1:(2-10).

[0017] In some more preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O′-benzenesulfonate to the halogenated alkane in S1 is 1:(0.1-0.3); more preferably 1:(0.1-0.25). Within this ratio range, the three-phase system can achieve better stratification, achieve significant impurity removal and further ensure the yield.

[0018] In some more preferred embodiments of the present invention, after the primary extraction and before the carboxylic acid ester extraction in S1, a secondary extraction step is further included; the secondary extraction is to add a halogenated alkane for extraction after the primary extraction; further preferably, the amount of the halogenated alkane added in the secondary extraction does not exceed 25% of the amount of the halogenated alkane added in the primary extraction in S1. The secondary extraction is performed depending on the effect of the primary extraction. When the primary extraction effect is good, the secondary extraction may not be required.

[0019] In some more preferred embodiments of the present invention, the halogenated alkane is selected from chloroform, dichloromethane, carbon tetrachloride, dichloroethane or a combination thereof.

[0020] In some more preferred embodiments of the present invention, the carboxylate in S1 is selected from ethyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate or a combination thereof.

[0021] In some more preferred embodiments of the present invention, the mass ratio of the solution containing crude paeoniflorin-6-O′-benzenesulfonate to the carboxylate in S1 is 1:(1-10); preferably 1:(2-8).

[0022] In some more preferred embodiments of the present invention, the number of water washing in S1 is 1 to 5 times; further preferably, the number of water washing in S1 is 2 to 3 times.

[0023] In some more preferred embodiments of the present invention, the amount of water used for washing in S1 is about 1 / 2 of the amount of the carboxylic acid ester.

[0024] In some more preferred embodiments of the present invention, the eluent of the column chromatography in S2 is a halogenated alkane-alcohol mixed solvent; further preferably, the halogenated alkane is selected from chloroform, dichloromethane or a combination thereof; the alcohol is selected from lower alcohols; preferably methanol, ethanol or a combination thereof.

[0025] In some more preferred embodiments of the present invention, the volume ratio of the halogenated alkane to the alcohol is (10-15):1; further preferably (10-13):1.

[0026] In some more preferred embodiments of the present invention, the mass volume ratio of the crude paeoniflorin-6-O′-benzenesulfonate A to the eluent is 1: (45-65) g / mL; further preferably 1: (50-60) g / mL.

[0027] In some more preferred embodiments of the present invention, the recrystallization in a mixed solvent in S3 refers to dissolving the crude product B of paeoniflorin-6-O′-benzenesulfonate in a good solvent and then adding a poor solvent for crystallization.

[0028] In some more preferred embodiments of the present invention, the mass volume ratio of the crude paeoniflorin-6-O′-benzenesulfonate B to the good solvent is 1: (8-16) g / mL; more preferably 1: (10-13) g / mL.

[0029] In some more preferred embodiments of the present invention, the mass volume ratio of the crude paeoniflorin-6-O′-benzenesulfonate B to the poor solvent is 1: (5-8) g / mL.

[0030] In some more preferred embodiments of the present invention, the good solvent is selected from any one of isopropyl acetate and isobutyl acetate.

[0031] In the present invention, the crude product B of paeoniflorin-6-O'-benzenesulfonate is recrystallized by using any one of isopropyl acetate and isobutyl acetate as a good solvent, which can successfully crystallize paeoniflorin-6-O'-benzenesulfonate, while the recrystallization by using conventional solvents such as ethyl acetate will cause paeoniflorin-6-O'-benzenesulfonate to be unable to crystallize in a poor solvent or even after crystallization, its physical state is not conducive to subsequent processing. This is mainly due to the extremely high solubility of paeoniflorin-6-O'-benzenesulfonate in ethyl acetate. When the poor solvent is recrystallized, no matter how the temperature is controlled or the amount of the poor solvent is controlled, the precipitation state is always turbid or viscous paste. Further, whether it is standing, freezing or centrifuging, it is impossible to achieve a good separation of paeoniflorin-6-O'-benzenesulfonate from the solvent, which is extremely unfavorable for the subsequent filtering, drying and other processes of paeoniflorin-6-O'-benzenesulfonate. On the contrary, through screening of a large number of solvent types, dosages, crystallization conditions, etc., the inventors unexpectedly discovered that only isopropyl acetate and isobutyl acetate are capable of achieving the recrystallization effect of paeoniflorin-6-O′-benzenesulfonate, that is, when encountering a poor solvent, crystallization can be completed in a crystalline or flocculent state, which can be well separated from the solvent system, facilitating subsequent filtration, drying and other processes.

