Starfish saponin extraction method and application thereof

Abstract

The invention belongs to the field of biological separation, and particularly relates to an extraction method and application of asterosaponin. The extraction method of the starfish saponin comprises the following steps: S1, freeze-drying and crushing fresh starfishes to obtain starfish powder; s2, the starfish powder is subjected to enzymolysis through chitinase, collagenase and hyaluronidase, and enzymatic hydrolysate is obtained; and S3, adsorbing the enzymatic hydrolysate by adopting a chromatographic column filled with anion exchange resin, and then eluting to obtain a crude purified starfish saponin eluent. And S4, performing supercritical CO2 degreasing and refining on the coarsely purified asterosaponin eluent, and performing freeze drying to obtain asterosaponin powder. According to the method disclosed by the invention, through an integrated process of multi-enzyme synergistic targeted enzymolysis, anion exchange chromatography and supercritical CO2 degreasing, not only can the problem of low extraction rate caused by poor permeability of a traditional solvent be overcome, but also the use activity of asterosaponin can be kept at a relatively high level.

Description

Technical Field

[0001] The present invention relates to the technical field of biological separation, and in particular to a method for extracting starfish saponin and application thereof. Background Art

[0002] Asterosaponins are a class of steroidal saponin compounds with unique biological activity. They are widely present in the body wall and epidermis of starfish and have anti-tumor, anti-inflammatory, and immunomodulatory effects. However, asterosaponins form tightly bound complexes with chitin, collagen, and sulfated polysaccharides in the body wall, making their extraction difficult.

[0003] Traditional starfish saponin extraction relies primarily on organic solvent extraction, such as reflux extraction with methanol, ethanol, or chloroform, followed by purification via silica gel column chromatography. While this method is simple to operate, it suffers from poor solvent permeability, low extraction yields, and significant saponin loss due to polarity mismatch with the solvent.

[0004] To improve the extraction rate of starfish saponins, ultrasound-assisted extraction technology has been introduced. This method accelerates solvent penetration through ultrasonic treatment based on organic solvent extraction. This method can significantly improve the extraction rate of starfish saponins. However, in ultrasound-assisted extraction, the ultrasonic cavitation effect easily creates a localized high temperature environment. This high temperature condition can easily lead to the degradation of saponin molecules, thereby reducing the activity of starfish saponins.

[0005] Therefore, the current extraction process of starfish saponins generally faces the problem of contradiction between efficient extraction and activity preservation. It is necessary to provide a starfish saponin extraction method that has both efficient extraction and activity retention. Summary of the Invention

[0006] (1) Technical issues to be solved

[0007] In order to solve the contradiction between efficient extraction and activity preservation commonly faced by the extraction process of starfish saponins in the prior art, the present invention provides a method for extracting starfish saponins and applications thereof.

[0008] (2) Technical solution

[0009] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] The present invention provides a method for extracting starfish saponins, comprising the following steps:

[0011] S1: freeze-dry fresh starfish and then crush them to obtain starfish powder;

[0012] S2: enzymatically hydrolyzing the starfish powder with chitinase, collagenase, and hyaluronidase to obtain an enzymatic hydrolyzate;

[0013] S3: using a chromatography column filled with anion exchange resin to adsorb the enzymatic hydrolyzate, and then eluting the chromatography column to obtain a crude purified starfish saponin eluate;

[0014] S4: The crude purified starfish saponin eluate is defatted and purified by supercritical CO2, and then freeze-dried to obtain starfish saponin powder.

[0015] In the above-mentioned method for extracting starfish saponins, preferably, in step S1, fresh starfish are cut into pieces and then immersed in liquid nitrogen for 5-10 minutes, and then crushed to obtain starfish powder with a particle size of ≤0.5 mm.

[0016] In the above-mentioned method for extracting starfish saponins, preferably, in step S2, the starfish powder is added to a first buffer solution containing chitinase, collagenase and hyaluronidase, and the reaction is carried out at 40-50° C. for 1.5-2 hours to obtain an enzymatic hydrolyzate;

[0017] The solid-liquid ratio of the starfish powder to the first buffer is 1 g: (9-12) mL, and the first buffer is a citric acid-disodium hydrogen phosphate buffer with a pH of 5.5-6.5;

[0018] The concentration of chitinase in the first buffer is 40-80 U / g, the concentration of collagenase is 50-100 U / g, and the concentration of hyaluronidase is 30-50 U / g.

