Preparation method of triterpenoid saponin

Abstract

The invention discloses a preparation method of triterpenoid saponin, which comprises the following steps: firstly, taking saponin as an initial raw material, dissolving the saponin raw material by using a dissolving solution, and filtering to remove insoluble impurities in the solution; and diluting the saponin dissolved and filtered sample, loading the diluted saponin dissolved and filtered sample to a polymer chromatographic medium, carrying out linear gradient elution on a target peak from 30% of B phase to 50% of B phase, and collecting the elution peak, so as to obtain a one-step purified solution. According to the method, VQ-SAP is used as a raw material, the recovery rate of QS-21 is 50% or above through two-step chromatography, the purity of QS-21 is 97% or above, and compared with QS-21 of DK, the product does not contain isomers with molecular weights of 1865 and 1988. The method not only can be used for efficiently preparing the triterpenoid saponin with high peak product purity, but also is suitable for saponin raw materials from various sources. The QS-21 obtained through the method is high in purity and recovery rate, raw materials are easy to obtain, meanwhile, the product safety is higher, the production cost is lower, and the international monopoly from raw materials to finished products is broken.

Description

Technical Field

[0001] This invention relates to the field of adjuvant preparation technology, specifically a method for preparing triterpenoid saponins. Background Technology

[0002] Adjuvants enhance immunogenicity by initiating and modulating innate inflammatory and adaptive immune responses. These components play a crucial role in determining the kinetics, breadth, and depth of acute vaccine responses, as well as the quality and duration of memory immune responses. Adjuvants facilitate the development of precise vaccines tailored to specific immune mechanisms, significantly enhancing antigenic protective efficacy and broadening the therapeutic scope of vaccines, including for newborns, the elderly, and immunocompromised individuals.

[0003] Despite the recognized advantages of adjuvants, only a few sufficiently effective adjuvants with acceptable levels of toxicity are used regularly. The most effective adjuvant used in licensed vaccines is an adjuvant formulated with QS-21 saponin. AS01 and ALFQ-containing adjuvant mixtures are liposomal formulations composed of QS-21 and 3-O-deacylated-4′-monophosphoryllipid A (MPLA); QS saponins (including QS-21) were initially identified and isolated from the aqueous extract of QS bark, primarily from soap trees growing at the foot of the Chilean Andes. These complex natural products consist of a hydrophobic triterpenoid core, aliphatic side chains, and complex, well-defined carbohydrate functional groups. However, the source of raw materials for the domestic production of QS saponins (including QS-21) is currently limited by the tree species (Chilean soapberry) and processing techniques. The lack of corresponding domestic preparation methods leads to a monopoly of raw materials by international manufacturers, hindering supply security and thus impacting the industry's development.

[0004] To address this problem, a method for preparing triterpenoid saponins is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a simple, efficient, high-capacity, low-cost, and high-purity method for preparing triterpenoid saponins (QS-21) to break the foreign technology monopoly and ensure the effective supply of QS saponin raw materials and auxiliary materials in China.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing triterpenoid saponins, comprising the following steps: Step 1: Dissolving and filtering saponin powder: Using saponin saponins as the starting material, the saponin raw material is dissolved in a dissolving solution and the insoluble impurities inside the solution are removed by filtration. Step 2: One-step chromatography: Take the saponin dissolution and filtration solution obtained in Step 1, and separate the triterpenoid saponins according to the following steps: First, sample loading: Dissolve the saponin, filter the sample, dilute it, and then load it onto the polymer chromatography medium; Elution followed: The target peak was eluted with a linear gradient from 30% B phase to 50% B phase, and the elution peaks were collected to obtain a one-step purified solution; Step 3: Two-step chromatography: Take the purified solution obtained in Step 2 and perform the following purification steps: First, load the sample: dilute the one-step purification solution with water to an acetonitrile concentration of 15-25%, and then load the sample onto the chromatographic medium; Elution: The target peak was eluted with a linear gradient from 35% organic phase to 50% organic phase. The elution peaks were collected in segments, and the samples with high purity were combined to obtain a two-step purification solution. Step 4: Rotary evaporation and drying: Take the purified solution from step 3 and remove the organic phase by rotary evaporation using a rotary evaporator, thereby performing rotary evaporation and drying; Step 5: Freeze-drying: Take the solid after rotary evaporation and dry it, add water and filter it, then place it in a freeze-drying tray, freeze-dry it according to the set freeze-drying curve, and store it at 2~8℃ after freeze-drying.

