Preparation method of sea cucumber secondary saponin

Abstract

The invention discloses a method for preparing sea cucumber secondary saponin by enzymolysis of sea cucumber alkane saponin in apostichopus japonicus. According to the method, sea cucumber total saponins or sea cucumber saponin monomers are used as raw materials, helicase is adopted for hydrolysis, and obtained sea cucumber saponin zymolyte is subjected to silica gel and / or reversed-phase column chromatography separation and / or preparative high performance liquid chromatography separation and purification to obtain sea cucumber secondary saponin Cladoloside A monomers.

Description

Technical Field

[0001] The invention belongs to the research field of natural drug chemistry. Background Art

[0002] Apostichopus japonicus Selenka belongs to the phylum Echinopermata, class Holothuroidea, order Aspidochirotida, family Stichopodidae, and genus Apostichopus. It is a traditional food and Chinese medicinal material in my country. It is the main edible sea cucumber species in northern my country and the most precious sea cucumber species, known as the "crown of ginseng". Traditional medicine believes that Apostichopus japonicus Selenka has a tonic effect and can treat symptoms such as menstrual blood deficiency, weakness and fatigue, impotence, nocturnal emission, frequent urination, dry bowels and constipation. Modern medical research shows that Apostichopus japonicus is rich in essential nutrients for the human body, such as amino acids, vitamins, fatty acids, macro- and trace elements, which can regulate human growth and metabolism, enhance human immunity, and promote human brain development. The imitation sea cucumber also contains saponins, cerebrosides, gangliosides, acidic mucopolysaccharides, lectins and other novel secondary metabolites. These substances have multiple biological activities such as anti-tumor, antibacterial, antiviral, anticoagulant, and anti-radiation.

[0003] Sea cucumber saponins are important active ingredients in sea cucumbers. They are composed of aglycones and sugar chains connected by β-glycosidic bonds. Most of them belong to lanosterol-type triterpenoid oligosaccharides. The sugar chain of saponins in sea cucumbers is generally composed of 2-6 monosaccharides, such as xylose (Xyl), glucose (Glc), quinolose (Qui), 3-methyl glucose (MeGlc), etc. The first monosaccharide connecting the aglycone is often xylose. The aglycones of saponins in sea cucumbers are often tetracyclic triterpenes and pentacyclic triterpenes. According to the structure of the aglycone, they can be divided into two types: holostane-type and non-holostane-type. Holostane-type aglycones generally contain 18(20)-lactone rings, while non-holostane-type aglycones have 18(16)-lactone rings or no lactone ring structure. At present, there are 49 sea cucumber saponins reported in Simulanthus japonicus, among which, the majority are sea cucumber ane saponins, among which, Holotoxin A1, Holotoxin B1 and Cladoloside B are the three sea cucumber saponin compounds with higher content in Simulanthus japonicus. The sea cucumber saponins in Simulanthus japonicus have multiple biological activities such as anti-tumor 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 to discover potential anticancer drugs with excellent activity. The main methods for hydrolyzing saponins are acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Among them, the conditions of acid and alkaline hydrolysis are relatively drastic, which can easily cause changes in the nucleus of saponins, while the conditions of enzymatic hydrolysis are relatively mild and have strong specificity, making it easy to prepare target secondary saponins. Chen Jinglan et al. (Enzymatic hydrolysis of sea cucumber saponin Echinoside A and its product structure identification and hemolytic toxicity [J]. Food Industry Science and 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 hydrolysis effects on Echinoside A, while amyloglucosidase and β-galactosidase had no enzymatic hydrolysis effects on Echinoside A. The enzymatic hydrolysis products were analyzed by HPLC-MS, and it was speculated that there were two enzymatic hydrolysis products of Echinoside A, a disaccharide secondary sea cucumber saponin and a monosaccharide secondary sea cucumber saponin. Summary of the invention

[0005] The present invention provides a method for preparing sea cucumber secondary saponins by enzymatic hydrolysis of sea cucumber-like trelotanyl saponins. The specific contents of the present invention are as follows.