[0019] The above-mentioned method for extracting starfish saponins, preferably, the first buffer further contains 0.03-0.05wt% of an enzyme stabilizer;

[0020] The enzyme stabilizer is polyethylene glycol 6000 or polyethylene glycol 8000.

[0021] In the above-mentioned method for extracting starfish saponins, preferably, after step S2 and before step S3, the enzymatic hydrolyzate is ultrafiltered and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 10-12 kDa to obtain a concentrated solution.

[0022] In the above-mentioned method for extracting starfish saponins, preferably, in step S3, the concentrated solution is added to a chromatography column filled with anion exchange resin for adsorption treatment, and then the chromatography column is cleaned by a washing relative chromatography column, and then eluted by an elution relative chromatography column to obtain a crude purified starfish saponin eluate.

[0023] In the above-mentioned method for extracting starfish saponins, preferably, the anion exchange resin is a polystyrene resin functionalized with quaternary ammonium groups;

[0024] The cleaning phase is a Tris-HCl buffer containing 0.1-0.2 M NaCl, the pH of the cleaning phase is 8.0-8.5, the flow rate is 1.5-2.5 mL / min, and the cleaning volume is 3-5 times the volume of the chromatography column;

[0025] The elution phase is a Tris-HCl buffer containing NaCl at a concentration of 0.5-0.8 M, the pH of the elution phase is 8.0-8.5, the flow rate is 1.0-1.5 mL / min, and the elution volume is 2-3 times the volume of the chromatography column;

[0026] After the elution is completed, the pH of the eluate is adjusted to 6.5-7.5 to obtain a crude purified starfish saponin eluate.

[0027] In the above-mentioned method for extracting starfish saponins, preferably, in step S4, the supercritical CO2 degreasing treatment has a pressure of 25-35 MPa, a temperature of 35-45°C, a CO2 flow rate of 15-25 L / h, an entrainer of 1-3% by volume of an ethyl lactate aqueous solution, and a degreasing time of 1.5-2.5 h.

[0028] In the above-mentioned method for extracting starfish saponins, preferably, in step S4, the freeze-drying process adopts two-stage drying:

[0029] First, pre-freeze at -40--50 DEG C and vacuum degree ≤10Pa for 4-6 hours, then heat to 20-30 DEG C, maintain vacuum degree ≤15Pa, and dry to moisture ≤3% to obtain starfish saponin powder.

[0030] The present invention also provides an application of the starfish saponin prepared by the above extraction method in anticancer drugs, anti-inflammatory drugs or immunomodulatory drugs.

[0031] (3) Beneficial effects

[0032] The present invention, through an integrated process combining multi-enzyme targeted enzymolysis, anion exchange chromatography, and supercritical CO2 degreasing, effectively resolves the core contradiction in the existing art, which is the difficulty in balancing the efficient extraction and activity preservation of starfish saponins. Specifically, the present invention uses the synergistic action of chitinase, collagenase, and hyaluronidase to precisely destroy the chitin-collagen-polysaccharide complex structure in the starfish body wall, fully releasing the bound saponins in the starfish, and overcoming the low extraction rate caused by the poor permeability of traditional solvents.

[0033] The present invention also avoids the degradation of saponin heat-sensitive groups, such as sulfate groups, caused by the high-temperature environment formed by ultrasonic treatment through a mild process combining low-temperature enzymatic hydrolysis, chromatography column adsorption and supercritical CO2 degreasing. The retention rate of sulfate groups in the prepared starfish saponins is ≥90%, and the activity is well maintained. DETAILED DESCRIPTION

[0034] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with specific embodiments.

[0035] The present invention provides a method for extracting starfish saponins, comprising the following steps:

[0036] S1: freeze-dry fresh starfish and then crush them to obtain starfish powder.

[0037] S2: The starfish powder is enzymatically hydrolyzed by chitinase, collagenase, and hyaluronidase to obtain an enzymatic hydrolyzate.

[0038] S3: using a chromatography column filled with anion exchange resin to adsorb the enzymatic hydrolyzate, and then eluting the chromatography column to obtain a crude purified starfish saponin eluate.

[0039] S4: The crude purified starfish saponin eluate is defatted and purified by supercritical CO2, and then freeze-dried to obtain starfish saponin powder.