[0007] Preferably, in step 1, the dissolving solution is one of the following: a 0% to 60% ethanol solution containing 1‰ to 5‰ acid.

[0008] Preferably, in step 1, the saponin powder is one of VQ-SAP, VET-SAP, Quil-A, Vax Sap, or SuperSap.

[0009] Preferably, in step 1, the ratio of saponin powder to solution is 1:10 to 1:50 (W / V). After dissolution, the solution is filtered through a 5μm filter membrane and then filtered through a 0.45+0.2μm filter (wherein, VQ-SAP can be directly loaded after dissolution without filtration).

[0010] Preferably, in step 2, the polymer chromatography medium is one of PolyRP 30-300, UniPS 30-300, or Monomix 30-500.

[0011] Preferably, in step 2, the sample loading is 1~100 mg / ml, the linear flow rate is 150~300 cm / h, after the sample loading is completed, it is rinsed with aqueous phase, and then balanced with 30% organic phase to remove pigments and other impurities. During gradient elution, the elution peak is collected, and the sample with a purity greater than 50% is collected. Preferably, the sample with a purity greater than 80% is collected.

[0012] Preferably, in step 2, the aqueous phase is an aqueous solution containing 1‰ to 5‰ acid, including but not limited to formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, and citric acid.

[0013] Preferably, in step 3, the chromatographic medium is one of Poly PRP10-300, UniPS 5-300, UniSil 8~120, or UniSil 5~120 Polar.

[0014] Preferably, in step 3, the sample loading is 0.2~10 mg / ml, the linear flow rate is 100~300 cm / h, after the sample loading is completed, the sample is rinsed with aqueous phase, then equilibrated with 20% organic phase, and during gradient elution, the sample with a purity greater than 95% is collected.

[0015] Preferably, in step 4, the rotary evaporation parameters are: rotary evaporation temperature: 50~85℃, rotary evaporation speed: 10~80rpm, and vacuum parameters: -0.04~-0.1 MPa.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention patent utilizes VQ-SAP as a raw material, achieving a QS-21 recovery rate of over 50% and a QS-21 purity of over 97% through a two-step chromatography process. Compared to DK's QS-21, the product does not contain isomers with molecular weights of 1865 and 1988. This method not only enables the efficient preparation of triterpenoid saponins with high peak product purity but is also applicable to saponin raw materials from various sources. The QS-21 obtained using this method has high purity, high recovery rate, readily available raw materials, higher product safety, and lower production costs, thus breaking the international monopoly from raw material to finished product. Attached Figure Description

[0017] Figure 1 The images show the properties of VQ-SAP, VET-SAP, and Quil-A after dissolution according to the present invention. Figure 2 The above are HPLC chromatograms of VQ-SAP, VET-SAP, and Quil-A after dissolution according to the present invention. Figure 3 This is a one-step chromatography spectrum of Poly PRP 30-300 according to the present invention; Figure 4 This is a one-step chromatography spectrum of UniPS 30-300 according to the present invention; Figure 5 This is a one-step chromatography spectrum of Monomix 30-500 according to the present invention; Figure 6 This is a two-step chromatography pattern of Poly PRP 10-300 according to the present invention; Figure 7 This is the two-step chromatography pattern of UniPS 5-100 of the present invention; Figure 8 This is the two-step chromatography pattern of UniSil 8-120 C18 of the present invention; Figure 9 This is the UniSil 10-120 Polar two-step tomography spectrum of the present invention; Figure 10 This is the RP-HPLC detection chromatogram of the present invention; Figure 11 This is the HPLC-MS chromatogram of the present invention. Detailed Implementation

[0018] The present invention will now be described in more detail by way of examples. These examples are merely illustrative and do not limit the scope of the present invention in any way.

