Application of sea cucumber saponins and derivatives thereof in anti-helicobacter pylori preparations

Abstract

The application discloses application of sea cucumber saponins and / or sea cucumber saponin derivatives in anti-helicobacter pylori products. The application specifically 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 desulfurized products of the sea cucumber saponin monomers in anti-helicobacter pylori products. The application also discloses preparation methods of the sea cucumber total saponins, the sea cucumber saponin monomers, the disaccharide-removed products of the sea cucumber saponin monomers, the tetrasaccharide-removed products of the sea cucumber saponin monomers and the desulfurized products of the sea cucumber saponin monomers.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to the application of sea cucumber saponins. Background Technology

[0002] Helicobacter pylori (Hp) is a Gram-negative, microaerophilic spiral bacterium. It is a common gastrointestinal pathogen and a Group 1 carcinogen that causes chronic gastritis, gastric ulcers, and gastric cancer.

[0003] Currently, routine treatment for Helicobacter 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. Furthermore, Helicobacter pylori can form biofilms in the acidic environment of the stomach, further enhancing its resistance. Statistics show that the global resistance rate of Helicobacter pylori to clarithromycin has exceeded 20%, and in some regions it is as high as 50%.

[0004] Traditional antibiotic treatments not only have limited effectiveness but can also disrupt the balance of gut microbiota, causing side effects such as diarrhea and allergies. Therefore, developing novel, highly effective, and low-toxicity anti-Helicobacter pylori products, especially those based on natural products, is of great significance.

[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 aglycones (triterpenes or steroids). Due to their broad-spectrum antibacterial properties, low toxicity, and environmental friendliness, they have become a hot topic in the research of natural antibacterial agents.

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

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

[0008] Compared with plant-derived saponins (such as ginsenosides, tea saponins, and soybean saponins) and microbial saponins, holothurian saponins have a unique sulfated structure (sulfate group, -OSO3H) in their molecular structure, which makes them more likely to bind to the lipopolysaccharides of the outer membrane of Gram-negative bacteria, thereby enhancing their membrane-targeting and disruptive effects on Gram-negative bacteria (such as Escherichia coli).

[0009] Currently, there is limited research on the antibacterial properties of sea cucumber saponins and their derivatives, and no studies have been reported on the antibacterial activity of sea cucumber saponins against Helicobacter pylori. Summary of the Invention

[0010] This invention clarifies the effects of sea cucumber saponins and their derivatives on anti-Helicobacter pylori, 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] Furthermore, the aforementioned sea cucumber saponins can be total sea cucumber saponins or sea cucumber saponin monomers.

[0012] Furthermore, the aforementioned sea cucumber saponin monomers can be sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A.

[0013] Furthermore, the above-mentioned sea cucumber saponin derivatives can be desaccharified sea cucumber saponin products and / or desulfurized sea cucumber saponin products.

[0014] Furthermore, the above-mentioned desaccharified sea cucumber saponins can be desaccharified sea cucumber saponins and / or detetrasaccharified sea cucumber saponins (aglycones).

[0015] The present invention also provides an anti-Helicobacter pylori product, wherein the active ingredients of the product include sea cucumber saponins and / or sea cucumber saponin derivatives.

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

[0017] Furthermore, the aforementioned sea cucumber saponin monomers can be sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A.

[0018] Furthermore, the above-mentioned sea cucumber saponin derivatives can be derivatives with sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A as structural units.

[0019] Furthermore, the above-mentioned sea cucumber saponin derivatives can be desaccharified sea cucumber saponin products and / or desulfurized sea cucumber saponin products.

[0020] Furthermore, the above-mentioned desaccharified sea cucumber saponins can be desaccharified sea cucumber saponins and / or detetrasaccharified sea cucumber saponins (aglycones).

[0021] Furthermore, the above-mentioned sea cucumber saponin derivatives can be derivatives with desugared and / or desulfurized products of sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A as structural units.

[0022] Furthermore, the dosage of the above-mentioned sea cucumber saponins and / or sea cucumber saponin derivatives can be 1 to 10 mg / kg body weight.

[0023] Furthermore, the preparation method of total saponins from sea cucumber may include the following steps:

[0024] 1) The sea cucumber body wall is vacuum freeze-dried and then pulverized;

[0025] 2) Extract the crushed sea cucumber from step 1) with 60% ethanol at room temperature, and concentrate under vacuum to obtain sea cucumber extract;

[0026] 3) Dissolve the sea cucumber extract from step 2) in water, extract with water-saturated n-butanol, and concentrate under vacuum;

[0027] 4) Dissolve the sea cucumber n-butanol extract concentrated in vacuum in step 3) in water, place it on a macroporous adsorption resin column, and then elute with water and 70% ethanol as mobile phases in sequence, and collect the 70% ethanol eluent.

[0028] 5) The 70% ethanol eluent collected in step 4) is concentrated under vacuum and dried to obtain total sea cucumber saponins.

