Method for removing polysaccharides and polyphenols in saponin of gleditsia sinensis

Abstract

The method comprises the following steps: adding a non-alcohol polar solvent into a concentrated soapberry bark powder water extract, stirring at low temperature to separate out impurities, and filtering to obtain a soapberry saponin concentrated solution from which the polysaccharide and polyphenol are removed. According to the method, a non-alcohol polar solvent low-temperature precipitation mode is adopted, and polysaccharides and polyphenols in the saponin of the gleditsia sinensis can be effectively removed. Part of polysaccharides and polyphenols are removed from the obtained saponin concentrated solution, the risk of blocking a preparative column can be reduced, solvent replacement is not needed, the influence on the saponin concentration in the process is small, and saponin compounds such as QS-21 and the like can be directly prepared.

Description

Technical Field

[0001] This invention belongs to the field of vaccine adjuvant preparation, specifically relating to a method for removing polysaccharides and polyphenols from saponins. Background Technology

[0002] Saponins are mainly extracted from the bark of the South American soap tree (Quillaja saponaria Molinatree). They exhibit significant immune adjuvant activity, such as QS-7, QS-17, QS-18, and QS-21. Among them, QS-21 is a key component of immune adjuvants for vaccines and has been used in adjuvants such as AS01 and AS02.

[0003] Saponins from soap trees are mostly water-soluble substances, primarily extracted from soap bark using water extraction. However, the water extract contains a large number of impurities such as pigments, polysaccharides, and polyphenols that need to be removed. If subsequent separation of saponin compounds is required, these impurities also need to be pretreated and removed before separation. Failure to remove them can easily clog the preparation column and severely damage the preparation system.

[0004] According to relevant literature and patent reports, the main methods for removing polysaccharides and polyphenols from saponins are as follows: CN111372604A mentions a pretreatment process for soap bark extract, mainly using cross-linked polyvinylpyrrolidone (PVPP) adsorption under acidic conditions to remove most of the impurities such as pigments, polysaccharides, and polyphenols. After obtaining the pretreated extract, subsequent separation and preparation of related saponin compounds are carried out. However, if a large amount of PVPP is added during the adsorption process, the filtration operation becomes difficult.

[0005] CN118852314A discloses a method for preparing saponin 21 from saponins. The method involves pretreatment of saponin raw materials by adding an alcohol solvent with a volume fraction of 75% to 90% to remove insoluble substances (polysaccharides, proteins, tannins, polyphenols, etc.) to obtain pretreated and purified substances. Saponin 21 is then separated and prepared. However, the addition of alcohol solvent requires solvent replacement. The preparation and separation of saponins mainly uses acetonitrile as the eluent, which has a significant impact on subsequent operations and is not easy to recover and reuse.

[0006] The method for extracting saponins from plants disclosed in WO2017 / 091333A1 mentions a processing technique for extracting saponins from soap bark. This technique primarily involves adding calcium salts under alkaline conditions to adsorb and remove polysaccharides, followed by adding cross-linked polyvinylpyrrolidone (PVPP) under acidic conditions to adsorb and remove remaining polysaccharides and polyphenols, and then using nanofiltration to remove other impurities. This yields saponins with a purity of 83-91%. This process is relatively cumbersome, the filtration operation is difficult, and it introduces metal ions, which is not conducive to industrial production. Summary of the Invention

[0007] To address the above problems, this invention provides a method for removing polysaccharides and polyphenols from saponins. This method uses a non-alcoholic polar solvent for low-temperature precipitation, which can effectively remove polysaccharides and polyphenols from saponins.

[0008] The objective of this invention is achieved through the following solution: A method for removing polysaccharides and polyphenols from saponins, comprising the following steps: A non-alcoholic polar solvent was added to the concentrated soap bark powder aqueous extract, and after stirring at low temperature to precipitate impurities, the mixture was filtered to obtain a concentrated soap saponin solution with polysaccharides and polyphenols removed.

[0009] The saponin concentrate obtained by this invention, which has had polysaccharides and polyphenols removed, can be directly used to prepare saponin compounds such as QS-21.

[0010] Preferably, the concentrated soap bark powder aqueous extract is obtained by water extraction, filtration and concentration of soap bark powder, with 1L of the concentrated soap bark powder aqueous extract formed from 1kg of soap bark powder.