[0019] This invention provides a technical solution: a method for preparing triterpenoid saponins, comprising the following steps: Step 1: Dissolving and filtering saponin powder: Using saponin saponins as the starting material, the saponin raw material is dissolved in a dissolving solution and the insoluble impurities inside the solution are removed by filtration. Step 2: One-step chromatography: Take the saponin dissolution and filtration solution obtained in Step 1, and separate the triterpenoid saponins according to the following steps: First, sample loading: Dissolve the saponin, filter the sample, dilute it, and then load it onto the polymer chromatography medium; Elution followed: The target peak was eluted with a linear gradient from 30% B phase to 50% B phase, and the elution peaks were collected to obtain a one-step purified solution; Step 3: Two-step chromatography: Take the purified solution obtained in Step 2 and perform the following purification steps: First, load the sample: dilute the one-step purification solution with water to an acetonitrile concentration of 15-25%, and then load the sample onto the chromatographic medium; Elution: The target peak was eluted with a linear gradient from 35% organic phase to 50% organic phase. The elution peaks were collected in segments, and the samples with high purity were combined to obtain a two-step purification solution. Step 4: Rotary evaporation and drying: Take the purified solution from step 3 and remove the organic phase by rotary evaporation using a rotary evaporator, thereby performing rotary evaporation and drying; Step 5: Freeze-drying: Take the solid after rotary evaporation and dry it, add water and filter it, then place it in a freeze-drying tray, freeze-dry it according to the set freeze-drying curve, and store it at 2~8℃ after freeze-drying.

[0020] This embodiment provides a method for preparing triterpenoid saponin (QS-21). First, the saponin powder was dissolved and filtered: using saponin saponins as the starting material, the powder was dissolved in a dissolving solution and then filtered to obtain a one-step chromatography loading solution. Then, one-step chromatography was performed using the loading solution. The chromatographic packing material for one-step chromatography was polymer packing material. The elution conditions were as follows: gradient elution was performed by mixing solution A (1‰~5‰ aqueous solution of acid) and solution B (1‰~5‰ acetonitrile solution of acid) in a volume ratio, and high-purity samples were collected. The high-purity sample collected by the first-step chromatography was then subjected to a second-step chromatography. The chromatographic packing material for the second-step chromatography was a chromatographic packing material. The elution conditions were as follows: gradient elution was performed by mixing solution A (1‰~5‰ aqueous solution of acid) and solution B (1‰~5‰ acetonitrile solution of acid) in a volume ratio, and the high-purity sample was collected. The high-purity sample collected by the two-step chromatography was then concentrated by rotary evaporation until dry. The rotary evaporation parameters were: evaporation temperature: 50~85℃, rotary evaporation speed: 10~80rpm, and vacuum parameters: -0.04~-0.1 MPa. Finally, freeze-dry the solid after rotary evaporation: add water, filter, and place in a freeze-drying pan. Freeze-dry according to the set freeze-drying curve. Store at 2-8℃ after freeze-drying.

[0021] The saponin raw material (VQ-SAP) was prepared by Chengdu Jicang Biotechnology Co., Ltd., with minimal batch-to-batch variation, low levels of impurities such as plant polyphenols, tannins, polysaccharides, and proteins, high QS-21 purity and content, and readily available raw materials. Using VQ-SAP as the starting material eliminates the need for pretreatment; the sample can be dissolved in solution A and then subjected to one-step chromatography, reducing production costs.

[0022] Other saponin raw materials besides VQ-SAP (such as VET-SAP, Quil-A, etc.) require pretreatment before chromatography due to their high impurity content (including high concentrations of plant polysaccharides, etc.). After dissolution with a dissolving solution (50% ethanol), insoluble impurities are visible in the solution. These impurities can be removed by natural sedimentation, centrifugation, filtration, etc.