[0040] The present invention, through an integrated process combining multi-enzyme targeted enzymolysis, anion exchange chromatography, and supercritical CO2 degreasing, effectively resolves the core contradiction in the existing art, which is the difficulty in balancing the efficient extraction and activity preservation of starfish saponins. Specifically, the present invention uses the synergistic action of chitinase, collagenase, and hyaluronidase to precisely destroy the chitin-collagen-polysaccharide complex structure in the starfish body wall, fully releasing the bound saponins in the starfish, and overcoming the low extraction rate caused by the poor permeability of traditional solvents.

[0041] The present invention also avoids the degradation of saponin heat-sensitive groups, such as sulfate groups, caused by the high-temperature environment formed by ultrasonic treatment through a mild process combining low-temperature enzymatic hydrolysis, chromatography column adsorption and supercritical CO2 degreasing. The retention rate of sulfate groups in the prepared starfish saponins is ≥90%, and the activity is well maintained.

[0042] Preferably, in the above step S1, the fresh starfish is cut into pieces and then immersed in liquid nitrogen for 5-10 minutes. After being freeze-dried with liquid nitrogen, the water content of the starfish is ≤5%, and then it is crushed to obtain starfish powder with a particle size of ≤0.5 mm. The starfish used in the present invention is Asterix rosenbergii or Asterix polyspinosa produced in the Yellow Sea / Bohai Sea area of ​​Dalian area. Sulfate groups are commonly found in the saponins contained in Asterix rosenbergii and Asterix polyspinosa. Sulfate groups in starfish saponins are their main characteristic functional groups, which are widely present on sugar chains or steroid skeletons and play a decisive role in activity, solubility and stability. This structural characteristic is not only the basis of its pharmacological efficacy, but also the core mark that distinguishes it from other saponins.

[0043] Liquid nitrogen instantly freezes starfish tissue, preventing the slow growth of ice crystals that could damage cells, thereby preserving the molecular structure of saponins. Furthermore, starfish pieces frozen in liquid nitrogen become dehydrated and brittle, making them easier to crush into a fine powder ≤0.5mm. This increases the surface area for enzymatic hydrolysis and improves the extraction rate of saponins.

[0044] Preferably, in the above step S2, the starfish powder is added to a first buffer solution containing chitinase, collagenase and hyaluronidase, and the mixture is reacted at 40-50° C. for 1.5-2 hours to obtain an enzymatic hydrolyzate.

[0045] Wherein, the solid-to-liquid ratio of starfish powder and the first buffer solution is 1g:(9-12)mL, and the first buffer solution is citric acid-disodium hydrogen phosphate buffer solution, and its pH is 5.5-6.5. In the first buffer solution, the concentration of chitinase is 40-80U / g, the concentration of collagenase is 50-100U / g, and the concentration of hyaluronidase is 30-50U / g. The present invention, through the synergistic effect of chitinase-collagenase-hyaluronidase, accurately cuts off the composite network of chitin-collagen-polysaccharide in the starfish body wall, fully releases bound saponin. Compared to traditional single enzymolysis, multi-enzyme combination can significantly improve saponin release rate, and it is also possible to avoid the excessive problem of cell debris caused by mechanical fragmentation, while avoiding the saponin structure destruction caused by ultrasound-assisted method or strong solvent extraction.

[0046] Further preferably, the first buffer solution also contains 0.03-0.05 wt% of an enzyme stabilizer, which may be polyethylene glycol 6000 or polyethylene glycol 8000. 0.03-0.05 wt% of polyethylene glycol (PEG 6000 / 800) as an enzyme stabilizer can inhibit the aggregation and inactivation of enzyme molecules through a steric hindrance effect, thereby maintaining a high activity of the enzyme during the enzymatic hydrolysis reaction, and further improving the enzymatic hydrolysis efficiency and process stability.

[0047] Preferably, after step S2 and before step S3, the present invention further performs ultrafiltration and concentration treatment on the enzymatic hydrolyzate through an ultrafiltration membrane with a molecular weight cutoff of 10-12 kDa to obtain a concentrated solution.

