Application of sea cucumber saponin and derivatives thereof in anti-helicobacter pylori product

Abstract

The invention discloses an application of sea cucumber saponin and / or a sea cucumber saponin derivative in an anti-helicobacter pylori product. The invention particularly discloses application of sea cucumber total saponins, sea cucumber saponin monomers, disaccharide-removed products of the sea cucumber saponin monomers, tetrasaccharide-removed products of the sea cucumber saponin monomers and desulfurization products of the sea cucumber saponin monomers in anti-helicobacter pylori products. The invention also discloses a preparation method of the sea cucumber total saponins, the sea cucumber saponin monomer, the disaccharide-removed product of the sea cucumber saponin monomer, the tetrasaccharide-removed product of the sea cucumber saponin monomer and the desulfurization product of the sea cucumber saponin monomer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological products, and particularly relates to an application of holothurian saponins. BACKGROUND

[0002] Helicobacter pylori (Hp) is a common gastrointestinal pathogen, a class I carcinogen that causes chronic gastritis, gastric ulcer, and gastric cancer. It is a gram-negative, microaerophilic spiral-shaped bacterium.

[0003] Currently, the conventional treatment of H. pylori mainly relies on antibiotics (such as clarithromycin and amoxicillin) and proton pump inhibitors. However, antibiotic resistance is becoming increasingly serious and has become a global health challenge. In addition, H. pylori can form biofilms in the stomach acid environment, further enhancing its drug resistance. According to statistics, the global resistance rate of H. pylori to clarithromycin has exceeded 20%, and in some areas, it has even reached 50%.

[0004] Traditional antibiotic therapy not only has limited effect, but also can disrupt the balance of intestinal flora, causing diarrhea, allergies and other side effects. Therefore, it is of great significance to develop new high-efficiency, low-toxicity anti-H. pylori products, especially those based on natural products.

[0005] Saponins are a class of natural active compounds widely found in plants, marine organisms, and some microorganisms. Their molecular structure consists of hydrophilic sugar chains and hydrophobic aglycons (triterpenoids or steroids). Due to their broad-spectrum antibacterial properties, low toxicity, and environmental friendliness, saponins have become a hot topic in the research of natural antibacterial agents.

[0006] The length of the sugar chain of saponins affects their binding ability to targets, and the hydrophobic aglycon can disrupt the integrity of microbial cell membranes. Among them, triterpenoid saponins generally have a broader antibacterial spectrum than steroidal saponins. For example, tea saponin has a stronger inhibitory effect on Staphylococcus aureus than steroidal saponins.

[0007] For sulfated saponins, the sulfate group binds to the phosphate group of the lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria through electrostatic interaction, which can disrupt the integrity of the outer membrane and cause the inactivation of membrane proteins such as porin OmpF.

[0008] Compared with plant-derived saponins (such as ginsenosides, tea saponin, and soybean saponin) and microbe-derived saponins, holothurian saponins contain a unique sulfated structure (sulfate group, -OSO3H) in their molecular structure, making them more easily bind to the lipopolysaccharide in the outer membrane of gram-negative bacteria, enhancing their membrane-targeting damage to gram-negative bacteria (such as Escherichia coli).

[0009] At present, there are few studies on the antibacterial activity of sea cucumber saponins and their derivatives, and no study on the anti-Helicobacter pylori activity of sea cucumber saponins has been reported. SUMMARY

[0010] The application discloses the effect of sea cucumber saponins and their derivatives on the anti-Helicobacter pylori activity, and provides the application of sea cucumber saponins and / or sea cucumber saponin derivatives as active ingredients in the preparation of anti-Helicobacter pylori products.

[0011] Further, the sea cucumber saponins can be total sea cucumber saponins or sea cucumber saponin monomers.

[0012] Further, the sea cucumber saponin monomers can be sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A.

[0013] Further, the sea cucumber saponin derivatives can be sea cucumber saponin desugar products and / or sea cucumber saponin desulfurization products.

[0014] Further, the sea cucumber saponin desugar products can be sea cucumber saponin desugar products and / or sea cucumber saponin desugar products (aglycone).

[0015] The application also provides an anti-Helicobacter pylori product, and the effective components of the product include sea cucumber saponins and / or sea cucumber saponin derivatives.

[0016] Further, the sea cucumber saponins can be total sea cucumber saponins or sea cucumber saponin monomers.

[0017] Further, the sea cucumber saponin monomers can be sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A.

[0018] Further, the sea cucumber saponin derivatives can be derivatives with sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A as structural units.

[0019] Further, the sea cucumber saponin derivatives can be sea cucumber saponin desugar products and / or sea cucumber saponin desulfurization products.

[0020] Further, the sea cucumber saponin desugar products can be sea cucumber saponin desugar products and / or sea cucumber saponin desugar products (aglycone).

[0021] Further, the sea cucumber saponin derivatives can be derivatives with sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A as structural units.

[0022] Further, the sea cucumber saponin and / or sea cucumber saponin derivative can be used in an amount of 1-10 mg / kg body weight.

[0023] Further, the sea cucumber saponin and / or sea cucumber saponin derivative can be used in an amount of 1-10 mg / kg body weight.