[0023] This method yields high-purity QS-21 through two-step chromatography. Gradient elution is used. One-step chromatography achieves: (1) separation of multiple components in saponins, such as QS-7, QS-17, QS-18, and QS-21; (2) high purity of each saponin component obtained in one-step chromatography; (3) continuous loading, allowing for reloading after equilibration in one-step chromatography; and (4) high loading capacity and good separation purity. Two-step chromatography achieves: (1) low-cost, reusable, and stable packing materials; (2) readily available and controllable quality packing materials; (3) packing materials not limited to polymer packing materials or reversed-phase packing materials; and (4) high sample recovery rate. High-purity QS-21 can be obtained through two purification steps, with fewer purification steps and a high QS-21 recovery rate.

[0024] The QS-21 product obtained using the technical route of this invention has a purity of ≥95% for "1988 component" and a yield of ≥50%, and does not contain "2002 component or 2018 component". The product has high safety and good quality.

[0025] In step 1, the saponin raw material is dissolved and filtered. The saponin raw material is added to the dissolving solution and stirred until it is fully dissolved. The dissolving solution is an alcohol solution containing 1‰ to 5‰ acid (the volume fraction of the alcohol solvent is 0% to 60%), preferably 50% ethanol.

[0026] (1) The saponin raw material in step 1 is at least one of VQ-SAP, VET-SAP, Quil-A, Vax SAP and Super SAP.

[0027] (2) The ratio of saponin powder to solution in step 1 is 1:10 to 1:50 (W / V), preferably, the ratio of saponin powder to solution is 1:20 (W / V).

[0028] (3) When the saponin raw material mentioned in step 1 is VQ-SAP, the VQ-SAP becomes a clear solution after being dissolved in the dissolving solution, and can be directly loaded onto the sample without filtration. Preferably, the dissolving solution is an aqueous solution of 1‰~5‰ acid.

[0029] (4) When the saponin raw material mentioned in step 1 is at least one of VET-SAP, QuilA, Vax Sap, and Super Sap, the saponin raw material is dissolved in a dissolving solution. Preferably, the dissolving solution is a 0% to 60% ethanol solution of 1‰ to 5‰ acid. After adding the dissolving solution, the solution becomes turbid (containing insoluble impurities). To extend the service life of the packing material and increase the loading capacity, impurities must be removed before the sample can be loaded. The methods for removing impurities include, but are not limited to, natural sedimentation, centrifugation, and filtration. Natural sedimentation can be carried out at 2 to 8°C for at least 8 hours, and the supernatant is poured off. Centrifugation can be carried out using a low-temperature refrigerated centrifuge, a tubular centrifuge, etc., with a centrifugation speed of at least 2000 rpm. Filtration can be carried out in two stages: first, a 5 to 0.5 μm filter bag is used to remove large particulate impurities, and then a 0.45 + 0.2 μm filter is used to remove small particulate impurities. Preferably, after filtering with a 5 μm filter bag and collecting the filtrate, the 0.45 + 0.2 μm filter is used to collect the supernatant.

[0030] In step 2, a one-step chromatography was performed. The chromatography packing material for the one-step chromatography was Poly PRP 30-300. The elution conditions were as follows: solution A (aqueous solution of 1‰~5‰ acid) and solution B (acetonitrile solution of 1‰~5‰ acid) were mixed in a volume ratio and then gradient elution was performed to collect the high-purity sample.

[0031] (1) The chromatography packing material in step 2 is at least one of Poly PRP 30-300, UniPS 30-300, and Monomix 30-500.

[0032] (2) In step 2, solution A is an aqueous solution containing 1‰ to 5‰ acid, and solution B is an acetonitrile solution containing 1‰ to 5‰ acid. The volume fraction of the acid is one of 1‰, 2‰, 2.5‰, 3‰, 4‰, or 5‰.

[0033] (3) The acid in step 2 is one of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, or citric acid.

