Preparation method of a sea cucumber secondary saponin

Abstract

The application discloses a method for preparing sea cucumber secondary saponin by enzymolysis of sea cucumber saponin of the holothurian type. The method uses total sea cucumber saponin or sea cucumber saponin monomer as raw material, adopts snail enzyme for hydrolysis, and obtains sea cucumber saponin enzymolysis product. The sea cucumber secondary saponin Cladoloside A monomer is obtained by separation and / or preparation high performance liquid chromatography separation and purification of the sea cucumber saponin enzymolysis product through silica gel and / or a reverse phase column.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry research. Background Technology

[0002] The spiny sea cucumber (Apostichopus japonicus Selenka) belongs to the phylum Echinopermata, class Holothuroidea, order Aspidochirotida, family Stichopodidae, and genus Apostichopus. It is a traditional food ingredient and medicinal herb in my country, and a major edible sea cucumber species in northern my country. It is considered the most prized sea cucumber, often referred to as the "King of Sea Cucumbers." Traditional medicine believes that the spiny sea cucumber has nourishing properties and can treat various symptoms such as menstrual blood loss, weakness, impotence, nocturnal emission, frequent urination, and constipation. Modern medical research shows that the spiny sea cucumber is rich in essential nutrients such as amino acids, vitamins, fatty acids, and macro and micro elements, which can regulate human growth and metabolism, enhance immunity, and promote brain development. The sea cucumber also contains novel secondary metabolites such as saponins, cerebrosides, gangliosides, acidic mucopolysaccharides, and lectins, which have various biological activities such as anti-tumor, antibacterial, antiviral, anticoagulant, and anti-radiation.

[0003] Sea cucumber saponins are important active components in sea cucumber cultivars, composed of aglycones and sugar chains linked by β-glycosidic bonds, and mostly belong to the lanosterol-type triterpenoid oligosaccharides. The sugar chains of saponins in sea cucumber cultivars are generally composed of 2-6 monosaccharides, typically xylose, glucose (Glc), quinolose (Qui), 3-methylglucose (MeGlc), etc., with xylose often being the first monosaccharide linking the aglycone. The aglycones of saponins in sea cucumber cultivars are often tetracyclic or pentacyclic triterpenoids, and can be classified into two types according to their structure: holostane-type and non-holostane-type. Holostane-type aglycones generally contain an 18(20)-lactone ring, while non-holostane-type aglycones have an 18(16)-lactone ring or no lactone ring structure. Currently, 49 types of sea cucumber saponins have been reported in *Stichopus japonicus*, of which the vast majority are of the scutellarin type. Among these, scutellarin-type saponins Holotoxin A1, Holotoxin B1, and Cladoloside B are the three most abundant sea cucumber saponin compounds in *Stichopus japonicus*. Sea cucumber saponins in *Stichopus japonicus* possess various biological activities, including antitumor activity, antioxidant activity, antibacterial activity, and anticoagulant activity.

[0004] Network pharmacology and molecular docking studies have shown that sea cucumber secondary saponins with shorter sugar chains have better anticancer activity than those with longer sugar chains. The preparation of sea cucumber secondary saponins with shorter sugar chains is helpful for discovering potential anticancer drugs with excellent activity. The main methods of saponin hydrolysis are acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Among them, acid and alkaline hydrolysis are relatively harsh and can easily cause changes in the saponin nucleus, while enzymatic hydrolysis is relatively mild and has strong specificity, making it easy to prepare target secondary saponins. Chen Jinglan et al. (Enzymatic hydrolysis of sea cucumber saponin Echinoside A and identification of its product structure and hemolytic toxicity[J]. Food Industry Technology, 2018, 39(10):95-99,109.) studied the enzymatic hydrolysis of sea cucumber saponin Echinoside A by five commercial enzymes. The results showed that pectinase, pectinase Ultra SP-L, and cellulase had enzymatic hydrolytic effects on Echinoside A, while amylase and β-galactosidase had no enzymatic hydrolytic effect on Echinoside A. HPLC-MS analysis of the hydrolysis products suggested that there are two types of hydrolysis products of Echinoside A: a disaccharide-based secondary sea cucumber saponin and a monosaccharide-based secondary sea cucumber saponin. Summary of the Invention

[0005] This invention provides a method for preparing secondary saponins from sea cucumber by enzymatic hydrolysis of sea cucumber saponins of the genus *Acanthopanax*. The specific details of this invention are as follows.