[0048] Ultrafiltration membranes selectively retain undegraded collagen fragments and some polysaccharide aggregates, reducing the content of macromolecular impurities in the feed by 60-70%, preventing nonspecific adsorption of anion exchange resins and clogging of chromatography columns, and increasing the dynamic adsorption capacity of the columns. Ultrafiltration membranes allow starfish saponins with a molecular weight of less than 10 kDa to pass freely and concentrate them, reducing the volume of the feed to 1 / 3-1 / 5 of its original volume. This increased saponin concentration reduces the load on subsequent chromatography treatments and prevents the hydrolysis of the saponin sulfate groups caused by high-temperature concentration.

[0049] In addition, the ultrafiltration process can be completed at room temperature without the addition of organic solvents or acid-base regulators. The waste liquid contains only a small amount of large molecular impurities, which significantly improves environmental protection.

[0050] Residual enzymes in the hydrolyzate can also be captured and removed by the ultrafiltration membrane, completely terminating the enzymatic reaction and preventing excessive cleavage of saponin sugar chains, thereby ensuring the structural integrity and pharmacological activity of the saponins. Furthermore, traditional processes require high temperatures or extreme pH levels to inactivate enzymes, which can easily lead to the degradation of heat-sensitive saponin groups, such as sulfate groups. Ultrafiltration physically captures the enzyme protein under mild conditions, improving the retention of sulfate groups.

[0051] Preferably, in the above step S3, the concentrated solution obtained by ultrafiltration through ultrafiltration membrane is added to a chromatography column containing an anion exchange resin for adsorption treatment, and the sulfate groups (-OSO3 - ) has a negative charge, so it is adsorbed to the resin through electrostatic action, and then it is cleaned by a low salt concentration washing relative chromatography column to elute impurities with weaker adsorption (such as some peptides), followed by an elution relative chromatography column with a high salt concentration. The eluent competitively replaces the saponin molecules bound to the resin to obtain a crude purified starfish saponin eluate.

[0052] Ultrafiltration cannot distinguish charged properties, while chromatography columns can separate negatively charged saponins from neutral or weakly negatively charged impurities (such as lipids and some pigments) through the specific adsorption of positively charged resins. Furthermore, the concentrated solution after ultrafiltration still contains a variety of saponin homologues (such as high- and low-sulfated saponins). Chromatographic columns achieve polarity fractionation through salt gradients, enabling the selective enrichment of highly active components.

[0053] Further preferably, the anion exchange resin is a polystyrene resin functionalized with a quaternary ammonium group, i.e., a quaternized polystyrene resin, the cleaning phase is a Tris-HCl buffer containing 0.1-0.2 M NaCl, the pH of the cleaning phase is 8.0-8.5, the flow rate is 1.5-2.5 mL / min, and the cleaning volume is 3-5 times the volume of the chromatography column. The elution phase is a Tris-HCl buffer containing 0.5-0.8 M NaCl, the pH of the elution phase is 8.0-8.5, the flow rate is 1.0-1.5 mL / min, and the elution volume is 2-3 times the volume of the chromatography column.

[0054] After the elution step, the pH of the eluate needs to be adjusted to prevent degradation of the active groups of the saponins due to long-term exposure to a high pH environment. Specifically, the pH of the eluate is adjusted to 6.5-7.5 to obtain a crude purified starfish saponin eluate.

[0055] Step S3 is mainly based on charge interaction, preferentially adsorbing negatively charged starfish saponins to effectively remove impurities with charge properties that differ greatly from those of saponins, such as residual polysaccharides and proteins. However, neutral or weakly polar lipids are difficult to be effectively separated by ion exchange due to their lack of strong charge properties and will still be co-eluted with saponins. Supercritical CO2 has a solubility for lipids that is more than 800 times that of saponins, and can achieve targeted degreasing through polarity differences, reducing lipid residues to <0.1%.

[0056] Preferably, in step S4, the supercritical CO2 degreasing treatment has a pressure of 25-35 MPa, a temperature of 35-45°C, a CO2 flow rate of 15-25 L / h, an entrainer of 1-3% by volume ethyl lactate aqueous solution, and a degreasing time of 1.5-2.5 h.

[0057] The present invention does not use toxic solvents such as chloroform and n-butanol throughout the process, CO2 can be recycled, and ethyl lactate is used as an edible-grade entrainer with a residual amount of ≤10ppm.