[0032] In some more preferred embodiments of the present invention, the poor solvent is selected from n-hexane, n-heptane, petroleum ether, isopropyl ether, isooctane or a combination thereof.

[0033] In some more preferred embodiments of the present invention, the dissolving is heating dissolving, and preferably, the dissolving temperature is 30°C to 70°C.

[0034] In some more preferred embodiments of the present invention, the crystallization temperature is 10°C to 30°C; further preferably, the crystallization time is not less than 1 hour.

[0035] In some more preferred embodiments of the present invention, S3 further comprises a step of further purification after the recrystallization.

[0036] In some more preferred embodiments of the present invention, the purification is carried out by filtration and washing; further preferably, the filtration is selected from any one of vacuum filtration, pressure filtration, and centrifugal filtration; the washing is carried out by the poor solvent; further preferably, the washing is carried out 2 to 4 times.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention improves the reaction conditions and adopts a new purification and refining process. The purification method of paeoniflorin-6-O′-benzenesulfonate provided by the present invention can effectively remove impurities such as benzenesulfonyl chloride and its derivatives, iron, heavy metals, etc. in the preparation of paeoniflorin-6-O′-benzenesulfonate crude product, and obtain paeoniflorin-6-O′-benzenesulfonate with a content of more than 98%.

[0039] 2. The method for purifying paeoniflorin-6-O′-benzenesulfonate provided by the present invention can purify crude paeoniflorin-6-O′-benzenesulfonate with a paeoniflorin-6-O′-benzenesulfonate content of more than 70%, and obtain paeoniflorin-6-O′-benzenesulfonate with a content of at least 98%. Therefore, it has a wide range of applications and has no high requirements on the content of raw materials, thereby reducing the production cost of paeoniflorin-6-O′-benzenesulfonate.

[0040] 3. The purification method of paeoniflorin-6-O′-benzenesulfonate provided by the present invention has fewer steps and takes less time, and can be easily applied to the industrial production of paeoniflorin-6-O′-benzenesulfonate. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0042] Figure 1 This is the HPLC test result of crude paeoniflorin-6-O′-benzenesulfonate S1 in Test Example 1 of the present invention.

[0043] Figure 2 The HPLC test results of the products of each step of Test Example 1 of the present invention are shown.

[0044] Figure 3 The figure shows the effect of different proportions of chloroform on the content and yield of paeoniflorin-6-O′-benzenesulfonate in Experimental Example 2 of the present invention.

[0045] Figure 4 This is the HPLC test result after refinement of S4 ethyl acetate and isopropyl acetate in Test Example 3 of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0047] Example 1

[0048] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0049] S1: Take 10 g of paeoniflorin with a content of 76.3%, add 50 mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 71.5%.

[0050] S2: Add 100mL purified water and 3mL chloroform to the reaction solution for the first extraction, and extract for 50min at room temperature. After 50min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 0.6mL chloroform to the reaction solution for the second extraction, extract for 30min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 100mL ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 50mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing, and obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 80.4%. The extraction step is now complete.

[0051] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with chloroform-methanol (10:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 95.2%.

[0052] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 100 mL of isobutyl acetate at 65°C to dissolve it, cool it to 25°C after dissolution, add 50 mL of isopropyl ether, and after all paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final finished product of paeoniflorin-6-O′-benzenesulfonate with a content of 98.1%.

[0053] Example 2

[0054] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0055] S1: Take 10 g of paeoniflorin with a content of 98%, add 50 mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 90.2%.

[0056] S2: Add 100mL purified water and 3mL chloroform to the reaction solution for the first extraction, and extract for 50min at room temperature. After 50min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 0.6mL chloroform to the reaction solution for the second extraction, extract for 30min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 100mL ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 50mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing, and obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 94.4%. The extraction step is now complete.

[0057] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with chloroform-methanol (10:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 98.3%.

[0058] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 100 mL of isobutyl acetate at 65°C to dissolve it, cool it to 25°C after dissolution, add 50 mL of isopropyl ether, and after all paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final finished product of paeoniflorin-6-O′-benzenesulfonate with a content of 99.6%.