[0034] (4) The volume ratio of A solution and B solution for gradient elution in step 2 is: A solution (50%~80%), B solution (20%~50%). For example, the volume ratio of solution A to solution B is: 80%:20%, 79%:21%, 78%:22%, 77%:23%, 76%:24%, 75%:25%, 74%:26%, 73%:27%, 72%:28%, 71%:29%, 70%:30%, 69%:31%, 68%:32%, 67%:33%, 66%:34%, 65%:33%, 64%:36%, 63%:37%, 62%:38%, 61%:39%, 60%:40%, 59%:41%, 58%:42%, 57%:43%, 56%:44%, 55%:45%, 53%:47%, 52%:48%, 51%:49%, 50%:50%.

[0035] (5) The gradient elution volume in step 2 is one of 5~20CV.

[0036] (6) The linear flow rate in step 2 is 100~300cm / h.

[0037] (7) The loading amount of saponin raw material in step 2: the column volume (m:V) of the one-step chromatography is one of 1mg / ml to 100mg / ml.

[0038] (8) In step 2, the purity of QS-21 in the sample collected by one-step chromatography is above 50%, preferably above 80%.

[0039] In step 3, a two-step chromatography was performed. The chromatography packing material for the two-step chromatography was Poly PRP 10-300. The elution conditions were as follows: solution A (aqueous solution of 1‰~5‰ acid) and solution B (acetonitrile solution of 1‰~5‰ acid) were mixed in a volume ratio and then gradient elution was performed to collect the high-purity sample.

[0040] (1) The chromatography packing material in step 3 is at least one of Poly PRP 10-300, UniPS 5-300, UniSil 8-120 C18, and UniSil 10-120 Polar.

[0041] (2) In step 3, solution A is an aqueous solution containing 1‰ to 5‰ acid, and solution B is an acetonitrile solution containing 1‰ to 5‰ acid. The volume fraction of the acid is one of 1‰, 2‰, 2.5‰, 3‰, 4‰, or 5‰.

[0042] (3) The type of acid in step 3 is one of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, or citric acid.

[0043] (4) The volume ratio of A solution and B solution for gradient elution in step 3 is: A solution (50%~80%), B solution (20%~50%). For example, the volume ratio of solution A to solution B is: 80%:20%, 79%:21%, 78%:22%, 77%:23%, 76%:24%, 75%:25%, 74%:26%, 73%:27%, 72%:28%, 71%:29%, 70%:30%, 69%:31%, 68%:32%, 67%:33%, 66%:34%, 65%:33%, 64%:36%, 63%:37%, 62%:38%, 61%:39%, 60%:40%, 59%:41%, 58%:42%, 57%:43%, 56%:44%, 55%:45%, 53%:47%, 52%:48%, 51%:49%, 50%:50%.

[0044] (5) The gradient elution volume in step 3 is one of 5~20CV.

[0045] (6) The linear flow rate in step 3 is 100~300cm / h.

[0046] (7) The amount of saponin raw material loaded in step 3: the content (m:V) of QS-21 is one of 0.2mg / ml~10mg / ml.

[0047] (8) The purity of QS-21 in the sample collected by two-step chromatography in step 3 is above 95%.

[0048] In step 4, rotary evaporation concentration is performed: the high-purity sample collected by the two-step chromatography is concentrated by rotary evaporation.

[0049] (1) In step 4, the rotary evaporation temperature is one of 50~85℃.

[0050] (2) In step 4, the rotary evaporation speed is one of 10~80 rpm.

[0051] (3) In step 4, the vacuum parameter is one of -0.04 to -0.1 MPa.

[0052] (4) In step 4, after rotary evaporation to concentrate until the organic phase is removed, ethanol is added to remove the water in the solution until it is dried by rotary evaporation. Step 5: Freeze-drying: Dissolve the solid obtained from rotary evaporation in Step 4 in water, filter, and place in a freeze-drying pan. Freeze-dry according to the set freeze-drying curve. Store at 2-8℃ after freeze-drying.

[0053] (1) In step 5, weigh the solid after rotary evaporation and add water for injection to dissolve it to a concentration of 2~20 mg / ml of QS-21.

[0054] (2) In step 5, after complete dissolution, use a 0.2μm filter for sterilization filtration.

[0055] (3) In step 5, the filtered solution is dispensed into vials of 2ml, 10ml or 100ml.