[0006] A method for preparing sea cucumber secondary saponins comprises the following steps: (1) dissolving sea cucumber total saponins or sea cucumber saponin monomers in a buffer solution and ultrasonically dissolving them 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 with the enzyme solution to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzate; (4) cooling the enzymatic hydrolyzate, precipitating the enzymatic hydrolyzate with alcohol, and recovering the solvent from the supernatant to obtain a sea cucumber saponin enzymatic hydrolyzate; and (5) subjecting the sea cucumber saponin enzymatic hydrolyzate to silica gel column chromatography and / or reverse phase column chromatography separation and / or preparative high performance liquid chromatography separation and purification to obtain a sea cucumber secondary saponin Cladoloside A monomer.

[0007] The selected total saponin of sea cucumber is the total saponin of sea cucumber imitation, and the selected sea cucumber saponin monomer is the sea cucumber saponin HolotoxinB1 or the sea cucumber saponin Cladoloside B.

[0008] The enzyme selected was snail enzyme.

[0009] The buffer solution used was sodium dihydrogen phosphate-citric acid buffer solution at pH 5.0.

[0010] The alcohol precipitation reagent selected is ethanol.

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

[0012] The stationary phase of the reverse phase column chromatography 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.

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

[0014] The sea cucumber secondary saponin Cladoloside A is used for preparing anticancer drugs.

[0015] The sea cucumber secondary saponin Cladoloside A and medically acceptable pharmaceutical excipients form the medicine.

[0016] Sea cucumber secondary saponin Cladoloside A is used to prepare health products with anti-cancer function.

[0017] Sea cucumber secondary saponin Cladoloside A and other auxiliary materials are prepared into health products.

[0018] The enzymatic hydrolysis method of sea cucumber saponins provided by the present invention can be used to enzymatically hydrolyze various saponin components in sea cucumbers and other sea cucumbers to obtain various sea cucumber secondary saponins. Molecular docking simulation results indicate that sea cucumber secondary saponins have better cytotoxic effects on triple-negative breast cancer MDA-MB-231 cells and liver cancer HepG2 cells than sea cucumber saponins.

[0019] The purity of the Cladoloside A monomer prepared by the invention can reach more than 99%, and can be used for quality control of imitation sea cucumber and related products and for preparation of anticancer drug compositions and other products.

[0020] The present invention uses MS and NMR to identify the chemical structure of the enzymatic hydrolyzate MJ-1 as Cladoloside A. The chemical structure of MJ-1, the structural identification process and related data are as follows:

[0021]