[0059] Example 3

[0060] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0061] S1: Take 20 g of paeoniflorin with a content of 84.5%, add 120 mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 79.2%.

[0062] S2: Add 150mL purified water and 3mL chloroform to the reaction solution for the first extraction, and extract for 40min at room temperature. After 40min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 0.3mL chloroform to the reaction solution for the second extraction, extract for 20min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 160mL ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 80mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 83.9%. The extraction step is now complete.

[0063] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with dichloromethane-methanol (15:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 95.7%.

[0064] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 320 mL of isobutyl acetate at 55°C to dissolve it, cool it to 20°C after dissolution, add 120 mL of n-hexane, and after all the paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final finished product of paeoniflorin-6-O′-benzenesulfonate with a content of 98.4%.

[0065] Example 4

[0066] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0067] S1: Take 100 g of paeoniflorin with a content of 88.3%, add 500 mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 85.6%.

[0068] S2: Add 100mL purified water and 5mL chloroform to the reaction solution for the first extraction, and extract for 35min at room temperature. After 35min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 0.75mL chloroform to the reaction solution for the second extraction, extract for 10min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 100mL ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 50mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 91.8%, and the extraction step is completed.

[0069] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with chloroform-methanol (13:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 96.0%.

[0070] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 1200 mL of isobutyl acetate at 45° C. to dissolve it, cool it to 10° C. after dissolution, add 500 mL of n-heptane, and after all the paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final paeoniflorin-6-O′-benzenesulfonate product with a content of 98.8%.

[0071] Example 5

[0072] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0073] S1: Take 500 g of paeoniflorin with a content of 88.3%, add 2.5 L of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 84.9%.

[0074] S2: Add 1.5L purified water and 450mL chloroform to the reaction solution for the first extraction, and extract for 10 minutes at room temperature. After 10 minutes, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 45mL chloroform to the reaction solution for the second extraction, extract for 15 minutes at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 1.5L ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 750mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 90.6%. The extraction step is now complete.

[0075] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with chloroform-methanol (10:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 95.5%.

[0076] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 5L of isopropyl acetate at 35°C to dissolve it, cool it to 15°C after dissolution, add 3L of isopropyl ether, and after all the paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final finished product of paeoniflorin-6-O′-benzenesulfonate with a content of 98.6%.

[0077] Example 6

[0078] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0079] S1: Take 1 kg of paeoniflorin with a content of 92.5%, add 5 L of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 88.1%.

[0080] S2: Add 5L purified water and 750mL chloroform to the reaction solution for the first extraction, and extract at room temperature for 15 minutes. After 15 minutes, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 37.5mL chloroform to the reaction solution for the second extraction, extract at room temperature for 20 minutes, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 5L ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 2.5L purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 93.1%, and the extraction step is completed.

[0081] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with chloroform-methanol (14:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 97.5%.

[0082] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 12L of isopropyl acetate at 40°C to dissolve it, cool it to 25°C after dissolution, add 7L of isooctane, and after all the paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final paeoniflorin-6-O′-benzenesulfonate product with a content of 99.3%.

[0083] Example 7

[0084] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0085] S1: Take 10 kg of paeoniflorin with a content of 92.5%, add 50 L of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 87.4%.

[0086] S2: Add 100L purified water and 3L chloroform to the reaction solution for the first extraction, and extract for 60min at room temperature. After 60min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 0.75L chloroform to the reaction solution for the second extraction, extract for 60min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 100L ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 50L purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 92.5%, and the extraction step is completed.

[0087] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with dichloromethane-methanol (12:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 96.9%.

[0088] S4: Heat the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with 80 L of isopropyl acetate at 45° C. to dissolve it, cool it to 30° C. after dissolution, add 80 L of n-hexane, and after all the paeoniflorin-6-O′-benzenesulfonate is precipitated, filter it, and dry it to obtain the final paeoniflorin-6-O′-benzenesulfonate product with a content of 99.0%.

[0089] Example 8

[0090] This example prepares a peonyside-6-O′-benzenesulfonate, and the specific process is as follows:

[0091] S1: Take 100 kg of paeoniflorin with a content of 90%, add 500 L of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react, monitor the reaction, and the obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate with a content of 85.4%.