[0056] (4) In step 5, the dispensed sample is placed in a freeze-drying tray and freeze-dried.

[0057] (5) In step 5, the freeze-drying parameters are: Freezing: Freezing and cooling the temperature to -20 to -40°C within 60 to 120 minutes, and maintaining it for 1 to 24 hours until the liquid is completely frozen.

[0058] Primary drying: During operation, the temperature is increased to -2 to -20℃ within 60 to 120 minutes, and maintained for 50 to 80 hours until the sample is initially dried.

[0059] Secondary drying: Run the cooling process to raise the temperature to 20-50℃ within 60-120 minutes, and maintain it for 8-24 hours until the sample is freeze-dried and formed.

[0060] (6) In step 5, use a matching capping machine or manually capping.

[0061] The QS-21 product obtained using the technical route of this invention has a purity of ≥97.0% for "1988 component" and a yield of ≥50%, and does not contain impurities of "2018 component" and "2002 component".

[0062] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0063] Main ingredient: VQ-SAP is self-made by Chengdu Jicang Biotechnology Co., Ltd.

[0064] Tests showed that the raw material contained no less than 8% QS-21; QS-21 accounted for no less than 10% of the total saponins; moisture content was no more than 5%; total ash content was no more than 3%; endotoxin content was less than 10 EU / g; residues of five heavy metals were no more than 20 ppm; and pesticide residues met the standards of the Chinese Pharmacopoeia.

[0065] Example 1: This embodiment provides a one-step chromatography method for saponins: In this embodiment, VQ-SAP was used as the raw material, and a dissolving solution (0.25% acetic acid aqueous solution) was used to dissolve VQ-SAP. Specifically, VQ-SAP was mixed thoroughly and dissolved completely at a feed ratio of VQ-SAP:dissolving solution = 1:15 (w:v). 100g of VQ-SAP was used with 1.5L of dissolving solution. After mixing, this was the sample for one-step chromatography loading.

[0066] Example 2: Compared to Example 1, the only difference is that VET-SAP was used as the raw material, and a dissolving solution (0.25% acetic acid + 50% ethanol aqueous solution) was used to dissolve the VET-SAP. Specifically, the VET-SAP was mixed thoroughly and dissolved completely at a ratio of VET-SAP:dissolving solution = 1:15 (w:v). 100g of VET-SAP was used with 1.5L of dissolving solution. After mixing, the solution was first filtered through a 5μm filter bag, then through a 0.5μm filter bag. The supernatant was collected as the sample for one-step chromatography loading.

[0067] Example 3: The only difference from Example 2 is that Quil-A is used as the raw material.

[0068] Example 4: The only difference from Example 2 is that Vax Sap is used as the raw material.

[0069] Example 5: The only difference from Example 2 is that Super Sap is used as the raw material.

[0070] Example 6: This embodiment provides a method for preparing triterpenoid saponin (QS-21) using VQ-SAP as the saponin raw material: (1) Mix the saponins according to the ratio of VQ-SAP:solution = 1:15 (w:v). After mixing and dissolving thoroughly, the sample is ready for one-step chromatography.

[0071] (2) First Chromatography Take a sample for one-step chromatography and operate according to the following steps: Dynamic axial compression column: DAC-100; Chromatography packing material: Poly PRP 30-300; Eluent: Solution A - 0.25% acetic acid; Solution B - 0.25% acetic acid + acetonitrile solution; Flow rate: 240 mL / min; After sample loading, equilibrate with 20% Solution B, remove impurities with 20%~30% Solution B, and gradient elute the target peak of QS-21 with 30%~50% Solution B. Collect the target peak in segments, perform QS-21 purity analysis, and collect samples with a purity ≥50% to obtain one-step chromatography QS-21.

[0072] (3) Second Chromatography Take one-step chromatography QS-21 and dilute it with water at a ratio of 1:1 (v:v) to obtain the sample for two-step chromatography. Dynamic axial compression column: DAC-100; chromatography packing material: Poly PRP 10-300; eluent: Solution A - 0.25% acetic acid; Solution B - 0.25% acetic acid + acetonitrile solution; flow rate: 240 mL / min; after sample loading, equilibrate with 20% B solution, remove impurities with 20%~35% B solution, and gradient elute the target peak of QS-21 with 35%~50% B solution. Collect the target peaks in segments for QS-21 purity analysis. Collect samples with a purity ≥95% to obtain the two-step chromatography QS-21.