[0024] 1) freeze-drying and crushing the sea cucumber body wall;

[0025] 2) extracting the crushed sea cucumber of step 1) with 60% ethanol at room temperature, vacuum concentrating to obtain a sea cucumber extract;

[0026] 3) dissolving the sea cucumber extract of step 2) in water, extracting with water-saturated n-butanol, vacuum concentrating;

[0027] 4) dissolving the vacuum-concentrated sea cucumber n-butanol extract of step 3) in water, placing on a macroporous adsorption resin column, and then eluting with water and 70% ethanol as mobile phases in sequence, collecting the 70% ethanol eluate;

[0028] 5) vacuum-concentrating and drying the collected 70% ethanol eluate of step 4) to obtain sea cucumber total saponin.

[0029] Further, the sea cucumber saponin monomer Holothrium A can be prepared by the following steps: dissolving sea cucumber total saponin in water, placing on a normal-phase silica gel column, and gradient eluting with chloroform:methanol:water as mobile phases, collecting the 7:2.5:0.2 (v / v / v) eluate; vacuum-concentrating and freeze-drying the collected eluate to obtain sea cucumber saponin monomer Holothrium A.

[0030] Further, the sea cucumber saponin monomer Echinoside A can be prepared by the following steps: dissolving sea cucumber total saponin in water, placing on a reverse-phase silica gel column, and eluting with 50% methanol as mobile phase, collecting the eluate; vacuum-concentrating and freeze-drying the eluate to obtain sea cucumber saponin monomer Echinoside A.

[0031] Further, the sea cucumber saponin monomer Holothrium A or Echinoside A can be prepared by the following steps: dissolving sea cucumber saponin monomer (Holothrium A or Echinoside A) as substrate, oscillating with pectinase and sodium acetate buffer solution (pH=4.6) at 40°C for 72h, then centrifuging the reaction solution to obtain a precipitate, and drying the precipitate to obtain the desugared product of sea cucumber saponin monomer (Holothrium A or Echinoside A).

[0032] Further, the preparation method of the desulfurization product of the holothurin monomer can include the following steps: taking the holothurin monomer (Holothrium A or Echinoside A), adding 0.2 mol / L sulfuric acid aqueous solution, oscillating and hydrolyzing at 80°C for 22 hours, neutralizing the hydrolysis solution with NaOH solution, adding n-butanol for extraction, taking the n-butanol layer solution, and removing the solvent by rotary evaporation to obtain the desulfurization product of the holothurin monomer (Holothrium A or Echinoside A).

[0033] Further, the preparation method of the desulfurization product of the holothurin monomer can include the following steps: taking the holothurin monomer (Holothrium A or Echinoside A), adding 0.2 mol / L sulfuric acid aqueous solution, oscillating and hydrolyzing at 80°C for 22 hours, neutralizing the hydrolysis solution with NaOH solution, adding n-butanol for extraction, taking the n-butanol layer solution, and removing the solvent by rotary evaporation to obtain the desulfurization product of the holothurin monomer (Holothrium A or Echinoside A).

[0034] Specifically, the present application is as follows.

[0035] 1. Application of holothurin and / or holothurin derivative in anti-Helicobacter pylori preparation.

[0036] 2. The application according to item 1, characterized in that the holothurin is total holothurin or holothurin monomer; the holothurin monomer is holothurin monomer Holothrium A and / or holothurin monomer Echinoside A; and the holothurin derivative is desibasic product of holothurin monomer and / or desitetrose product of holothurin monomer and / or desulfurization product of holothurin monomer.

[0037] 3. The application according to item 1, characterized in that the Helicobacter pylori is Helicobacter pylori in the stomach of human or animal.

[0038] 4. The application according to any one of items 1 to 3, characterized in that the amount of holothurin or holothurin derivative used is 1-10 mg / kg body weight.

[0039] 5. An anti-Helicobacter pylori preparation, characterized in that the effective component of the preparation comprises holothurin and / or holothurin derivative.

[0040] 6. The preparation against H. pylori according to item 4, wherein the holothurian saponin is total holothurian saponin or holothurian saponin monomer; the holothurian saponin monomer is holothurian saponin monomer Holothrium A and / or holothurian saponin monomer Echinoside A; and the holothurian saponin derivative is a disaccharide-removing product of the holothurian saponin monomer and / or a tetrasaccharide-removing product of the holothurian saponin monomer, a sulfur-removing product of the holothurian saponin monomer.

[0041] 7. The preparation against H. pylori according to item 5, wherein the H. pylori is H. pylori in the stomach of human or animal.

[0042] 8. The preparation against H. pylori according to any one of items 5 to 7, wherein the holothurian saponin and / or the holothurian saponin derivative is used in an amount of 1 to 10 mg / kg of body weight.

[0043] 9. A method for preparing holothurian saponin monomer, comprising the following steps:

[0044] 1) freeze-drying and crushing the body wall of sea cucumber;

[0045] 2) extracting the crushed sea cucumber of step 1) with 60% ethanol at room temperature, and vacuum-concentrating to obtain sea cucumber extract;

[0046] 3) dissolving the sea cucumber extract of step 2) in water, extracting with water-saturated n-butanol, and vacuum-concentrating;

[0047] 4) dissolving the vacuum-concentrated sea cucumber n-butanol extract of step 3) in water, and loading onto a macroporous adsorption resin column, and then eluting with water and 70% ethanol as mobile phases in sequence, and collecting the 70% ethanol eluate;

[0048] 5) vacuum-concentrating and drying the collected 70% ethanol eluate of step 4) to obtain total holothurian saponin;

[0049] 6) loading the total holothurian saponin of step 5) onto a normal-phase silica gel column, and gradient-eluting with chloroform:methanol:water as mobile phases, and collecting the eluate of 7:2.5:0.2 (v / v / v);

[0050] 7) vacuum-concentrating and freeze-drying the collected eluate of step 6) to obtain holothurian saponin monomer Holothrium A.