[0092] S2: Add 1000L purified water and 30L chloroform to the reaction solution for the first extraction, and extract for 60min at room temperature. After 60min, discard the lower layer (chloroform layer) and retain the upper layer (acetone-water). Add 7.5L chloroform to the reaction solution for the second extraction, extract for 60min at room temperature, discard the lower layer (chloroform layer), and continue to retain the upper layer (acetone-water). Add 1000L ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 500L purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, with a content of 91.4%, and the extraction step is completed.

[0093] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction was separated by column chromatography and eluted with dichloromethane-methanol (12:1) to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate with a content of 96.5%.

[0094] S4: Heat 800L of isopropyl acetate at 55°C to dissolve the crude product 2 of paeoniflorin-6-O′-benzenesulfonate, cool to 30°C after dissolution, add 500L of isooctane, and after all paeoniflorin-6-O′-benzenesulfonate is precipitated, filter and dry to obtain the final finished product of paeoniflorin-6-O′-benzenesulfonate with a content of 98.7%.

[0095] Test Example 1

[0096] This test example monitors the impurity removal effect during the refining process of paeoniflorin-6-O′-benzenesulfonate. Samples are taken for HPLC monitoring after the reaction, primary extraction, secondary extraction, column chromatography separation, and recrystallization, with a focus on monitoring the changes in impurities in the material. The results are as follows: Figure 1 and Figure 2 The specific process is as follows:

[0097] S1: Take 90% paeoniflorin (containing 1 g of paeoniflorin), add 4 mL of acetone, and simultaneously add benzenesulfonyl chloride, triethylamine, etc. to react. The obtained reaction solution contains crude paeoniflorin-6-O′-benzenesulfonate, and sampling is performed for HPLC monitoring.

[0098] S2: Add 4.5mL purified water and 0.2mL chloroform to the reaction solution for the first extraction, and extract for 30min at room temperature. After 30min, discard the lower layer (chloroform layer), retain the upper layer (acetone-water), and collect samples for HPLC monitoring. Add 0.04mL chloroform to the reaction solution for the second extraction, extract for 30min at room temperature, discard the lower layer (chloroform layer), continue to retain the upper layer (acetone-water), and collect samples for HPLC monitoring. Add 5mL ethyl acetate to the above acetone-water layer for full extraction, discard the water layer after standing, and retain the organic layer (ethyl acetate). Add 2.5mL purified water to the above ethyl acetate layer and wash twice, discard the water layer after standing to obtain the crude product 1 of paeoniflorin-6-O′-benzenesulfonate, and the extraction step is completed.

[0099] S3: The crude product 1 of paeoniflorin-6-O′-benzenesulfonate obtained after extraction is separated by column chromatography to obtain the crude product 2 of paeoniflorin-6-O′-benzenesulfonate, and sampling is performed for HPLC monitoring.

[0100] S4: Dissolve the crude product 2 of paeoniflorin-6-O′-benzenesulfonate 2 by heating with 15 mL of isopropyl acetate at 50°C, cool to 30°C after dissolution, add 7.5 mL of n-hexane, and after all paeoniflorin-6-O′-benzenesulfonate is precipitated, filter and dry to obtain the final product of paeoniflorin-6-O′-benzenesulfonate, and take samples for HPLC monitoring.

[0101] Figure 1 and Figure 2It shows that after the paeoniflorin extract reacts with benzenesulfonyl chloride, a total of 15 impurities are generated, of which except for impurity 7 which is a known impurity (peoniflorin-6-O′-benzenesulfonate), the remaining 14 impurities are unknown impurities after the reaction. From the research results, the extraction process of the technical solution of the present invention has a good removal effect on impurities 1, 2, 3, 5, and 8 after the reaction of paeoniflorin-6-O′-benzenesulfonate. After extraction, the types of impurities are reduced from 15 to 11, among which impurity 1 is reduced from 3.69% to 0.23%. Overall, the extraction method can obviously remove the impurity types before the main peak, while the removal effect of the impurity types after the main peak is poor (except impurity 8). It can be seen that the refining effect brought by the extraction method is mainly concentrated before the main peak.