[0011] More preferably, the concentrated soapberry bark powder aqueous extract is prepared by the following method: Add soap bark powder to water and extract it by stirring at 50-70°C, preferably 60-70°C, for 1-10 hours, preferably 2-6 hours. Then filter the solution and concentrate the filtrate under reduced pressure to obtain the final product.

[0012] More preferably, the weight of the water is 10 to 30 times the weight of the soap bark powder, and even more preferably 10 to 20 times.

[0013] Preferably, the non-alcoholic polar solvent is one of acetonitrile, acetone, or tetrahydrofuran, with acetonitrile being the most preferred.

[0014] Preferably, the temperature of the low-temperature stirring is 0~15℃, more preferably 5~10℃.

[0015] Preferably, the volume of the non-alcoholic polar solvent is 0.7 to 1.5 times that of the concentrated soap bark powder aqueous extract.

[0016] The beneficial effects of this invention are as follows: 1. This invention uses a non-alcoholic polar solvent for low-temperature precipitation, which can effectively remove polysaccharides and polyphenols from saponins in soap trees.

[0017] 2. The saponin concentrate obtained by this invention removes some polysaccharides and polyphenols, which can reduce the risk of column blockage and eliminates the need for solvent replacement. The process has little impact on the saponin concentration and can be used to directly prepare saponin compounds such as QS-21. Attached Figure Description

[0018] Figure 1 This is the UPLC spectrum of the aqueous extract of soap bark powder obtained in Example 1.

[0019] Figure 2 This is the UPLC spectrum of the concentrated soap bark powder aqueous extract obtained in Example 1.

[0020] Figure 3 This is the UPLC spectrum of the concentrated saponin solution obtained in Example 1.

[0021] Figure 4 This is the UPLC spectrum of the concentrated saponin solution obtained in Example 2.

[0022] Figure 5 This is the UPLC spectrum of the concentrated saponin solution obtained in Example 3.

[0023] Figure 6 This is the UPLC spectrum of the concentrated saponin solution obtained in Example 4. Detailed Implementation

[0024] The following specific embodiments illustrate the technical solution of the present invention, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained through market procurement.

[0025] Example 1 (1) Add 4 kg of soap bark powder to a reaction vessel, add 80 kg of purified water for water extraction, heat to 70℃, and keep warm for 6 hours. The water-soluble substances are basically dissolved and extracted. Filter to obtain the soap bark powder water extract. Detect the extract using ultra-high performance liquid chromatography (UPLC). Figure 1 The effective saponin concentration is approximately 3.59 mg / ml, and the QS-21 concentration is approximately 0.29 mg / ml.

[0026] (2) The obtained soap bark powder aqueous extract was concentrated to 4L under reduced pressure and detected by ultra-high performance liquid chromatography (UPLC). Figure 2The effective saponin concentration was approximately 34.45 mg / ml, and the QS-21 concentration was approximately 2.84 mg / ml. Acetonitrile, at a volume equal to one unit of the concentrated solution volume, was added. The mixture was stirred at 5-10°C until insoluble impurities precipitated, then filtered to obtain a concentrated saponin solution. Detection was performed using ultra-high performance liquid chromatography (UPLC). Figure 3 The effective saponin concentration was approximately 14.94 mg / ml, with a saponin yield of approximately 86.72%. The QS-21 concentration was approximately 1.09 mg / ml, with a QS-21 yield of approximately 76.39%. This concentrated solution was directly used for subsequent QS-21 preparation. The mobile phase used in the preparation process was acetonitrile and water, therefore solvent replacement was not required, and direct injection was possible. The injection system functioned normally with no insoluble precipitation, and the preparative chromatography operated normally. After preparation, QS-21 with a purity of over 80% was obtained.

[0027] Example 2 (1) Take 5 kg of soap bark powder and add it to the reaction vessel. Add 90 kg of purified water for water extraction. Heat the water to 70°C and keep it warm for 3 hours. The water-soluble substances are basically dissolved and extracted. Filter to obtain soap bark powder water extract. According to the test, the effective saponin concentration is about 3.63 mg / ml.

[0028] (2) The obtained soap bark powder aqueous extract was concentrated to 5L under reduced pressure. The effective saponin concentration was calculated to be approximately 34.32 mg / ml. 0.7 times acetonitrile was added, and the mixture was stirred at a low temperature of 5-10℃. After the insoluble impurities precipitated, the mixture was filtered to obtain the concentrated soap bark powder solution. The solution was analyzed by ultra-high performance liquid chromatography (UPLC) as shown in the figure. Figure 4 The effective saponin concentration is approximately 19.56 mg / ml, and the saponin yield is approximately 94.65%.