[0051] 8) dissolving the total holothurian saponin of step 5) in water, and loading onto a reversed-phase silica gel column, and eluting with 50% methanol as mobile phase, and collecting the eluate;

[0052] 9) vacuum-concentrating and freeze-drying the eluate of step 8) to obtain holothurian saponin monomer Echinoside A.

[0053] 10. A method for preparing a double sugar-removed product of a holothurian glycoside monomer, comprising the following steps:

[0054] 1) reacting the holothurian glycoside monomer of item 9 as a substrate with pectinase and a sodium acetate buffer solution (pH = 4.6) in a 40℃ constant-temperature shaking incubator for 72 hours;

[0055] 2) centrifuging the reaction solution to obtain a precipitate;

[0056] 3) drying the precipitate obtained in step 2).

[0057] 11. A method for preparing a tetrasaccharide-removed product of a holothurian glycoside monomer, comprising the following steps:

[0058] 1) adding 2 mol / L trifluoroacetic acid aqueous solution to the holothurian glycoside monomer of claim 9 as a substrate, sealing, and hydrolyzing at 110℃ for 3 hours;

[0059] 2) neutralizing the hydrolysis solution of step 1) with a NaOH solution and adding n-butanol for extraction;

[0060] 3) taking the n-butanol layer solution of step 2), removing the solvent by rotary evaporation to obtain the tetrasaccharide-removed product of the holothurian glycoside monomer.

[0061] 12. A method for preparing a desulfurized product of a holothurian glycoside monomer, comprising the following steps:

[0062] 1) adding 0.2 mol / L sulfuric acid aqueous solution to the holothurian glycoside monomer of claim 9 as a substrate, and hydrolyzing at 80℃ for 22 hours;

[0063] 2) neutralizing the hydrolysis solution of step 1) with a NaOH solution and adding n-butanol for extraction;

[0064] 3) taking the n-butanol layer solution of step 2), removing the solvent by rotary evaporation to obtain the desulfurized product of the holothurian glycoside monomer.

[0065] The holothurian glycoside and derivatives thereof provided by the present application can inhibit Helicobacter pylori colonization, promote the clearance of Helicobacter pylori in the stomach, and inhibit the activity of Helicobacter pylori. In vitro bacteriostatic experiments show that the bacteriostatic effect of the holothurian glycoside is proportional to the concentration of the holothurian glycoside, and is better than that of ginsenoside monomer Rd; the bacteriostatic ability of the holothurian glycoside depends on the presence of sugar chains and sulfate groups, and the shorter the sugar chain, the weaker the bacteriostatic effect. In vivo animal experiments show that the holothurian glycoside and derivatives thereof can effectively reduce the number of Helicobacter pylori colonization in the stomach of mice. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 The molecular structure of the holothurian glycoside and derivatives thereof used in the embodiments of the present application is shown.

[0067] Figure 2 Figure 1 shows the liquid chromatogram of total sea cucumber saponins and sea cucumber saponin monomer Holothurin A and sea cucumber saponin monomer Echinoside A according to an embodiment of the present application.

[0068] Figure 3 Figure 4 shows the results of urease activity determination of H. pylori in the stomach of mice according to an embodiment of the present application. DETAILED DESCRIPTION

[0069] In order to better understand the present application, the following examples are given in conjunction with the accompanying drawings. It should be understood that the examples of the present application are only used to explain the present application but not to limit the present application, and the protection scope of the present application is only defined by the claims of the present application. The examples provided by the present application are only some preferred examples, and do not limit the present application in any form. Those skilled in the art can make changes, equivalent substitutions or modifications according to the content of the present application to form different embodiments. However, any changes and modifications made to the method of the present application, any equivalent substitutions, are within the protection scope of the present application without departing from the concept of the present application.

[0070] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] Example 1, Preparation of Sea Cucumber Saponins and Derivatives Thereof

[0072] 1.1, Extraction of Sea Cucumber Saponins

[0073] 1.1.1, Extraction of Total Sea Cucumber Saponins

[0074] The sea cucumbers were purchased from Nanshan Seafood Market in Qingdao. The body wall of the sea cucumbers was vacuum freeze-dried by a freeze dryer (Alpha 1-4 LSC, Marin Christ, Germany) under the following conditions: temperature -50°C, vacuum degree <10 kPa, and time 48 h. After freeze-drying, the freeze-dried sea cucumber body wall was ground by a grinder, and then extracted twice with 60% ethanol (v / v) at room temperature with a solid-liquid ratio of 1:4. The extraction solution was combined, and the solvent was removed by a rotary evaporator (RE-2000A, Shanghai Yalong Biochemical Instrument Factory) until the sea cucumber extract was in the form of an extract residue on the inner wall of the rotary evaporation flask.

[0075] The specific conditions for removing the solvent by rotary evaporation were as follows: rotary speed 30-50 rpm / min, water bath temperature 45-50°C, and vacuum degree 0.09 MPa.

