Method for extracting QS21 series saponin from peeled soap tree trunk

Abstract

The invention provides a method for extracting QS21 series saponin from a peeled gleditsia sinensis trunk. The QS21 series saponin is prepared by the following steps: performing water extraction on the peeled gleditsia sinensis trunk, adding a stabilizer into an extracting solution, concentrating, separating a concentrated solution by virtue of a multi-stage combined chromatographic column, eluting by virtue of an organic solvent, and collecting and drying eluent. The content of the QS21 saponin extracted by the method disclosed by the invention is 10-35%.

Description

Technical Field

[0001] This invention relates to the field of saponin separation and purification technology, specifically to a method for extracting QS21 series saponins from the bark of soapberry trees. Background Technology

[0002] The bark of the Chilean saponaria (Quillaja Saponaria) contains QS-21 saponin, a potent vaccine adjuvant that plays a crucial role in the development of new vaccines, particularly for major diseases such as malaria and influenza. Extracting the QS component from Chilean saponaria bark is a laborious and expensive process: one gram of QS-21 can cost up to $100,000; furthermore, only one-fifth of Chilean saponaria trees contain sufficient QS-21. Chile harvests 240 tons of Chilean saponaria bark annually to produce 158,000 grams of QS-21, or 3.2 billion to 6.4 billion doses of adjuvant (depending on vaccine requirements; the Shingrix vaccine requires 50 micrograms of QS-21 per dose, while other vaccines require only 25 micrograms). However, the bark takes up to 25 years to mature and become harvestable, and mature Chilean saponaria trees are becoming increasingly scarce due to climate change, illegal logging, and over-peeling. With the ever-increasing demand for saponins, Chile's saponin forests may face depletion due to overexploitation, ultimately failing to meet the growing needs of vaccine manufacturers. Currently, the long maturation cycle and threatened habitat of Chilean saponin trees are the biggest challenges.

[0003] Therefore, there is an urgent need to develop new methods for extracting saponins, on the one hand to improve the yield of QS21 series components, and on the other hand to expand the available raw materials, such as the bark-peeled soap tree trunks and branches, and Chilean soap trees that have withered due to drought, to enrich the trace QS series components and maximize the utilization of resources. Summary of the Invention

[0004] QS-21 is an important vaccine adjuvant, mainly enriched in the bark of soap trees (such as the Chilean soap tree, Quillajasaponaria), while its content in the trunk is extremely low and contains a variety of analogues. These analogues are numerous and abundant, which greatly interfere with the extraction and purification of QS-21. Therefore, the trunk is usually not used as the main extraction source.

[0005] In addition, the QS-21 series components have poor stability in solution and are easily degraded or deactivated by environmental factors. Furthermore, they are prone to forming abundant foam during extraction and concentration, which increases the pressure and difficulty of concentration. When optimizing extraction, purification and drying processes, special attention should be paid to their stability.

[0006] To solve the above-mentioned technical problems, the present invention has the following approach: The first step is to optimize the extraction process: By improving extraction methods and techniques, such as using more efficient solvent systems and optimizing extraction temperature and time, the extraction efficiency and purity of QS-21 can be improved, and interference from similar substances can be reduced.

[0007] The second step is stabilizer screening: To improve the stability of QS-21 in solution, different stabilizers were screened for addition. These stabilizers can protect QS-21 from the effects of experimental solvents and environmental factors, prevent oxidation, improve solubility, maintain the extract within a specific pH range, and extend its shelf life. When selecting stabilizers, factors such as safety, effectiveness, and cost were considered. Stabilizers include, but are not limited to, common antioxidants, acid stabilizers, and alkali stabilizers. The third step is bubble control: During extraction and concentration, bubble formation is controlled by adjusting operating conditions (such as pressure, temperature, and stirring speed). Additionally, defoaming agents or bubble separation techniques can be used to further reduce the impact of bubbles.

[0008] The specific technical solution is as follows: A method for extracting QS21 series saponins from the bark of a soapberry tree, characterized by comprising the following steps: Step a: Extraction: Take the bark-removed soapberry trunk, crush it, add pure water or low-concentration alcohol solution and stir to extract, collect the extract and concentrate it appropriately; Step b: Purification by multi-stage combined chromatography column: Separate the concentrate onto a multi-stage combined chromatography column, eluting sequentially with 20-45%, 50-70%, and 95% ethanol gradients. Collect the 50-70% ethanol eluent, evaporate to dryness under reduced pressure, and obtain the QS21 series saponin extract.

[0009] Further, the extraction described in step a involves placing the crushed soapberry trunk in an extraction tank, adding 10-15 times the amount of pure water (w / v) equal to the amount of the medicinal material, extracting 1-3 times, each time for 1-2 hours, at an extraction temperature of 30-50℃, combining the extracts, adding a stabilizer, and concentrating.

[0010] The multi-stage combined chromatography column mentioned in step b is prepared by taking pretreated macroporous resin, packing it wet-process, taking 2 kg of medicinal material / L of resin as the sample solution, loading the sample at a flow rate of 1 BV / h until complete, and eluting sequentially with 40-45%, 50-55%, and 95% ethanol gradients, with each gradient being 3-10 BV and a flow rate of 2-3 BV / h. The 50-55% ethanol eluent is collected, concentrated, and dried.

[0011] Preferably, the multi-stage combined chromatography column in step b is a mixture of two or three of the following resins: AB-8, XAD, LX60, LX20SS, or D-101.