[0029] Example 3 (1) Take 5 kg of soap bark powder and add it to the reaction vessel. Add 100 kg of purified water for water extraction. Heat the water to 70°C and keep it warm for 3 hours. The water-soluble substances are basically dissolved and extracted. Filter to obtain soap bark powder water extract. According to the test, the effective saponin concentration is about 3.58 mg / ml.

[0030] (2) The obtained soap bark powder aqueous extract was concentrated to about 5L under reduced pressure. The effective saponin concentration was calculated to be approximately 34.43 mg / ml. 1.5 times the amount of acetonitrile was added, and the mixture was stirred at a low temperature of 5-10℃. After the insoluble impurities precipitated, the mixture was filtered to obtain the concentrated soap saponin solution. The solution was analyzed by ultra-high performance liquid chromatography (UPLC). Figure 5 The effective saponin concentration is approximately 11.62 mg / ml, and the saponin yield is approximately 84.41%.

[0031] Example 4 (1) Take 5 kg of soap bark powder and add it to the reaction vessel. Add 75 kg of purified water for water extraction. Heat the water to 70°C and keep it warm for 3 hours. The water-soluble substances are basically dissolved and extracted. Filter to obtain soap bark powder water extract. According to the test, the effective saponin concentration is about 3.71 mg / ml.

[0032] (2) The obtained soap bark powder aqueous extract was concentrated to 5L under reduced pressure. The effective saponin concentration was calculated to be approximately 34.45 mg / ml. 2.3 times the amount of acetonitrile was added, and the mixture was stirred at a low temperature of 5-10℃. After the insoluble impurities precipitated, the mixture was filtered to obtain a concentrated soap saponin solution. The solution was analyzed by ultra-high performance liquid chromatography (UPLC). Figure 6 The effective saponin concentration was approximately 6.36 mg / ml, with a saponin yield of approximately 61.55%.

[0033] Using the method of this invention to remove polysaccharides and polyphenols from saponins, the resulting solution removes some of the polysaccharides and polyphenols, which can reduce the risk of column blockage and eliminates the need for solvent replacement. The process has little impact on the saponin concentration and can directly prepare saponin compounds such as QS-21. The purification method of this invention is simple to operate and has high fault tolerance.

Claims

1. A method for removing polysaccharides and polyphenols from saponins, characterized in that, Includes the following steps: A non-alcoholic polar solvent was added to the concentrated soap bark powder aqueous extract, and after stirring at low temperature to precipitate impurities, the mixture was filtered to obtain a concentrated soap saponin solution with polysaccharides and polyphenols removed.

2. The method for removing polysaccharides and polyphenols from saponins according to claim 1, characterized in that, The concentrated soap bark powder aqueous extract is obtained by water extraction, filtration and concentration of soap bark powder, with 1L of the concentrated soap bark powder aqueous extract formed by 1kg of soap bark powder.

3. The method for removing polysaccharides and polyphenols from saponins according to claim 2, characterized in that, The concentrated soapberry bark powder aqueous extract is prepared by the following method: Add soap bark powder to water and stir at 50-70℃ for 1-10 hours for water extraction. Then filter, take the filtrate and concentrate under reduced pressure to obtain the final product.

4. The method for removing polysaccharides and polyphenols from saponins according to claim 3, characterized in that, The stirring temperature is 60~70℃.

5. The method for removing polysaccharides and polyphenols from saponins according to claim 3, characterized in that, The stirring time is 2-6 hours.

6. The method for removing polysaccharides and polyphenols from saponins according to claim 1, characterized in that, The weight of the water is 10 to 30 times the weight of the soap bark powder.

7. The method for removing polysaccharides and polyphenols from saponins according to claim 6, characterized in that, The weight of the water is 10 to 20 times the weight of the soap bark powder.

8. The method for removing polysaccharides and polyphenols from saponins according to claim 1, characterized in that, The non-alcoholic polar solvent is acetonitrile, acetone, or tetrahydrofuran, with acetonitrile being preferred.

9. The method for removing polysaccharides and polyphenols from saponins according to claim 1, characterized in that, The temperature of the low-temperature stirring is 0~15℃, preferably 5~10℃.

10. The method for removing polysaccharides and polyphenols from saponins according to claim 1, characterized in that, The volume of the non-alcoholic polar solvent is 0.7 to 1.5 times that of the concentrated soap bark powder aqueous extract.

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