[0022] The structure of Cladoloside A

[0023] Compound MJ-1 is a white powder. High resolution mass spectrometry (HR-ESI-MS) gives m / z 1077.5228 [M+Na] + The peak indicates that the molecular weight of compound MJ-1 is 1054.7545, and its molecular formula is estimated to be C 53 H82 O 21 Compound MJ-1 13 The CNMR (C5D5N, 150MHz) spectrum gives a total of 53 carbon signals, including 30 parent carbon signals and 23 sugar carbon signals. Combining the DEPT 90° spectrum and the DEPT 135° spectrum, it can be seen that the δ C 213.26 and δ C 176.14 The two quaternary carbon signals are carbonyl carbon signals. C 111.21 and δ C 151.54 is speculated to be the double bond carbon signal on the parent nucleus, where δ C 111.21 is the tertiary carbon signal, which indicates that the double bond on the parent nucleus is located at position 9 (11). C 105.93, δ C 105.81, δ C 105.65 and δ C 105.44 is speculated to be the terminal carbon signal of sugar. 13 C NMR spectrum gives 8 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, and it is speculated that the sugar group of compound MJ-1 may contain 3-methylglucose. C 18.21 is the characteristic signal of methyl group in quinolose, and it is speculated that the sugar group of compound MJ-1 may contain quinolose. 1 The H NMR spectrum (C5D5N, 600 MHz) gave eight methyl proton signals. Combined with the HSQC spectrum and H 1 -H 1 COSY spectrum 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 0.78 (s, H-30) These six H signals are characteristic methyl proton signals of the sea sambucil skeleton structure, and δ H1.64 (d, J = 6.0 Hz) is the methyl proton signal of quinolose on the sugar chain, δ H 3.74(s) is the signal of the methoxy proton in 3-methylglucose on the sugar chain. H 5.07 (d, J = 7.2 Hz), δ H 4.70(d,J=7.2Hz),δ H 5.20 (d, J = 7.8 Hz) and δ H 4.72 (d, J = 7.8 Hz) These four groups of proton signals can be attributed to the terminal proton signals of sugars. It can be inferred that there may be four molecules of sugar in compound MJ-1. Since their J values ​​are all greater than 7.2 Hz, it can be known that the configuration of sugars is β-D. In the HMBC spectrum, the terminal proton signal δ H 4.70 (d, J = 7.2 Hz) and δ C 89.06 (C-3) long-range correlation, it can be judged that the C-3 position of the parent nucleus is connected to a molecule of xylose. The terminal proton signal of quinolose δ H 5.07 (d, J = 7.2 Hz) and the C-2 carbon signal of xylose connected to the mother nucleus δ C 84.42 has a long-range correlation, indicating that glucose is connected to the 2-position of xylose. The terminal proton signal of another molecule of xylose δ H 4.72 (d, J = 7.2 Hz) and the C-4 carbon signal of quinolone δ C 86.21 has a long-range correlation, indicating that the xylose is connected to the 4-position of quinolose. The terminal proton signal of 3-methylglucose δ H 5.20 (d, J = 7.8 Hz) and the C-3 carbon signal of the above xylose C 87.59 has a long-range correlation, indicating that 3-methylglucose is connected to the 3-position of xylose. 1 H- 1 H COSY, HSQC, HMBC and literature corroborated each other, and finally determined that compound MJ-1 was 3β-O-[3-O-methyl-β-D-pyranose-(1→3)-β-D-pyranose-(1→4)-β-D-pyranose-quinol-(1→2)-β-D-pyranose]-sea sambucil-9(11),25(26)-diene-16-one. After literature search, it was confirmed that compound MJ-1 was Cladoloside A. 13 C NMR and 1 The H NMR chemical shift values ​​are shown in Table 1.

[0024] Table 1 Compound MJ-1 13 C NMR and 1H NMR data (C5D5N, 150 / 600 MHz, 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 (5 μm, 4.6 × 250 mm); mobile phase: methanol-water (64:36); flow rate: 1 mL / min; column temperature: 37.5 °C; injection volume: 20 μL;

[0029] ELSD detector drift tube temperature: 95°C; impact state: off; carrier gas pressure: 0.45 MPa; nebulizer flow rate: 2.0 L / min; magnification: 1.0 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 .Mass spectrum of Cladoloside A

[0031] Figure 2 .Cladoloside A 1 H-NMR graph

[0032] Figure 3 .Cladoloside A 13 C-NMR graph

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

[0034] Figure 5 .Cladoloside A 1 H- 1 H COSY plot

[0035] Figure 6 HMBC chart of .Cladoloside A

[0036] Figure 7 HSQC diagram of Cladoloside A DETAILED DESCRIPTION

[0037] The present invention is further explained below in conjunction with the preferred embodiments of the present invention. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, rather than to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0038] Example 1

[0039] Fresh sea cucumber was homogenized, anhydrous ethanol was used as solvent, heated and refluxed for extraction twice, the solid-liquid ratio was 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 passed through AB-8 macroporous adsorption resin, the loading rate was 4BV / h, 60% ethanol solution was selected for analysis, and the analysis solution was decompressed to recover the solvent, and the total saponin of sea cucumber was obtained.