[0076] The extract residue of the sea cucumber extract was dissolved with a small amount of water, and then extracted twice with water-saturated n-butanol. The n-butanol organic layer was combined, and the solvent was removed by a rotary evaporator to obtain the n-butanol extract of the sea cucumber.

[0077] The obtained sea cucumber n-butanol extract was dissolved in a small amount of water and placed on a DIAION macroporous adsorption resin (HP-20, Mitsubishi Chemical Corporation, Japan) chromatographic column, and then eluted with water and 70% ethanol as the mobile phase, respectively, and the 70% ethanol eluate was collected.

[0078] The 70% ethanol eluate was removed by rotary evaporation to obtain sea cucumber total saponins.

[0079] The Agilent liquid chromatography system (Agilent 1260 Infinity II, Agilent Technologies (China) Co., Ltd.) was used for detection. The sea cucumber saponin was sea cucumber alkane type triterpenoid saponin, and the general structure formula was as follows: Figure 1 A, and the liquid chromatography peak diagram of sea cucumber total saponins was as follows: Figure 2 A.

[0080] 1.1.2, Extraction of sea cucumber saponin monomer Holothrium A

[0081] The sea cucumber total saponin extract obtained by vacuum concentration was dissolved with methanol and placed on a normal phase silica gel chromatographic column (200-300 mesh column chromatography silica gel filler, provided by Yantai Zhifu Huangwu Silica Gel Development Test Factory), and gradient elution was performed using chloroform:methanol:water (10:1:0.1-7:3:0.3, v / v / v) as the mobile phase. The specific conditions were as follows: chloroform:methanol:water (10:1:0.1) eluted for 3 column volumes, chloroform:methanol:water (9:1.5:0.15) eluted for 5 column volumes, chloroform:methanol:water (8:2:0.2) eluted for 6 column volumes, chloroform:methanol:water (7.5:2.5:0.2) eluted for 7 column volumes, chloroform:methanol:water (7:2.5:0.2) eluted for 6 column volumes, and chloroform:methanol:water (7:3:0.3) eluted for 5 column volumes.

[0082] The eluate of chloroform:methanol:water (7:2.5:0.2) was collected and the solvent was removed by rotary evaporation.

[0083] The Agilent liquid chromatography system (Agilent 1260 Infinity II, Agilent Technologies (China) Co., Ltd.) was used for detection, which was sea cucumber saponin monomer Holothrium A (HA), and the general structure formula was as follows: Figure 1 B, and the liquid chromatography peak diagram was as follows: Figure 2 B.

[0084] 1.1.3, Extraction of sea cucumber saponin monomer Echinoside A

[0085] The sea cucumber total saponin extract obtained by vacuum concentration above was dissolved in a small amount of water and placed on a reversed-phase silica gel chromatography column (Chromatorex C18 SMB 100-20 / 45 reversed-phase filler, Fuji Chemical Co., Ltd., Japan), and then eluted with 50% methanol as the mobile phase.

[0086] The eluate was collected and the solvent was removed by rotary evaporation.

[0087] Detection was performed using an Agilent liquid chromatography system (Agilent 1260 Infinity II, Agilent Technologies (China) Co., Ltd.). The sea cucumber saponin monomer Echinoside A (EA) was detected, and the general structure is shown in Figure 1 C, and the liquid chromatography peak diagram is shown in Figure 2 C.

[0088] 1.1.4. Preparation of sea cucumber saponin desugaring product

[0089] The sea cucumber saponin monomer HA was mixed with Novozyme pectinase (Pectinex Ultra SP-L, Novozyme) and sodium acetate buffer solution (pH = 4.6) at a ratio of 25:1:12.5 (w / v / v) as a substrate, and incubated in a 40°C constant temperature shaking incubator at 130 r / min for 72 h. Then the reaction solution was centrifuged at 12000 r / min for 10 min, and the precipitate obtained by centrifugation was placed in a 65°C oven for drying to obtain the desugaring product HA-2 of sea cucumber saponin HA. The general structure of the sea cucumber saponin desugaring product HA-2 is shown in Figure 1 D.

[0090] The sea cucumber saponin monomer EA was mixed with pectinase (Novozyme, Pectinex Ultra SP-L) and sodium acetate buffer solution (pH = 4.6) at a ratio of 25:1:12.5 (w / v / v) as a substrate, and incubated in a 40°C constant temperature shaking incubator at 130 r / min for 72 h. Then the reaction solution was centrifuged at 12000 r / min for 10 min, and the precipitate obtained by centrifugation was placed in a 65°C oven for drying to obtain the desugaring product EA-2 of sea cucumber saponin EA. The general structure of the sea cucumber saponin desugaring product EA-2 is shown in Figure 1 E.

[0091] Take 0.5 g of sea cucumber saponin monomer HA, add 2 mol / L trifluoroacetic acid aqueous solution 500 mL, dissolve thoroughly, seal, hydrolysis reaction at 110℃ for 3 hours. The hydrolysis solution is neutralized with NaOH solution, and equal volume of n-butanol is added and extracted 4 times. Take the n-butanol layer solution, remove the solvent by rotary evaporation with a rotary evaporator, and obtain the desugar product of sea cucumber saponin HA, which is the aglycone HA-4 of sea cucumber saponin monomer HA. The structural formula of sea cucumber saponin aglycone HA-4 is as follows Figure 1 F.