[0012] The preferred total saponin extract from South American soap trees contains 10-35% QS21 saponins. Attached Figure Description

[0013] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort. Figure 1 is a high-performance liquid chromatogram of QS21 saponin prepared in Example 2 of the present invention. Detailed Implementation

[0014] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0015] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0016] Example 1. Preparation of QS21 series saponin extracts Take 1 kg of crushed soap tree trunk, place it in a 25L stainless steel bucket, add 10 kg of pure water, extract twice, 2 hours each time, at an extraction temperature of 30℃, combine the two extracts, and add a stabilizer; concentrate the extract under reduced pressure at 40℃ until there is no alcohol odor, and obtain the sample solution.

[0017] Half of the sample solution was purified by chromatography using an XAD macroporous resin column (3 cm in diameter, 25 cm in height, and 176 mL in volume). The column was loaded completely at a flow rate of 1 BV / h, followed by elution with 40% ethanol (5 BV), 45% ethanol (3 BV), 50% ethanol (10 BV), and 95% ethanol (3 BV) at a flow rate of 2.5 BV / h. The 50% ethanol eluent was collected, concentrated under reduced pressure at 40°C, and dried to powder, yielding 3.25 g of QS21 series saponin extract. The QS21 saponin content was determined to be 12.4% according to patent application number 2024111342858, "An HPLC-ELSD method for simultaneous determination of two saponin components from saponins."

[0018] Example 2. Preparation of QS21 series saponin extracts Take 1 kg of crushed soapberry trunk and place it in a 25 L stainless steel container. Add 15 kg of 20% ethanol aqueous solution and extract 3 times, 1 hour each time, at an extraction temperature of 50 °C. Combine the 3 extracts and add a stabilizer. Concentrate the extract under reduced pressure at 40 °C until there is no alcohol odor to obtain the loading solution.

[0019] Half of the sample solution was purified by chromatography using an LX20SS column (3 cm in diameter, 25 cm in height, and 176 mL in volume). The sample was loaded completely at a flow rate of 1 BV / h, followed by sequential elution with 40% ethanol (8 BV), 55% ethanol (8 BV), and 95% ethanol (3 BV) at a flow rate of 2 BV / h. The 55% ethanol eluent was collected, concentrated under reduced pressure at 40°C, and dried to powder, yielding 2.08 g of QS21 series saponin extract. The QS21 saponin content was determined to be 17.2% according to patent application number 2024111342858, "An HPLC-ELSD method for simultaneous determination of two saponin components from saponins."

[0020] Example 3. Preparation of QS21 series saponin extracts The saponin loading solutions obtained in Examples 1 and 2 were combined and purified by chromatography using a D101 macroporous resin column (the macroporous resin column has a diameter of 3.6 cm, a height of 35 cm, and a column volume of 356 mL). The sample was loaded to complete at a flow rate of 1 BV / h, and then eluted sequentially with 40% ethanol (5 BV) and 95% ethanol (3 BV) at a flow rate of 3 BV / h. The 95% ethanol eluent was collected and concentrated under reduced pressure at 40°C until no alcohol odor was detected, yielding the first chromatography column eluent. The eluent was purified by chromatography using an LX60 column (3 cm diameter, 25 cm height, 176 mL volume). The column was loaded with 1 BV / h until complete, followed by sequential elution with 45% ethanol (3 BV), 55% ethanol (6 BV), and 95% ethanol (3 BV) at a flow rate of 2 BV / h. The 55% ethanol eluent was collected, concentrated under reduced pressure at 40°C, and dried to powder, yielding 0.935 g of QS21 series saponin extract. The QS21 saponin content was determined to be 34.8% according to patent application number 2024111342858, "An HPLC-ELSD method for simultaneous determination of two saponin components from saponins."

[0021] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting QS21 series saponins from the bark of a soapberry tree, characterized in that, The method includes the following steps: Step a: Extraction: Take the bark-removed soapberry trunk, crush it, add pure water or low-concentration alcohol and stir to extract, collect the extract and concentrate it appropriately; Step b: Purification by multi-stage combined chromatography column: Separate the concentrate onto a multi-stage combined chromatography column, eluting sequentially with 20-45%, 50-70%, and 95% ethanol gradients. Collect the 50-70% ethanol eluent, evaporate to dryness under reduced pressure, and obtain the QS21 series saponin extract.

2. The method for extracting QS21 series saponins according to claim 1, characterized in that, The extraction described in step a involves placing the crushed soapberry trunk into an extraction tank, adding 10-15 times the amount of pure water (w / v) of the medicinal material, extracting 1-3 times, 1-2 hours each time, at an extraction temperature of 30-50℃, combining the extracts, adding a stabilizer, and concentrating.

3. The method for extracting QS21 series saponins according to claim 1, characterized in that, The multi-stage combined chromatography column mentioned in step b is prepared by taking pretreated macroporous resin, packing it wet-process, taking 2 kg of medicinal material / L of resin as the sample solution, loading the sample at a flow rate of 1 BV / h until complete, and eluting sequentially with 40-45%, 50-55%, and 95% ethanol gradients, with each gradient being 3-10 BV and a flow rate of 2-3 BV / h. The 50-55% ethanol eluent is collected, concentrated, and dried.

4. The method for extracting QS21 series saponins according to claim 1, characterized in that, The multi-stage combined chromatography column mentioned in step b is a mixture of two or three of the following resins: AB-8, XAD, LX60, LX20SS, or D-101.

5. A total saponin extract from South American soap trees, characterized in that, The total saponin extract from the South American soap tree was prepared using the method described in claim 1.

6. The South American soapberry total saponin extract according to claim 5, characterized in that, The content of QS21 saponins is 10-35%.

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