[0006] A method for preparing sea cucumber secondary saponins involves the following steps: (1) dissolving total sea cucumber saponins or sea cucumber saponin monomers in a buffer solution using ultrasonic dissolution to obtain a sea cucumber saponin solution; (2) dissolving an enzyme in a buffer solution to obtain an enzyme solution; (3) mixing the sea cucumber saponin solution and the enzyme solution to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate; (4) cooling the enzymatic hydrolysate, precipitating it with alcohol, and recovering the solvent from the supernatant to obtain the sea cucumber saponin enzymatic hydrolysis product; (5) separating and purifying the sea cucumber saponin enzymatic hydrolysis product by silica gel column chromatography and / or reversed-phase column chromatography and / or preparative high-performance liquid chromatography to obtain the sea cucumber secondary saponin Cladoloside A monomer.

[0007] The total saponins selected were sea cucumber saponins that were similar to those of sea cucumber, and the selected sea cucumber saponin monomers were sea cucumber saponin Holotoxin B1 or sea cucumber saponin Cladoloside B.

[0008] The selected enzyme is snail enzyme.

[0009] The selected buffer solution was a disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0.

[0010] The selected alcohol precipitation reagent is ethanol.

[0011] The mobile phase for silica gel column chromatography is a mixed solvent of dichloromethane, methanol, and water, with a gradient elution volume ratio of 13:2.5:0.3 to 13:3.3:0.3.

[0012] The stationary phase for reversed-phase column chromatography is ODS, and the mobile phase is a methanol-water mixture with a gradient elution volume ratio of 10:1-4:1 or an acetone-water mixture with a gradient elution volume ratio of 12:1-2:1.

[0013] The mobile phase for preparative high-performance liquid chromatography is an acetonitrile-water mixed solvent, preferably with a volume ratio of 43:57-38:62.

[0014] The sea cucumber secondary saponin Cladoloside A is used to prepare anticancer drugs.

[0015] Sea cucumber secondary saponin Cladoloside A is combined with medically acceptable pharmaceutical excipients to form a drug.

[0016] Cladoloside A, a secondary saponin from sea cucumber, is used to prepare health products with anti-cancer properties.

[0017] Sea cucumber secondary saponin Cladoloside A is prepared into health products with other excipients.

[0018] The sea cucumber saponin enzymatic hydrolysis method provided by this invention can be used to enzymatically hydrolyze various saponin components in sea cucumbers such as *Stichopus japonicus* and others to obtain a variety of secondary sea cucumber saponins. Molecular docking simulation results suggest that secondary sea cucumber saponins have better cytotoxic effects on triple-negative breast cancer MDA-MB-231 cells and liver cancer HepG2 cells compared to primary sea cucumber saponins.

[0019] The Cladoloside A monomer prepared by this invention has a purity of over 99%, and can be used for quality control of sea cucumber and related products, as well as for the preparation of anticancer drug compositions and other products.