[0058] Further preferably, the freeze-drying process adopts two-stage drying: first, pre-freeze at -40 to -50°C and vacuum degree ≤10Pa for 4-6 hours. This stage can quickly freeze to form micron-sized ice crystals to prevent large ice crystals from piercing the saponin molecular structure. Then, the temperature is raised to 20-30°C, the vacuum degree is maintained at ≤15Pa, and the moisture is dried to ≤3% to obtain starfish saponin powder.

[0059] The extraction method of the present invention uses low temperature throughout the entire process, which can better retain the heat-sensitive groups of saponins and significantly improve the efficacy compared to traditional ultrasound-assisted methods. The composite enzymatic hydrolysis-chromatography-ultrafiltration process makes the saponins more pure, removes impurities such as polysaccharides and proteins, and can significantly reduce the immunogenicity risk of saponins, with higher safety. In addition, supercritical CO2 defatting reduces the lipid residue in the product to less than 0.1%, avoiding lipid peroxide-induced saponin oxidation failure and ensuring the stability of the saponins. Therefore, the starfish saponins prepared by the extraction method of the present invention can be used in anti-cancer drugs, anti-inflammatory drugs and immunomodulatory drugs.

[0060] In order to further clarify the solution of the present invention and its technical advancement, the following description is made in conjunction with specific embodiments and technical effects.

[0061] Example 1

[0062] This embodiment provides a method for extracting starfish saponins, comprising the following steps:

[0063] S1: Cut fresh starfish into pieces and soak them in liquid nitrogen for 8 minutes. Then grind them to obtain starfish powder with an average particle size of 0.3 mm. The starfish used in this example is Asterix rosenbergii from Dalian.

[0064] S2: Add starfish powder to a first buffer containing chitinase, collagenase, and hyaluronidase, and react at 45°C for 1.8 hours to obtain an enzymatic solution. The solid-to-liquid ratio of starfish powder to the first buffer is 1g:10mL. The first buffer is a citric acid-disodium hydrogen phosphate buffer with a pH of 6. The concentration of chitinase in the first buffer is 60U / g, the concentration of collagenase is 75U / g, the concentration of hyaluronidase is 50U / g, and the concentration of polyethylene glycol 6000 is 0.04wt%.

[0065] After step S2 and before step S3, the enzymatic hydrolyzate is subjected to ultrafiltration and concentration treatment through an ultrafiltration membrane with a molecular weight cutoff of 11 kDa to obtain a concentrated solution.

[0066] S3: The concentrated solution is added to a chromatography column filled with quaternized polystyrene resin for adsorption treatment, and then the column is cleaned with a low salt concentration washing phase, and then the column is eluted with a high salt concentration elution phase, and the pH of the eluate is adjusted to 7 to obtain a crude purified starfish saponin eluate. Wherein, the washing phase is a Tris-HCl buffer containing NaCl at a concentration of 0.15M, the pH of the washing phase is 8.2, the flow rate is 2mL / min, and the washing volume is 4 times the volume of the chromatography column. The elution phase is a Tris-HCl buffer containing NaCl at a concentration of 0.6M, the pH of the elution phase is 8.2, the flow rate is 1.2mL / min, and the elution volume is 2.5 times the volume of the chromatography column.

[0067] S4: The crude purified starfish saponin eluate was subjected to supercritical CO2 degreasing. Specifically, the supercritical CO2 degreasing process was performed at a pressure of 30 MPa, a temperature of 40° C., a CO2 flow rate of 20 L / h, an entrainer of a 2% by volume aqueous solution of ethyl lactate, and a degreasing time of 2 hours. The eluate was then pre-frozen at -45° C. and a vacuum of 10 Pa for 5 hours, then heated to 25° C., maintained at a vacuum of 15 Pa, and dried to a moisture content of 3% to obtain starfish saponin powder.

[0068] Example 2

[0069] This embodiment provides a method for extracting starfish saponins, comprising the following steps:

[0070] S1: Cut fresh starfish into pieces and soak them in liquid nitrogen for 5 minutes. Then grind them to obtain starfish powder with an average particle size of 0.5 mm. The starfish used in this example is Polyscias multispinosa from Dalian.

[0071] S2: Add starfish powder to a first buffer solution containing chitinase, collagenase, and hyaluronidase, and react at 40°C for 2 hours to obtain an enzymatic solution. The solid-liquid ratio of starfish powder to the first buffer solution is 1g:9mL, and the first buffer solution is a citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.5. The concentration of chitinase in the first buffer solution is 80U / g, the concentration of collagenase is 100U / g, the concentration of hyaluronidase is 50U / g, and the concentration of polyethylene glycol 8000 is 0.03wt%.