[0092] Take 0.5 g of sea cucumber saponin monomer EA, add 2 mol / L trifluoroacetic acid aqueous solution 500 mL, dissolve thoroughly, seal, hydrolysis reaction at 110℃ for 3 hours. The hydrolysis solution is neutralized with NaOH solution, and equal volume of n-butanol is added and extracted 4 times. Take the n-butanol layer solution, remove the solvent by rotary evaporation with a rotary evaporator, and obtain the desugar product of sea cucumber saponin EA, which is the aglycone EA-4 of sea cucumber saponin monomer EA. The structural formula of sea cucumber saponin aglycone EA-4 is as follows Figure 1 G.

[0093] 1.1.5, Preparation of sea cucumber saponin desulfurization product

[0094] The sea cucumber saponin desulfurization product is prepared by mild acid hydrolysis method.

[0095] Take 1 g of sea cucumber saponin monomer HA, add 0.2 mol / L sulfuric acid aqueous solution 200 mL, dissolve thoroughly, and hydrolysis in 80℃ water bath shaker for 22 hours. The hydrolysis solution is neutralized with NaOH solution, and equal volume of n-butanol is added and extracted 4 times. Take the n-butanol layer solution, remove the solvent by rotary evaporation with a rotary evaporator, and obtain the desulfurization product of sea cucumber saponin HA, which is desulfurized saponin HA-dS. The structural formula of desulfurized saponin HA-dS of sea cucumber saponin monomer HA is as follows Figure 1 H.

[0096] Take 1 g of sea cucumber saponin monomer EA, add 0.2 mol / L sulfuric acid aqueous solution 200 mL, dissolve thoroughly, and hydrolysis in 80℃ water bath shaker for 22 hours. The hydrolysis solution is neutralized with NaOH solution, and equal volume of n-butanol is added and extracted 4 times. Take the n-butanol layer solution, remove the solvent by rotary evaporation with a rotary evaporator, and obtain the desulfurization product of sea cucumber saponin EA, which is desulfurized saponin EA-dS. The structural formula of desulfurized saponin EA-dS of sea cucumber saponin monomer EA is as follows Figure 1 I.

[0097] Example 2, In vitro bacteriostatic activity of sea cucumber saponin and its derivatives on Helicobacter pylori

[0098] 2.1, Culture of Helicobacter pylori

[0099] The standard strain of Helicobacter pylori, Sydney strain 1 (SS1), which was stored in glycerol tubes in a -80°C freezer, was taken out and rapidly thawed in a 37°C water bath.

[0100] Inside the biosafety cabinet, spread 50 μL of Helicobacter pylori bacterial suspension onto Columbia blood agar plates and incubate at 37°C for 3–5 days in a microaerophilic environment (7% oxygen, 10% carbon dioxide, 83% nitrogen).

[0101] After colonies grow on the plate, pick a single colony with a sterile inoculation loop and resuspend it in Brucellosis broth liquid medium containing 7%–10% fetal bovine serum. Incubate in a microaerophilic environment at 37°C and 180 rpm for 3–5 days.

[0102] Take Helicobacter pylori in the logarithmic growth phase and adjust the concentration to 1×10⁻⁶. 8 CFU / mL.

[0103] 2.2 Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Helicobacter pylori

[0104] The inhibitory effects of sea cucumber saponins and their derivatives on Helicobacter pylori were determined using the serial dilution method.

[0105] The samples to be tested included sea cucumber saponin monomers HA and EA, the desaccharide product HA-2 of sea cucumber saponin HA, the desaccharide product EA-2 of sea cucumber saponin EA, the aglycone HA-4 of sea cucumber saponin HA, the aglycone EA-4 of sea cucumber saponin EA, the desulfurization product HA-dS of sea cucumber saponin HA, and the desulfurization product EA-dS of sea cucumber saponin EA. The monomer Rd, which exhibited the strongest antibacterial activity among the ginsenoside monomers, was used as a control. Ginsenoside monomer Rd was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0106] Add 200 μL of the test sample solution with a concentration of 800 μg / mL to well A1 of a 96-well plate. Add 100 μL of Brucella broth liquid culture medium to wells A2 through A10 respectively. Then, transfer 100 μL of the test sample solution from well A1 to well A2, mix well, and then transfer 100 μL of the test sample solution from well A2 to well A3, and so on, until finally transferring 100 μL of the test sample solution from well A8 to well A9. Discard 100 μL of the solution from well A9. Add 100 μL of 1×10⁻⁶ mol / L of the test sample solution to wells A1 through A10 respectively. 8The concentration of the Helicobacter pylori bacterial solution was CFU / mL. The concentrations of the samples to be tested in the A1-A10 wells were 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0 μg / mL, respectively. The A11 well was a sterile control group, which contained no bacterial solution, no sample to be tested, and only 200 μL of the Broth Liquid Culture Medium. There were three biological replicates for each well.

[0107] After the 96-well plate was placed in a microaerobic environment (7% oxygen, 10% carbon dioxide, and 83% nitrogen) and incubated at 37°C for 48 hours, the lowest concentration without turbidity was the minimum inhibitory concentration (MIC) observed by naked eye. The suspension in each well without turbidity was taken and uniformly coated on a Columbia blood agar plate, which was then incubated at 37°C for another 48 hours. Colony counting was performed, and the lowest concentration without any colony growth was the minimum bactericidal concentration (MBC). The experimental results are shown in Table 1.