[0029] Furthermore, the preparation method of sea cucumber saponin monomer Holothurin A may include the following steps: dissolving total sea cucumber saponins in water, placing the solution on a normal-phase silica gel column, eluting with a gradient of chloroform:methanol:water as the mobile phase, and collecting the eluent in a 7:2.5:0.2 (v / v / v) ratio; concentrating the collected eluent under vacuum and freeze-drying it to obtain sea cucumber saponin monomer Holothurin A.

[0030] Furthermore, the preparation method of sea cucumber saponin monomer Echinoside A may include the following steps: dissolving total sea cucumber saponins in water, placing the solution on a reversed-phase silica gel column, eluting with 50% methanol as the mobile phase, collecting the eluent; concentrating the eluent under vacuum, and freeze-drying to obtain sea cucumber saponin monomer Echinoside A.

[0031] Furthermore, the preparation method of the dedisaccharide product of sea cucumber saponin monomer may include the following steps: using sea cucumber saponin monomer (Holothurin A or Echinoside A) as a substrate, reacting it with pectinase and sodium acetate buffer solution (pH=4.6) at 40°C with shaking for 72 h, then centrifuging the reaction solution to obtain a precipitate, drying the precipitate, and obtaining the dedisaccharide product of sea cucumber saponin monomer (Holothurin A or Echinoside A).

[0032] Furthermore, the preparation method of the detetrasaccharide product (aglycone) of sea cucumber saponin monomer may include the following steps: take sea cucumber saponin monomer (Holothurin A or Echinoside A), add 2 mol / L trifluoroacetic acid aqueous solution, seal, hydrolyze at 110℃ for 3 hours, neutralize the hydrolysate with NaOH solution, add n-butanol for extraction, take the n-butanol layer solution, remove the solvent by rotary evaporation, and obtain the detetrasaccharide product of sea cucumber saponin monomer (Holothurin A or Echinoside A).

[0033] Furthermore, the preparation method of the desulfurization product of sea cucumber saponin monomer may include the following steps: take sea cucumber saponin monomer (Holothurin A or Echinoside A), add 0.2 mol / L sulfuric acid aqueous solution, hydrolyze by shaking at 80°C for 22 hours, neutralize the hydrolysate with NaOH solution, add n-butanol for extraction, take the n-butanol layer solution, remove the solvent by rotary evaporation, and obtain the desulfurization product of sea cucumber saponin monomer (Holothurin A or Echinoside A).

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

[0035] 1. Application of sea cucumber saponins and / or sea cucumber saponin derivatives in anti-Helicobacter pylori products.

[0036] 2. The application as described in item 1, characterized in that: the sea cucumber saponin is total sea cucumber saponin or sea cucumber saponin monomer; the sea cucumber saponin monomer is sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A; the sea cucumber saponin derivative is a dedisaccharide product of sea cucumber saponin monomer and / or a detetrasaccharide product of sea cucumber saponin monomer, or a desulfurization product of sea cucumber saponin monomer.

[0037] 3. The application as described in item 1, characterized in that: the Helicobacter pylori is a Helicobacter pylori found in the stomach of a human or animal.

[0038] 4. The application as described in any one of items 1 to 3, characterized in that: the amount of sea cucumber saponin or sea cucumber saponin derivative used is 1 to 10 mg / kg body weight.

[0039] 5. A product for treating Helicobacter pylori, characterized in that the active ingredient of the product includes sea cucumber saponins and / or sea cucumber saponin derivatives.

[0040] 6. The anti-Helicobacter pylori product as described in item 4, characterized in that: the sea cucumber saponin is total sea cucumber saponin or sea cucumber saponin monomer; the sea cucumber saponin monomer is sea cucumber saponin monomer Holothurin A and / or sea cucumber saponin monomer Echinoside A; the sea cucumber saponin derivative is a dedisaccharide product of sea cucumber saponin monomer and / or a detetrasaccharide product of sea cucumber saponin monomer, or a desulfurization product of sea cucumber saponin monomer.

[0041] 7. The anti-Helicobacter pylori product as described in item 5, characterized in that: the Helicobacter pylori is the Helicobacter pylori found in the stomach of a human or animal.

[0042] 8. The anti-Helicobacter pylori product as described in any one of items 5 to 7, characterized in that: the amount of sea cucumber saponin and / or sea cucumber saponin derivative used is 1 to 10 mg / kg body weight.

[0043] 9. A method for preparing sea cucumber saponin monomers, comprising the following steps:

[0044] 1) The sea cucumber body wall is vacuum freeze-dried and then pulverized;

[0045] 2) Extract the crushed sea cucumber from step 1) with 60% ethanol at room temperature, and concentrate under vacuum to obtain sea cucumber extract;

[0046] 3) Dissolve the sea cucumber extract from step 2) in water, extract with water-saturated n-butanol, and concentrate under vacuum;

[0047] 4) Dissolve the sea cucumber n-butanol extract concentrated in vacuum in step 3) in water, place it on a macroporous adsorption resin column, and then elute with water and 70% ethanol as mobile phases in sequence, and collect the 70% ethanol eluent.