[0073] (4) Rotary distillation concentration The two-step chromatography QS-21 sample was concentrated by rotary evaporation at a temperature of 60℃, a rotation speed of 30 rpm, and a vacuum parameter of -0.8 MPa. The concentrated sample was concentrated to 1 / 3 to 1 / 2 of its volume. Ethanol was added repeatedly until the sample was dried.

[0074] (5) Freeze-drying Weigh the solid after rotary evaporation and dissolve it in water for injection to a QS-21 concentration of 5 mg / ml. After complete dissolution, filter sterilely using a 0.2 μm filter. Aliquot the filtered solution into 2 ml vials. Place the aliquoted sample in a lyophilization tray and lyophilize. The lyophilized product is QS-21 lyophilized powder.

[0075] The freeze-drying parameters are: Freezing: Freezing and cooling the temperature to -40°C within 120 minutes and maintaining it for 24 hours until the liquid is completely frozen.

[0076] Primary drying: The temperature is raised to -20°C within 90 minutes and maintained for 72 hours until the sample is initially dry.

[0077] Secondary drying: Run the cooling process to raise the temperature to 50°C within 90 minutes and maintain it for 24 hours until the sample is freeze-dried and formed.

[0078] After freeze-drying, the capsules are crimped using a matching crimping machine.

[0079] Example 7: Compared with Example 6, the only difference is that in step (2), the chromatography packing material is UniPS 30-300.

[0080] Example 8: Compared with Example 6, the only difference is that in step (2), the chromatography packing material is Monomix 30-500.

[0081] Example 9: Compared with Example 6, the only difference is that in step (3), the chromatography packing material is Poly PRP 10-300.

[0082] Example 10: Compared with Example 3, the only difference is that in step (3), the chromatography packing material is UniPS 5-100.

[0083] Example 11: Compared with Example 6, the only difference is that in step (3), the chromatography packing material is UniSil 8-120 C18.

[0084] Example 12: Compared with Example 6, the only difference is that in step (3), the chromatography packing material is UniSil 10-120 Polar.

[0085] Experimental Example 1: Calculate the recovery rate and purity of QS-21 in Examples 1-5 respectively: QS-21 recovery rate (%): calculated by HPLC external standard method.

[0086] Table 1: HPLC Detection Methods

[0087] Table 2: Statistical table of QS-21 recovery rate and purity after first-step chromatography for examples of different saponin raw materials.

[0088] As shown in Table 2, the recovery rate of VQ-SAP was higher than that of other saponin raw materials. This may be because the purity and content of QS-21 in VQ-SAP were both higher than those in other saponin raw materials. The phenomena after dissolution of the saponin raw materials and the HPLC detection results are as follows: Figure 2 As shown.

[0089] Experimental Example 2: Using VQ-SAP from Example 6 as raw material, the first chromatography after Examples 6 to 8 yielded the following results: the recovery rate and purity of QS-21 are shown in Table 3.

[0090] The first step of the chromatographic pattern is as follows: Figures 3-5 As shown. Example 6 is as follows. Figure 3 As shown, Example 7 is as follows Figure 4 As shown, Example 7 is as follows Figure 5 As shown.

[0091] Using VQ-SAP from Example 6 as raw material, after a second chromatography following Examples 9 to 12, the recovery rate and purity of QS-21 are shown in Table 4:

[0092] The second step is to obtain the chromatogram. Figures 6-9 As shown. Example 9 is as follows. Figure 6 As shown, Example 10 is as follows Figure 7 As shown, Example 11 is as follows Figure 8 As shown, Example 12 is as follows Figure 9 As shown.

[0093] Using VQ-SAP as raw material, after first-step chromatography and second-step chromatography, the recovery rate of QS-21 was greater than 50%, and the purity of QS-21 was higher than 95%.