[0072] After step S2 and before step S3, the enzymatic hydrolyzate is subjected to ultrafiltration and concentration treatment through an ultrafiltration membrane with a molecular weight cutoff of 12 kDa to obtain a concentrated solution.

[0073] S3: The concentrated solution is added to a chromatography column filled with quaternized polystyrene resin for adsorption treatment, and then the column is cleaned with a low salt concentration washing phase, and then the column is eluted with a high salt concentration elution phase, and the pH of the eluate is adjusted to 6.5 to obtain a crude purified starfish saponin eluate. Wherein, the washing phase is a Tris-HCl buffer containing NaCl at a concentration of 0.1M, the pH of the washing phase is 8.0, the flow rate is 1.5mL / min, and the washing volume is 3 times the volume of the chromatography column. The elution phase is a Tris-HCl buffer containing NaCl at a concentration of 0.5M, the pH of the elution phase is 8.0, the flow rate is 1.0mL / min, and the elution volume is 2 times the volume of the chromatography column.

[0074] S4: The crude purified starfish saponin eluate was subjected to supercritical CO2 degreasing. Specifically, the supercritical CO2 degreasing process was performed at a pressure of 25 MPa, a temperature of 35° C., a CO2 flow rate of 15 L / h, an entrainer of 1% by volume ethyl lactate aqueous solution, and a degreasing time of 1.5 hours. The eluate was then pre-frozen at -40° C. and a vacuum of 10 Pa for 4 hours, then heated to 20° C., maintained at a vacuum of 14 Pa, and dried to a moisture content of 3% to obtain starfish saponin powder.

[0075] Example 3

[0076] This embodiment provides a method for extracting starfish saponins, comprising the following steps:

[0077] S1: Cut fresh starfish into pieces and soak them in liquid nitrogen for 10 minutes. Then grind them to obtain starfish powder with an average particle size of 0.2 mm. The starfish used in this example is Asterix rosenbergii from Dalian.

[0078] S2: Add starfish powder to a first buffer solution containing chitinase, collagenase, and hyaluronidase, and react at 50°C for 1.5 hours to obtain an enzymatic solution. The solid-liquid ratio of starfish powder to the first buffer solution is 1g:12mL, and the first buffer solution is a citric acid-disodium hydrogen phosphate buffer solution with a pH of 6.5. The concentration of chitinase in the first buffer solution is 40U / g, the concentration of collagenase is 50U / g, the concentration of hyaluronidase is 30U / g, and the concentration of polyethylene glycol 6000 is 0.05wt%.

[0079] After step S2 and before step S3, the enzymatic hydrolyzate is subjected to ultrafiltration and concentration treatment through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a concentrated solution.

[0080] S3: The concentrated solution is added to a chromatography column filled with quaternized polystyrene resin for adsorption treatment, and then the column is cleaned with a low salt concentration washing phase, and then the column is eluted with a high salt concentration elution phase, and the pH of the eluate is adjusted to 7.5 to obtain a crude purified starfish saponin eluate. Wherein, the washing phase is a Tris-HCl buffer containing NaCl at a concentration of 0.2M, the pH of the washing phase is 8.5, the flow rate is 2.5mL / min, and the washing volume is 5 times the volume of the chromatography column. The elution phase is a Tris-HCl buffer containing NaCl at a concentration of 0.8M, the pH of the elution phase is 8.5, the flow rate is 1.5mL / min, and the elution volume is 3 times the volume of the chromatography column.

[0081] S4: The crude purified starfish saponin eluate was subjected to supercritical CO2 degreasing. Specifically, the supercritical CO2 degreasing process was performed at a pressure of 35 MPa, a temperature of 45° C., a CO2 flow rate of 25 L / h, an entrainer of a 3% by volume aqueous solution of ethyl lactate, and a degreasing time of 2.5 hours. The eluate was then pre-frozen at -50° C. and a vacuum of 10 Pa for 6 hours, then heated to 30° C., maintained at a vacuum of 15 Pa, and dried to a moisture content of 3% to obtain starfish saponin powder.

[0082] The extraction rate of starfish saponins of Examples 1-3, the retention rate of sulfate groups (ie, saponin activity) and the saponin content in the extracts (ie, saponin purity) were tested and compared with the prior art to obtain Table 1.