[0020] This invention used MS and NMR to identify the chemical structure of the isolated enzymatic hydrolysis product MJ-1 as Cladoloside A. The chemical structure of MJ-1, the structural identification process, and related data are as follows:

[0021]

[0022] The structural formula of Cladoloside A

[0023] Compound MJ-1 is a white powder, and high-resolution mass spectrometry (HR-ESI-MS) shows an m / z of 1077.5228 [M+Na]. + The peak indicates that the molecular weight of compound MJ-1 is 1054.7545, suggesting its molecular formula is C1. 53 H82 O 21 Compound MJ-1 13 The CNMR (C5D5N, 150MHz) spectrum yielded 53 carbon signals, including 30 nucleus carbon signals and 23 glycosyl carbon signals. Combined with the DEPT 90° and DEPT 135° spectra, it can be seen that δ0.05 appears in the low-field region. C 213.26 and δ C The two quaternary carbon signals at 176.14 are carbonyl carbon signals. δ C 111.21 and δ C 151.54 is presumed to be a double-bonded carbon signal on the parent nucleus, where δ C 111.21 is a tertiary carbon signal, indicating that the double bond on the parent nucleus is located at position 9(11). δ C 105.93, δ C 105.81, δ C 105.65 and δ C 105.44 is presumably a signal from the terminal carbon of the sugar. 13 The C NMR spectrum yielded eight primary carbon signals, namely δ C 60.98, δ C 18.21, δ C 22.30, δ C 26.96, δ C 22.45, δ C 16.88, δ C 28.68 and δ C 20.84, where δ C 60.98 should be the carbon signal of -OCH3, suggesting that the glycosyl group of compound MJ-1 may contain 3-methylglucose. C 18.21 is a characteristic signal of the methyl group in quinolones, suggesting that the glycosyl group of compound MJ-1 may contain quinolones. Compound MJ-1... 1 The 1H NMR (C5D5N, 600MHz) spectrum showed eight methyl proton signals, which, combined with the HSQC spectrum and H... 1 -H 1 COSY spectral analysis shows that δ H 1.30(s,H-19), δ H 1.27(s,H-21), δ H 1.57 (s, H-27), δ H 1.04 (s, H-28), δ H 1.46 (s, H-29) and δ H The six H signals at 0.78 (s, H-30) are characteristic methyl proton signals of the skeletal skeleton structure, while δ H1.64 (d, J = 6.0 Hz) represents the methyl proton signal in the quinolone sugar of the sugar chain, δ H 3.74(s) represents the methoxy proton signal in the 3-methylglucose of the sugar chain. Low-field region δ H 5.07 (d, J = 7.2 Hz), δ H 4.70 (d, J = 7.2 Hz), δ H 5.20 (d, J = 7.8 Hz) and δ H The four sets of proton signals at 4.72 (d, J = 7.8 Hz) can be attributed to the terminal proton signals of the sugar, suggesting that compound MJ-1 may contain tetramolecules of sugar. Since their J values ​​are all greater than 7.2 Hz, the sugar configuration is β-D. In the HMBC spectrum, the terminal proton signal δ of xylose... H 4.70 (d, J = 7.2 Hz) and δ C The 89.06 (C-3) long-range correlation indicates that a xylose molecule is attached to the C-3 position of the parent nucleus. The terminal proton signal δ of quinolone... H 5.07 (d, J = 7.2 Hz) and the C-2 carbon signal δ of xylose connecting the parent nucleus C The correlation at 84.42 indicates a long-range correlation, suggesting that glucose is linked to the xylose at position 2. The terminal proton signal δ of the other xylose molecule... H 4.72 (d, J = 7.2 Hz) and the C-4 carbon signal δ of quinolone C The presence of a long-range correlation at 86.21 indicates that the xylose is linked to position 4 of the quinolone. The terminal proton signal δ of 3-methylglucose... H 5.20 (d, J = 7.8 Hz) and the C-3 carbon signal δ of the above xylose C The presence of a long-range correlation at 87.59 indicates that 3-methylglucose is linked to the xylose at position 3. (This is followed by a conjugation / relationship to a specific value.) 1 H- 1 H COSY, HSQC, HMBC, and literature corroborate each other, ultimately identifying compound MJ-1 as 3β-O-[3-O-methyl-β-D-glucopyranose-(1→3)-β-D-xylpyranose-(1→4)-β-D-quinolose-(1→2)-β-D-xylpyranose]-scutellarin-9(11),25(26)-dien-16-one. Literature search confirms that compound MJ-1 is Cladoloside A. Cladoloside A... 13 C NMR and 1 The 1H NMR chemical shift values ​​are shown in Table 1.