[0094] Experimental Example 3: The QS-21 lyophilized powder obtained in Example 6 was subjected to quality testing. Mass spectrometry parameters: high-resolution mass spectrometry (ESI source, acquisition mode negative ion full scan, m / z scan range: 100-3000, CDL temperature: 300.0℃, Heatblock temperature: 300.0℃, carrier gas flow rate: 8L / min). The results showed that the main peak contained a component with a molecular weight of 1988 (QS-21 main peak).

[0095] Example 6: HPLC detection results of QS-21 chromatography are as follows Figure 10 As shown, the mass spectrometry results of the QS-21 lyophilized powder are as follows: Figure 11 As shown.

[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing triterpenoid saponins, characterized in that: Includes the following steps: Step 1: Dissolving and filtering saponin powder: Using saponin saponins as the starting material, the saponin raw material is dissolved in a dissolving solution and the insoluble impurities inside the solution are removed by filtration. Step 2: One-step chromatography: Take the saponin dissolution and filtration solution obtained in Step 1, and separate the triterpenoid saponins according to the following steps: First, sample loading: Dissolve the saponin, filter the sample, dilute it, and then load it onto the polymer chromatography medium; Elution followed: The target peak was eluted with a linear gradient from 30% B phase to 50% B phase, and the elution peaks were collected to obtain a one-step purified solution; Step 3: Two-step chromatography: Take the purified solution obtained in Step 2 and perform the following purification steps: First, load the sample: dilute the one-step purification solution with water to an acetonitrile concentration of 15-25%, and then load the sample onto the chromatographic medium; Elution: The target peak was eluted with a linear gradient from 35% organic phase to 50% organic phase. The elution peaks were collected in segments, and the samples with high purity were combined to obtain a two-step purification solution. Step 4: Rotary evaporation and drying: Take the purified solution from step 3 and remove the organic phase by rotary evaporation using a rotary evaporator, thereby performing rotary evaporation and drying; Step 5: Freeze-drying: Take the solid after rotary evaporation and dry it, add water and filter it, then place it in a freeze-drying tray, freeze-dry it according to the set freeze-drying curve, and store it at 2~8℃ after freeze-drying.

2. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 1, the dissolving solution is one of the following: a 0% to 60% ethanol solution containing 1‰ to 5‰ acid.

3. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 1, the saponin powder is one of VQ-SAP, VET-SAP, Quil-A, Vax Sap, or Super Sap.

4. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 1, the ratio of saponin powder to solution is 1:10 to 1:50 (W / V). After dissolution, the solution is filtered through a 5μm filter membrane and then filtered through a 0.45+0.2μm filter (VQ-SAP can be directly loaded after dissolution without filtration).

5. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 2, the polymer chromatography medium is one of PolyRP 30-300, UniPS 30-300, or Monomix 30-500.

6. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 2, the sample loading is 1~100mg / ml, the linear flow rate is 150~300cm / h, after the sample loading is completed, it is rinsed with aqueous phase, and then balanced with 30% organic phase to remove pigments and other impurities. During gradient elution, the elution peak is collected, and the sample with a purity greater than 50% is collected. Preferably, the sample with a purity greater than 80% is collected.

7. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 2, the aqueous phase is an aqueous solution containing 1‰ to 5‰ acid, including but not limited to formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, and citric acid.

8. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 3, the chromatographic medium is one of Poly PRP10-300, UniPS 5-300, UniSil 8~120, or UniSil 5~120 Polar.

9. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 3, the sample loading rate is 0.2~10 mg / ml, the linear flow rate is 100~300 cm / h, after the sample loading is completed, it is rinsed with aqueous phase, then equilibrated with 20% organic phase, and during gradient elution, the sample with a purity greater than 95% is collected.

10. The method for preparing a triterpenoid saponin according to claim 1, characterized in that: In step 4, the rotary evaporation parameters are: rotary evaporation temperature: 50~85℃, rotary evaporation speed: 10~80rpm, and vacuum parameters: -0.04~-0.1 MPa.

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