[0102] After extraction, the content of paeoniflorin-6-O′-benzenesulfonate can reach more than 91%, but only impurities 2, 3, 5, and 8 are completely removed. Through further column chromatography separation, impurities 1, 4, 9, 10, 12, and 13 can be further removed. Among them, impurity 10 is reduced from 0.44% after extraction to 0.13% after column chromatography. Column chromatography separation is mainly used to remove impurity 7, i.e., paeoniflorin-6-O′-benzenesulfonate. The results show that through column chromatography separation, impurity 7 is reduced from the original 1.06% to 0.48%. Other impurities are reduced to below the detection limit due to their own small content. After column chromatography, the content of paeoniflorin-6-O′-benzenesulfonate reaches more than 95%, and the types of impurities are further reduced from the original 15 to 6. Studies have shown that the column chromatography process is mainly used to reduce the content of impurity 7, i.e., paeoniflorin-6-O′-benzenesulfonate, and can further reduce the content of impurity 4, impurity 6, impurity 9, impurity 10, impurity 12, impurity 13, and impurity 14.

[0103] It can be clearly seen from the above results that neither extraction nor column chromatography can effectively reduce impurities 11 and 15. Therefore, in order to obtain a content of more than 98% of paeoniflorin-6-O′-benzenesulfonate, a recrystallization step is required to remove impurities 11 and 15. After recrystallization, the contents of impurities 11 and 15 are significantly reduced, among which impurity 15 can be completely removed, and the content of impurity 11 is significantly reduced from the original 2.56% to 0.65%, and the content of paeoniflorin-6-O′-benzenesulfonate reaches more than 99%.

[0104] In summary, the purification method of paeoniflorin-6-O'-benzenesulfonate of the present invention can finally obtain a finished product of paeoniflorin-6-O'-benzenesulfonate with a content of more than 98%. The extraction technology has an obvious removal effect on impurities 1, 2, 3, 5, and 8, and column chromatography can further reduce the content of impurities 4, 6, 9, 10, 12, 13, and 14, among which the removal effect on impurity 7, i.e., paeoniflorin-6-O'-benzenesulfonate, is the most obvious, and impurities 11, 14 and impurity 15 are further reduced by recrystallization.

[0105] Test Example 2

[0106] The technical solution of the present invention provides a method for purifying paeoniflorin-6-O′-benzenesulfonate, which involves a solvent extraction step. It can be seen from the above test example 1 that the extraction process can effectively remove impurities before the main peak and impurity 8. Considering that in the extraction process, the entire system is a ternary system, involving a reaction solvent, an extraction solvent and purified water. Since acetone and water can dissolve each other, acetone and halogenated alkanes can dissolve each other, and water and halogenated alkanes cannot dissolve each other. Therefore, adjusting the ratio of acetone-water-halogenated alkanes can effectively improve the effect of the initial extraction, so that it can be well layered and ensure obvious impurity removal and a higher yield. Therefore, this test example investigates the ratio of halogenated alkanes in acetone-water-halogenated alkanes, and uses content and yield as investigation indicators to screen the optimal amount of halogenated alkanes.

[0107] According to the experimental scheme of Experimental Example 1, taking chloroform as an example, the suitable dosage ratio of halogenated alkanes in the extraction process was screened by single factor investigation method. Based on the total volume of chloroform and purified water, the amount of chloroform added was set to different proportions of 5%, 10%, 15%, 20%, 25%, and 30%. Considering that the recrystallization process is mainly for impurities 11 and 15, and has little effect on the yield, the experimental steps are terminated after column chromatography, and other operations are the same as in Effect Example 1. The yield and content of samples obtained in different groups are measured respectively. The results are as follows Figure 3 shown.

[0108] according to Figure 3It can be clearly seen from the results that the chloroform ratio shows a negative correlation trend for the content and yield of paeoniflorin-6-O′-benzenesulfonate (sample after column chromatography). With the increase of chloroform dosage, the extraction effect is significantly improved, but at the same time the yield drops sharply. The main reason for using chloroform for extraction is that the impurities before the main peak can be dissolved in chloroform, but at the same time, paeoniflorin-6-O′-benzenesulfonate can also be dissolved in chloroform. Therefore, with the increase of chloroform dosage, more and more impurities are dissolved in the chloroform layer, but at the same time, a large amount of paeoniflorin-6-O′-benzenesulfonate can be dissolved in chloroform. This results in that with the increase of chloroform dosage, the sample content is significantly improved, but the yield also drops sharply. Therefore, in order to balance the relationship between yield and content, a suitable chloroform ratio needs to be adopted.