[0024] Table 1. Compound MJ-1 13 C NMR and 11H NMR data (C5D5N, 150 / 600MHz, δ, ppm)

[0025]

[0026]

[0027] The HPLC-ELSD chromatographic conditions for analyzing the purity of compound Cladoloside A are as follows:

[0028] Chromatographic column - Agilent Eclipse XDB-C18 (5μm, 4.6×250mm); mobile phase - methanol-water (64:36); flow rate - 1mL / min; column temperature - 37.5℃; injection volume - 20μL;

[0029] ELSD detector drift tube temperature 95℃; impact state - off; carrier gas pressure - 0.45Mpa; atomizer flow rate - 2.0L / min; amplification factor - 1.0x. Attached Figure Description

[0030] Figure 1 Mass spectrum of Cladoloside A

[0031] Figure 2 Cladoloside A 1 H-NMR image

[0032] Figure 3 Cladoloside A 13 C-NMR image

[0033] Figure 4 DEPT90°, 135° diagram of Cladoloside A

[0034] Figure 5 Cladoloside A 1 H- 1 H COSY diagram

[0035] Figure 6 HMBC diagram of Cladoloside A

[0036] Figure 7 HSQC diagram of Cladoloside A Detailed Implementation

[0037] The present invention is further illustrated below with reference to preferred embodiments. It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0038] Example 1

[0039] Fresh sea cucumber (Stichopus japonicus) was homogenized and extracted twice by reflux with anhydrous ethanol as solvent at a solid-liquid ratio of 1:4. The extracts were combined, centrifuged, and the supernatant was concentrated to obtain the total saponin extract of sea cucumber. The total saponin extract of sea cucumber was then passed through AB-8 macroporous adsorption resin at a loading rate of 4 BV / h. The extract was elute with 60% ethanol solution, and the solvent was recovered under reduced pressure to obtain the total saponins of sea cucumber.

[0040] 70.0 mg of total saponins from sea cucumber (Stichopus japonicus) was dissolved in 15 mL of a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution and sonicated. The solution was then preheated in a 55°C water bath. 70.0 mg of snail enzyme was dissolved in 10 mL of the same solution and incubated in a 55°C water bath for 20 min. The total saponins and snail enzyme solutions were mixed and reacted in a 55°C water bath for 60 h. The reaction solution was cooled to room temperature, precipitated with 100 mL of ethanol, and the solvent was recovered from the supernatant under reduced pressure. TLC analysis confirmed the presence of 46.2 mg of enzymatically hydrolyzed sea cucumber saponins. The enzymatically hydrolyzed product was purified by preparative high-performance liquid chromatography (PHPLC) using an acetonitrile-water system (43:57 v / v) to obtain 23.8 mg of cladoloside A monomer. The purity was 99.3% as determined by HPLC-ELSD.

[0041] Example 2

[0042] 50.0 mg of Holotoxin B1 monomer was dissolved in 12 mL of disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) and sonicated. The solution was then placed in a 55°C water bath. 100.0 mg of snail enzyme was dissolved in 15 mL of disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) and incubated in a 55°C water bath for 20 min. The Holotoxin B1 solution and snail enzyme solution were mixed and reacted in a 55°C water bath for 60 h. The reaction solution was cooled to room temperature, precipitated with 120 mL of ethanol, and the solvent was recovered from the supernatant under reduced pressure. TLC analysis confirmed that the product was a sea cucumber saponin-like enzymatic hydrolysis product (62.3 mg). The above sea cucumber saponin-like enzymatic hydrolysis product was purified by silica gel column chromatography using a dichloromethane-methanol-water system (volume ratio 13:2.8:0.3) as the mobile phase to obtain 54.8 mg of Cladoloside A monomer. The purity was 99.6% as determined by HPLC-ELSD detector.