[0083] Table 1 Comparison of the extraction rate, sulfate group retention rate and saponin content of starfish saponins in Examples 1-3 and the prior art

[0084]

[0085]

[0086] As shown in Table 1, compared with the organic solvent extraction and organic solvent extraction + ultrasound-assisted method, the starfish saponin extraction rate, sulfate group retention rate and saponin purity of Examples 1-3 are significantly improved.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting starfish saponins, characterized in that: The steps include: S1: freeze-dry fresh starfish and then crush them to obtain starfish powder; S2: enzymatically hydrolyzing the starfish powder with chitinase, collagenase, and hyaluronidase to obtain an enzymatic hydrolyzate; S3: using a chromatography column filled with anion exchange resin to adsorb the enzymatic hydrolyzate, and then eluting the chromatography column to obtain a crude purified starfish saponin eluate; S4: The crude purified starfish saponin eluate is defatted and purified by supercritical CO2, and then freeze-dried to obtain starfish saponin powder.

2. The method for extracting starfish saponins according to claim 1, wherein In step S1, fresh starfish are cut into pieces, then immersed in liquid nitrogen for 5-10 minutes, and then crushed to obtain starfish powder with a particle size of ≤0.5 mm.

3. The method for extracting starfish saponins according to claim 1, wherein In step S2, the starfish powder is added to a first buffer solution containing chitinase, collagenase, and hyaluronidase, and reacted at 40-50° C. for 1.5-2 hours to obtain an enzymatic hydrolyzate; The solid-liquid ratio of the starfish powder to the first buffer is 1 g: (9-12) mL, and the first buffer is a citric acid-disodium hydrogen phosphate buffer with a pH of 5.5-6.5; The concentration of chitinase in the first buffer is 40-80 U / g, the concentration of collagenase is 50-100 U / g, and the concentration of hyaluronidase is 30-50 U / g.

4. The method for extracting starfish saponins according to claim 3, wherein The first buffer further contains 0.03-0.05 wt% of an enzyme stabilizer; The enzyme stabilizer is polyethylene glycol 6000 or polyethylene glycol 8000.

5. The method for extracting starfish saponins according to claim 1, wherein After step S2 and before step S3, the enzymatic hydrolyzate is ultrafiltered and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 10-12 kDa to obtain a concentrated solution.

6. The method for extracting starfish saponins according to claim 5, wherein In step S3, the concentrated solution is added to a chromatography column filled with anion exchange resin for adsorption treatment, and then the chromatography column is cleaned by a cleaning relative column, and then eluted by an elution relative column to obtain a crude purified starfish saponin eluate.

7. The method for extracting starfish saponins according to claim 6, wherein The anion exchange resin is a polystyrene resin functionalized with quaternary ammonium groups; The cleaning phase is a Tris-HCl buffer containing 0.1-0.2 M NaCl, the pH of the cleaning phase is 8.0-8.5, the flow rate is 1.5-2.5 mL / min, and the cleaning volume is 3-5 times the volume of the chromatography column; The elution phase is a Tris-HCl buffer containing NaCl at a concentration of 0.5-0.8 M, the pH of the elution phase is 8.0-8.5, the flow rate is 1.0-1.5 mL / min, and the elution volume is 2-3 times the volume of the chromatography column; After the elution is completed, the pH of the eluate is adjusted to 6.5-7.5 to obtain a crude purified starfish saponin eluate.

8. The method for extracting starfish saponins according to claim 1, wherein In step S4, the supercritical CO2 degreasing treatment pressure is 25-35MPa, the temperature is 35-45°C, the CO2 flow rate is 15-25L / h, the entrainer is an ethyl lactate aqueous solution with a volume fraction of 1-3%, and the degreasing time is 1.5-2.5h.

9. The method for extracting starfish saponins according to claim 1, wherein In step S4, the freeze-drying process adopts two-stage drying: First, pre-freeze at -40--50 DEG C and vacuum degree ≤10Pa for 4-6 hours, then heat to 20-30 DEG C, maintain vacuum degree ≤15Pa, and dry to moisture ≤3% to obtain starfish saponin powder.

10. Use of asteraenin prepared by the extraction method according to any one of claims 1 to 9 in anticancer drugs, anti-inflammatory drugs or immunomodulatory drugs.

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