[0108] Table 1. Determination results of the minimum inhibitory concentration and the minimum bactericidal concentration of sea cucumber saponins and their derivatives on Helicobacter pylori Group Minimum inhibitory concentration (μg / mL) Minimum bactericidal concentration (μg / mL) HA 6.25 25 EA 12.5 50 HA-2 25 100 EA-2 50 200 HA-4 100 400 EA-4 200 >400 HA-dS 50 200 EA-dS 100 400 Rd 50 200

[0109] As shown in Table 1, the bacteriostatic and bactericidal effects of the two sea cucumber saponin monomers HA and EA on Helicobacter pylori were better than those of the ginsenoside monomer Rd.

[0110] Compared with the sea cucumber saponin monomer HA, the bacteriostatic and bactericidal effects of the product HA-2 obtained by removing two sugar groups from the sea cucumber saponin monomer HA were decreased, and the bacteriostatic and bactericidal effects of the aglycone HA-4 obtained by removing four sugar groups from the sea cucumber saponin monomer HA were further decreased. Compared with the sea cucumber saponin monomer EA, the bacteriostatic and bactericidal effects of the product EA-2 obtained by removing two sugar groups from the sea cucumber saponin monomer EA were decreased, and the bacteriostatic and bactericidal effects of the aglycone EA-4 obtained by removing four sugar groups from the sea cucumber saponin monomer EA were further decreased. The above results indicate that the bacteriostatic and bactericidal effects of sea cucumber saponins are positively correlated with the length of the sugar chain, and the shorter the length of the sugar chain, the weaker the bacteriostatic and bactericidal effects.

[0111] Compared with the sea cucumber saponin monomer HA and the sea cucumber saponin monomer EA, the bacteriostatic and bactericidal effects of the desulfurized products HA-dS and EA-dS of the sea cucumber saponin monomers HA and EA were significantly decreased, indicating that the bacteriostatic and bactericidal abilities of the sea cucumber saponin monomers are dependent on the presence of sulfate groups.

[0112] 2.3. Bacteriostatic circle experiment of Helicobacter pylori

[0113] The diameter of the bacteriostatic circle is a core index for quantifying the bacteriostatic effect of an antibacterial drug in a bacteriostatic experiment, and its significance lies not only in judging the sensitivity of bacteria but also in providing a key basis for the screening of bacteriostatic substances.

[0114] Take 100 μL of a concentration of 1×10 8 CFU / mL of Helicobacter pylori bacterial suspension was evenly spread onto a 90 mm diameter blood agar plate using a sterile spreader and allowed to stand for 10 minutes to allow absorption. Using sterile forceps, a 6 mm diameter sterile filter paper was placed on the plate. 20 μL of test samples at different concentration gradients (0–200 μg / mL) (including sea cucumber saponin monomers HA and EA) were dropped onto each filter paper. 20 μL of 25 μg / mL test samples (including HA-2 (after removing two sugars from HA), EA-2 (after removing two sugars from EA), HA-4 (after removing four sugars from HA), EA-4 (after removing four sugars from EA), HA-dS (desulfurized from HA), EA-dS (desulfurized from EA), and ginsenoside Rd) were dropped onto each filter paper. Clarithromycin at a concentration of 0.5 μg / mL was used as a positive control, and sterile water as a negative control. Plates were incubated at 37°C for 48 hours under microaerophilic conditions, and the diameter of the inhibition zone was measured using the cross-hatching method. Each experiment was repeated three times, and the average of the three experiments was used to obtain the inhibition zone diameter. The standard deviation was calculated. The evaluation criteria for the antibacterial effect are shown in Table 2. The measurement results of the inhibition zone diameter are shown in Table 3.

[0115] Table 2. Evaluation criteria for antibacterial effect Inhibitory zone diameter (cm) Inhibitory effect 0 − (0-5] + (5-10] ++ (10-15] +++ (15-20] ++++ (20-25] +++++ >25 ++++++

[0116] Table 3. Results of inhibition zone diameter determination of sea cucumber saponins and their derivatives against Helicobacter pylori

[0117]

[0118] As shown in Table 3, except for the sterile water negative control group, sea cucumber saponins and their derivatives, ginsenosides, and clarithromycin positive control groups all produced inhibition zones of different sizes, indicating that sea cucumber saponins and their derivatives can inhibit the growth of Helicobacter pylori, and the antibacterial effect of sea cucumber saponin monomers is better than that of Rd, the ginsenoside monomer with the strongest antibacterial ability. The antibacterial effect of sea cucumber saponin monomers shows a dose-dependent relationship, positively correlated with the effective concentration. At the same dose, the antibacterial effect of sea cucumber saponin monomer HA is better than that of sea cucumber saponin monomer EA. When the effective concentration of sea cucumber saponin monomer HA is 200 μg / mL, its antibacterial effect is comparable to that of the positive control clarithromycin (0.5 μg / mL). Compared with sea cucumber saponin monomers HA and EA, the antibacterial ability of desaccharified and desulfurized sea cucumber saponin monomers is significantly reduced, indicating that the sugar chain and sulfate group of sea cucumber saponins play a key role in their antibacterial ability.