[0043] The above embodiments are merely preferred embodiments of the present invention and cannot be used to limit the scope of the essential technical content of the present invention. The essential technical content of the present invention is broadly defined within the scope of the claims. Any technical entity or method completed by a person skilled in the art that is completely identical to the one defined in the scope of the claims, or that is an equivalent modification, equivalent substitution, or improvement, should be included within the scope of the claims.

Claims

1. A method for preparing secondary saponins from sea cucumber, characterized in that... Sea cucumber secondary saponins were obtained through the following steps: (1) total saponins of sea cucumber were dissolved in a buffer solution by ultrasonic dissolution to obtain a sea cucumber saponin solution; (2) an enzyme was dissolved in a buffer solution to obtain an enzyme solution; (3) the sea cucumber saponin solution and the enzyme solution were mixed and subjected to enzymatic hydrolysis to obtain an enzymatic hydrolysate; (4) the enzymatic hydrolysate was cooled and precipitated with alcohol, and the solvent was recovered from the supernatant to obtain the sea cucumber saponin enzymatic hydrolysis product; (5) the sea cucumber saponin enzymatic hydrolysis product was separated and purified by silica gel column chromatography and / or reversed-phase column chromatography and / or preparative high performance liquid chromatography to obtain sea cucumber secondary saponin Cladoloside. A monomer; the selected enzyme is snail enzyme; the selected buffer solution is pH 5.0 disodium hydrogen phosphate-citric acid buffer solution; the selected alcohol precipitation reagent is ethanol; the total saponins of the sea cucumber were prepared as follows: fresh sea cucumber was homogenized, and extracted twice by heating and reflux using anhydrous ethanol as solvent, with a material-to-liquid ratio of 1:

4. The extracts were combined, centrifuged, and the supernatant was concentrated to obtain the total saponin extract of the sea cucumber; the total saponin extract of the sea cucumber was passed through AB-8 macroporous adsorption resin at a loading rate of 4 BV / h, and 60% ethanol solution was used for elution. The solvent was recovered under reduced pressure from the elution solution to obtain the total saponins of the sea cucumber.

2. A method for preparing secondary saponins from sea cucumber, characterized in that... (1) Dissolve sea cucumber saponin Holotoxin B1 in a buffer solution and sonicate to obtain a sea cucumber saponin solution; (2) Dissolve the enzyme in a buffer solution to obtain an enzyme solution; (3) Mix the sea cucumber saponin solution and the enzyme solution and carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate; (4) Cool the enzymatic hydrolysate, precipitate it with alcohol, and recover the solvent from the supernatant to obtain the sea cucumber saponin enzymatic hydrolysis product; (5) Separate and purify the sea cucumber saponin enzymatic hydrolysate by silica gel column chromatography and / or reversed-phase column chromatography and / or preparative high performance liquid chromatography to obtain the sea cucumber secondary saponin Cladoloside A monomer; the enzyme used is snail enzyme; the buffer solution used is a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution; the alcohol precipitation reagent used is ethanol.

3. The method for preparing sea cucumber secondary saponins according to claim 1 or 2, characterized in that... The mobile phase for silica gel column chromatography is a mixed solvent of dichloromethane, methanol, and water, with a gradient elution volume ratio of 13:2.5:0.3 to 13:3.3:0.

3.

4. The method for preparing sea cucumber secondary saponins according to claim 1 or 2, characterized in that... The reversed-phase column chromatography stationary phase is ODS, and the mobile phase is a methanol-water mixed solvent with a gradient elution volume ratio of 10:1-4:1 or an acetone-water mixed solvent with a gradient elution volume ratio of 12:1-2:

1.

5. The method for preparing sea cucumber secondary saponins according to claim 1 or 2, characterized in that... The mobile phase for the preparative high-performance liquid chromatography is an acetonitrile-water mixed solvent with a volume ratio of 43:57-38:62.

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