[0040] Take 70.0 mg of total saponins of imitation sea cucumber, add 15 mL of disodium hydrogen phosphate-citric acid buffer solution of pH 5.0, dissolve by ultrasonication, and place in a 55°C water bath for preheating; take 70.0 mg of snail enzyme, add 10 mL of disodium hydrogen phosphate-citric acid buffer solution of pH 5.0 to dissolve, and place in a 55°C water bath for incubation for 20 min; mix the above-mentioned total saponins solution of imitation sea cucumber and snail enzyme solution, and react in a 55°C water bath for 60 h; cool the reaction solution to room temperature, add 100 mL of ethanol for alcohol precipitation, and recover the solvent from the supernatant under reduced pressure. After TLC inspection, it is the enzymatic hydrolysis product of imitation sea cucumber saponins (46.2 mg). Take the above-mentioned enzymatic hydrolysis product of imitation sea cucumber saponins, use acetonitrile-water system (volume ratio of 43:57) as the mobile phase, and use preparative high performance liquid chromatography (PHPLC) to separate and purify, and obtain 23.8 mg of compound Cladoloside A monomer. The purity of the HPLC-ELSD detector is 99.3%.

[0041] Example 2

[0042] Take 50.0 mg of sea cucumber saponin Holotoxin B1 monomer, add 12 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution, ultrasonically dissolve, and place in a 55°C water bath; take 100.0 mg of snail enzyme, add 15 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution to dissolve, and place in a 55°C water bath for incubation for 20 min; mix the above Holotoxin B1 solution and snail enzyme solution, and react in a 55°C water bath for 60 h; cool the reaction solution to room temperature, add 120 mL of ethanol to precipitate, and recover the solvent under reduced pressure in the supernatant. After TLC inspection, it is the enzymatic hydrolysis product of sea cucumber saponin (62.3 mg). Take the above sea cucumber saponin enzymatic hydrolysis product, use dichloromethane-methanol-water system (volume ratio of 13::2.8:0.3) as the mobile phase to separate and purify by silica gel column chromatography to obtain 54.8 mg of compound Cladoloside A monomer. The purity was 99.6% as determined by HPLC-ELSD detector.

[0043] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of the substantial technical content of the present invention. The substantial technical content of the present invention is broadly defined in the scope of the claims of the application. Any technical entity or method completed by relevant technical personnel, if it is exactly the same as that defined in the scope of the claims of the application, or an equivalent modification, equivalent replacement, improvement, etc., should be included in the scope of the claims.

Claims

1. A method for preparing sea cucumber secondary saponins, characterized in that The sea cucumber secondary saponin is obtained by the following steps: (1) dissolving the total sea cucumber saponin or the sea cucumber saponin monomer in a buffer solution and ultrasonically dissolving it to obtain a sea cucumber saponin solution; (2) dissolving the enzyme in the buffer solution to obtain an enzyme solution; (3) mixing the sea cucumber saponin solution with the enzyme solution to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzate; (4) cooling the enzymatic hydrolyzate, precipitating it with alcohol, and recovering the solvent from the supernatant to obtain a sea cucumber saponin hydrolyzate; (5) subjecting the sea cucumber saponin hydrolyzate to silica gel column chromatography and / or reverse phase column chromatography separation and / or preparative high performance liquid chromatography separation and purification to obtain a sea cucumber secondary saponin Cladoloside A monomer.

2. The method for preparing the sea cucumber secondary saponin according to claim 1, characterized in that The selected total saponin of sea cucumber is the total saponin of sea cucumber imitation, and the selected sea cucumber saponin monomer is the sea cucumber saponin Holotoxin B1 or the sea cucumber saponin Cladoloside B.

3. The method for preparing the sea cucumber secondary saponin according to claim 1, characterized in that The enzyme selected was snail enzyme.

4. The method for preparing the sea cucumber secondary saponin according to claim 1, characterized in that The buffer solution used was disodium hydrogen phosphate-citric acid buffer solution at pH 5.

0.

5. The method for preparing the sea cucumber secondary saponin according to claim 1, characterized in that The alcohol precipitation reagent selected is ethanol.

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

3.

7. The method for preparing sea cucumber secondary saponin according to claim 1, characterized in that The stationary phase of the reverse phase column chromatography 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.

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

62.

9. Use of the sea cucumber secondary saponin Cladoloside A according to claim 1 in the preparation of anticancer drugs.

10. A medicine consisting of the sea cucumber secondary saponin Cladoloside A according to claim 1 and medically acceptable pharmaceutical excipients.

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