[0109] Depend on Figure 3 It can be clearly seen that when the chloroform ratio exceeds 25%, the degree of increasing the content of paeoniflorin-6-O'-benzenesulfonate is significantly reduced. For example, when the chloroform dosage is 25%, the content of paeoniflorin-6-O'-benzenesulfonate is 96.13%. When the chloroform ratio is further increased to 30%, the content is 96.86%, and the content is only increased by 0.73%, but the yield is lost by nearly 20%. Therefore, from the perspective of content and yield, the chloroform ratio in the acetone-water-chloroform three-phase system is preferably controlled at 10% to 25%, and 30% chloroform can also meet the content requirements, but its yield is relatively low.

[0110] Test Example 3

[0111] The technical solution of the present invention provides a method for purifying paeoniflorin-6-O′-benzenesulfonate, which involves a solvent refining step. It can be seen from the above Experimental Example 1 that the extraction process can effectively remove impurities 11, 14 and impurity 15. Using any one of isopropyl acetate and isobutyl acetate as a good solvent to recrystallize the crude product B of paeoniflorin-6-O′-benzenesulfonate can successfully crystallize paeoniflorin-6-O′-benzenesulfonate, while using conventional solvents such as ethyl acetate for recrystallization will cause paeoniflorin-6-O′-benzenesulfonate to be unable to crystallize in a poor solvent or its physical state after precipitation is turbid or viscous paste. Further, whether it is standing, freezing or centrifuging, it is impossible to achieve a good separation of paeoniflorin-6-O′-benzenesulfonate from the solvent, and it is difficult to increase the content of paeoniflorin-6-O′-benzenesulfonate to more than 98% (such as Figure 4 shown).

[0112] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for purifying paeoniflorin-6-O'-benzenesulfonate, characterized in that: The following steps are involved: S1: extraction: adding water and alkyl halide to a solution containing crude peonyside-6-O'-benzenesulfonate for primary extraction, discarding the alkyl halide layer; adding carboxylate for extraction to obtain a carboxylate layer; washing the carboxylate layer with water to obtain a crude peonyside-6-O'-benzenesulfonate product A; the crude peonyside-6-O'-benzenesulfonate has a peonyside-6-O'-benzenesulfonate content of at least 75%; S2: separation: separating the crude product A of paeoniflorin-6-O'-benzenesulfonate by column chromatography to obtain a crude product B of paeoniflorin-6-O'-benzenesulfonate having a content of at least 95%; S3: Recrystallization: dissolving the crude product B of paeoniflorin-6-O'-benzenesulfonate in a good solvent, and then adding a poor solvent for crystallization to obtain paeoniflorin-6-O'-benzenesulfonate with a content of at least 98%; The mass volume ratio of the crude product B of paeoniflorin-6-O'-benzenesulfonate to the good solvent is 1: (8-16) g / mL; the good solvent is selected from isopropyl acetate, isobutyl acetate or a combination thereof; The mass volume ratio of the crude product B of paeoniflorin-6-O'-benzenesulfonate to the poor solvent is 1: (5-8) g / mL; the poor solvent is selected from n-hexane, n-heptane, petroleum ether, isopropyl ether, isooctane or a combination thereof.

2. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The solution containing crude paeoniflorin-6-O'-benzenesulfonate in S1 refers to an acetone solution.

3. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate in S1 to the water is 1:(2-10).

4. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate in S1 to the halogenated alkane is 1:(0.1-0.3).

5. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: In S1, after the primary extraction and before the carboxylic acid ester extraction, a secondary extraction step is also included; the secondary extraction is to add halogenated alkane for extraction after the primary extraction.

6. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The halogenated alkane is selected from chloroform, dichloromethane, carbon tetrachloride, dichloroethane or a combination thereof.

7. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The mass ratio of the solution containing crude paeoniflorin-6-O'-benzenesulfonate to the carboxylate in S1 is 1:(1-10); the carboxylate is selected from ethyl acetate, propyl acetate, and butyl acetate.

8. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 1, characterized in that: The eluent for the column chromatography in S2 is a halogenated alkane-alcohol mixed solvent.

9. The method for purifying paeoniflorin-6-O'-benzenesulfonate according to claim 8, characterized in that: The volume ratio of the halogenated alkane to the alcohol is (8-16):1.

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