[0119] Example 3, in vivo study on the ability of holothurin and its derivatives to colonize and clear Helicobacter pylori in the stomach

[0120] 3.1, animal experiment

[0121] Eighty-four SPF male BALB / c mice (7 weeks old, weighing 30.0 ± 1.0 g) were purchased from Shandong Poney Experimental Animal Technology Co., Ltd. The animal feeding environment was maintained at 21-23°C and 45-55% humidity, with a 12h light / 12h dark cycle. The mice could freely eat AIN-93G standard full nutrient feed and drink water during the experiment. After 7 days of adaptive feeding, the mice were randomly divided into 12 groups, with 7 mice in each group. The experimental period was 5 weeks. The animal experiment grouping and administration method are shown in Table 4.

[0122] Table 4, animal experiment grouping and administration method Group 1st-2nd week 3rd-4th week 5th week Normal control group Normal saline Normal saline+normal saline Normal saline Model group Normal saline Normal saline+bacterial solution Normal saline HA group HA HA+bacterial solution HA EA group EA EA+bacterial solution EA HA-2 group HA-2 HA-2+bacterial solution HA-2 EA-2 group EA-2 EA-2+bacterial solution EA-2 HA-4 group HA-4 HA-4+bacterial solution HA-4 EA-4 group EA-4 EA-4+bacterial solution EA-4 HA-dS group HA-dS HA-dS+bacterial solution HA-dS EA-dS group EA-dS EA-dS+bacterial solution EA-dS Rd group Rd Rd+bacterial solution Rd Positive control group Quadruple drug Quadruple drug+bacterial solution Quadruple drug

[0123] During the first and second weeks, each mouse in the normal control group and the model group was given 100 μL of normal saline by gavage; each mouse in each experimental group was given 100 μL of the corresponding saponin or saponin derivative by gavage, with the gavage amount being 1 mg / kg of body weight. The positive control group was given 100 μL of quadruple drugs by gavage, with the gavage amount being esomeprazole (purchased from Shandong New Day Pharmaceutical Co., Ltd.) 3.03 μg / g of body weight, amoxicillin (purchased from Shandong Lu Kang Pharmaceutical Co., Ltd.) 151.67 μg / g of body weight, clarithromycin (purchased from Jiangsu Hengrui Medicine Co., Ltd.) 75.83 μg / g of body weight, and colloidal bismuth pectin (purchased from Anhui Yongshengtang Pharmaceutical Co., Ltd.) 45.5 μg / g of body weight.

[0124] From the third week, each group continued to be given normal saline, saponin or saponin derivative, and quadruple drugs by gavage, and each mouse was given Helicobacter pylori liquid by gavage, with the total volume of the sample and the liquid being 100 μL, and the inoculation amount of Helicobacter pylori being about 1 × 10 8 CFU, with gavage being performed every 2 days for a total of 7 times. Before each gavage of the liquid, the mice were fasted for 12 hours, and after gavage, they were fasted and watered for 4 hours.

[0125] From the fifth week, the corresponding solution was continued to be given by gavage according to the method of the first and second weeks, in order to empty the Helicobacter pylori that did not colonize in the stomach as much as possible.

[0126] After the end of the last gavage day, the mice were fasted for 12 hours without water. After isoflurane anesthesia, the mice were sacrificed by enucleation and decapitation, and the mouse stomach tissue and gastric juice were collected.

[0127] 3.2, determination of urease activity:

[0128] The urease (UE) activity detection kit (Solarbio, item number: BC4110, Beijing Solarbio Science and Technology Co., Ltd.) was used to determine the colonization of H. pylori in the stomach of mice in each group, and the clearance rate of H. pylori was calculated. The whole process was operated according to the kit instructions. 0.1 g of mouse stomach tissue was taken and added to 1 mL of extraction solution, homogenized on ice and centrifuged at 4℃, 12000g for 15 min. Take 100 μL of supernatant, add 200 μL of reagent one and 400 μL of reagent two, mix well, and react at 37℃ for 1 h. Take 400 μL of the above reaction mixture, add 80 μL of reagent three and 60 μL of reagent four, mix well, and stand at room temperature for 20 min. Then add 460 μL of distilled water, mix well, and use a visible spectrophotometer to measure the absorbance value at 630 nm. The results are shown in Figure 3 . OD 630nm The higher the OD value, the higher the urease activity of H. pylori, the more the colonization of H. pylori in the stomach, and the lower the clearance rate.

[0129] Figure 3 The results show that sea cucumber saponin monomer HA has the strongest H. pylori clearance ability in the stomach, which is better than sea cucumber saponin monomer EA and the strongest bacteriostatic monomer Rd in ginseng saponin, and is equivalent to the effect of the four-drug antibiotic. Sea cucumber saponin monomers HA and EA both show that the shorter the sugar chain, the weaker the bactericidal effect, that is, the bactericidal effect is in the order of saponin monomer > desugar saponin > saponin aglycone. In addition, the bactericidal effect of saponin desulfurization product is greatly reduced, that is, saponin monomer > desulfurized saponin. The above results show that the sugar chain and sulfate group in the structure of sea cucumber saponin play an important role in enhancing the clearance of H. pylori in the gastric mucosa.

[0130] In summary, the sea cucumber saponin and its derivatives provided by the present application can inhibit the colonization of H. pylori, promote the clearance of H. pylori in the stomach, and inhibit the activity of H. pylori. The in vitro bacteriostatic experiment shows that the bacteriostatic effect of sea cucumber saponin is proportional to the concentration, and is better than ginseng saponin monomer Rd; the bacteriostatic ability of sea cucumber saponin depends on the presence of sugar chain and sulfate group, and the shorter the sugar chain, the weaker the bacteriostatic effect. The in vivo animal experiment shows that sea cucumber saponin and its derivatives can effectively reduce the colonization of H. pylori in the stomach of mice.

Claims

1. Application of sea cucumber saponin and / or sea cucumber saponin derivative in anti-Helicobacter pylori products.

2. Use according to claim 1, characterized in that: The sea cucumber saponin is sea cucumber total saponin or sea cucumber saponin monomer; the sea cucumber saponin monomer is sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A; the sea cucumber saponin derivative is a disaccharide-removing product, a tetrasaccharide-removing product or a sulfur-removing product of the sea cucumber saponin monomer.

3. Use according to claim 1, characterized in that: The Helicobacter pylori is Helicobacter pylori in the stomach of human or animal.

4. Use according to any one of claims 1 to 3, characterized in that: The sea cucumber saponin or sea cucumber saponin derivative is used in an amount of 1-10 mg / kg body weight.

5. An article of manufacture against H. pylori, characterized in that, The effective component of the product comprises sea cucumber saponin and / or sea cucumber saponin derivative.

6. The anti-H. pylori preparation according to claim 5, wherein: The sea cucumber saponin is sea cucumber total saponin or sea cucumber saponin monomer; the sea cucumber saponin monomer is sea cucumber saponin monomer Holothrium A and / or sea cucumber saponin monomer Echinoside A, and the sea cucumber saponin derivative is a disaccharide-removing product, a tetrasaccharide-removing product or a sulfur-removing product of the sea cucumber saponin monomer.

7. The anti-H. pylori preparation of claim 5, wherein the anti-H. pylori preparation is a pharmaceutical composition for oral administration. The Helicobacter pylori is Helicobacter pylori in the stomach of human or animal.

8. The anti-H. pylori preparation according to any one of claims 5 to 7, wherein: The sea cucumber saponin or sea cucumber saponin derivative is used in an amount of 1-10 mg / kg body weight.

9. A preparation method of sea cucumber saponin monomer, comprising the following steps: 1) freeze-drying sea cucumber body wall under vacuum and crushing; 2) extracting the crushed sea cucumber of step 1) with 60% ethanol at room temperature, and vacuum-concentrating to obtain sea cucumber extract; 3) dissolving the sea cucumber extract of step 2) in water, and extracting with water-saturated n-butanol, and vacuum-concentrating; 4) dissolving the n-butanol extract of step 3) in water, and placing on a macroporous adsorption resin column, and then eluting with water and 70% ethanol as mobile phases in sequence, and collecting the 70% ethanol eluate; 5) vacuum-concentrating and drying the 70% ethanol eluate collected in step 4) to obtain sea cucumber total saponin; 6) placing the sea cucumber total saponin of step 5) on a normal-phase silica gel column, and gradient-eluting with chloroform:methanol:water as mobile phases, and collecting the eluate with chloroform:methanol:water as 7:2.5:0.2; 7) vacuum-concentrating the eluate collected in step 6), and freeze-drying to obtain sea cucumber saponin monomer Holothrium A. 8) dissolving the sea cucumber total saponin of step 5) in water, and placing on a reversed-phase silica gel column, and eluting with 50% methanol as mobile phase, and collecting the eluate; 9) vacuum-concentrating and freeze-drying the eluate of step 8) to obtain sea cucumber saponin monomer Echinoside A.

10. A preparation method of disaccharide-removing product of sea cucumber saponin monomer, comprising the following steps: 1) taking the sea cucumber saponin monomer of claim 9 as substrate, and reacting with pectinase and sodium acetate buffer solution (pH=4.6) at 40℃ for 72 hours; 2) centrifuging the reaction solution to obtain precipitate; 3) drying the precipitate obtained in step 2) to obtain disaccharide-removing product of sea cucumber saponin monomer.

11. A preparation method of tetrasaccharide-removing product of sea cucumber saponin monomer, comprising the following steps: 1) taking the sea cucumber saponin monomer of claim 9 as substrate, and adding 2 mol / L trifluoroacetic acid aqueous solution, sealing, and hydrolyzing at 110℃ for 3 hours; 2) The hydrolysis solution of step 1) is neutralized with NaOH solution and extracted with n-butanol; 3) The n-butanol layer solution of step 2) is taken, and the solvent is removed by rotary evaporation to obtain the tetrasaccharide-removed product of sea cucumber saponin monomer.

12. A method for preparing a desulfurized product of sea cucumber saponin monomer, comprising the following steps: 1) Sea cucumber saponin monomer of claim 9 is used as a substrate, and 0.2 mol / L aqueous sulfuric acid solution is added for oscillation hydrolysis at 80°C for 22 hours; 2) The hydrolysis solution of step 1) is neutralized with NaOH solution and extracted with n-butanol; 3) The n-butanol layer solution of step 2) is taken, and the solvent is removed by rotary evaporation to obtain the desulfurized product of sea cucumber saponin monomer.

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