[0048] 5) The 70% ethanol eluent collected in step 4) is concentrated under vacuum and dried to obtain total sea cucumber saponins;

[0049] 6) Place the total saponins from sea cucumber obtained in step 5) onto a normal-phase silica gel column and elute with a gradient of chloroform:methanol:water as the mobile phase, and collect the eluent in a ratio of 7:2.5:0.2 (v / v / v).

[0050] 7) The eluent collected in step 6) was concentrated under vacuum and freeze-dried to obtain the sea cucumber saponin monomer Holothurin A.

[0051] 8) Dissolve the total saponins from sea cucumber obtained in step 5) in water, place it on a reversed-phase silica gel column, elute with 50% methanol as the mobile phase, and collect the eluent.

[0052] 9) The eluent from step 8) was concentrated under vacuum and freeze-dried to obtain sea cucumber saponin monomer Echinoside A.

[0053] 10. A method for preparing sea cucumber saponin monomer dedisaccharide products, comprising the following steps:

[0054] 1) Using the sea cucumber saponin monomer described in item 9 as a substrate, react with pectinase and sodium acetate buffer solution (pH=4.6) in a constant temperature shaking incubator at 40℃ for 72h;

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

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

[0057] 11. A method for preparing sea cucumber saponin monomer detetrasaccharide products, comprising the following steps:

[0058] 1) Using the sea cucumber saponin monomer described in claim 9 as a substrate, add 2 mol / L trifluoroacetic acid aqueous solution, seal, and hydrolyze at 110°C for 3 hours;

[0059] 2) Neutralize the hydrolysate from step 1) with NaOH solution and extract with n-butanol;

[0060] 3) Take the n-butanol layer solution from step 2) and remove the solvent by rotary evaporation to obtain the detetrasaccharide product of sea cucumber saponin monomer.

[0061] 12. A method for preparing desulfurized sea cucumber saponin monomers, comprising the following steps:

[0062] 1) Using the sea cucumber saponin monomer described in claim 9 as a substrate, add 0.2 mol / L sulfuric acid aqueous solution and hydrolyze by shaking at 80°C for 22 hours;

[0063] 2) Neutralize the hydrolysate from step 1) with NaOH solution and extract with n-butanol;

[0064] 3) Take the n-butanol layer solution from step 2) and remove the solvent by rotary evaporation to obtain the desulfurized product of sea cucumber saponin monomer.

[0065] The sea cucumber saponins and their derivatives provided by this invention can inhibit Helicobacter pylori colonization, promote the clearance of Helicobacter pylori from the stomach, and inhibit the activity of Helicobacter pylori. In vitro antibacterial experiments show that the antibacterial effect of sea cucumber saponins is directly proportional to the effective concentration and is superior to that of ginsenoside monomer Rd; the antibacterial ability of sea cucumber saponins depends on the presence of sugar chains and sulfate groups, and the shorter the sugar chain, the weaker the antibacterial effect. In vivo animal experiments show that sea cucumber saponins and their derivatives can effectively reduce the number of Helicobacter pylori colonies in the stomach of mice. Attached Figure Description

[0066] Figure 1 The diagram shows the molecular structure of sea cucumber saponins and their derivatives used in the embodiments of this invention.

[0067] Figure 2 The figures represent the liquid chromatograms of total sea cucumber saponins and sea cucumber saponin monomers Holothurin A and Echinoside A in the embodiments of the present invention.

[0068] Figure 3 This represents the urease activity assay results of Helicobacter pylori in the mouse stomach according to an embodiment of the present invention. Detailed Implementation

[0069] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

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

[0071] Example 1: Preparation of sea cucumber saponins and their derivatives

[0072] 1.1 Extraction of sea cucumber saponins

[0073] 1.1.1 Extraction of total saponins from sea cucumber

[0074] Sea cucumbers were purchased from Nanshan Seafood Market in Qingdao. The sea cucumber body walls were freeze-dried under vacuum using an Alpha 1-4LSC freeze dryer (MarinChrist GmbH, Germany) at -50℃, vacuum <10kPa, and for 48 hours. After freeze-drying, the sea cucumbers were pulverized and extracted twice at room temperature with 60% ethanol (v / v) at a material-to-liquid ratio of 1:4. The extracts were combined, and the solvent was removed by rotary evaporation using a RE-2000A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory) until the sea cucumber extract was a paste-like substance remaining on the inner wall of the rotary evaporation flask.

[0075] The specific conditions for solvent removal by rotary evaporation are: rotation speed of 30-50 rpm / min, water bath temperature of 45-50℃, and vacuum degree of 0.09 MPa.

[0076] The sea cucumber extract in paste form was dissolved in a small amount of water and extracted twice with water-saturated n-butanol. The n-butanol organic layers were combined and the solvent was removed by rotary evaporation to obtain the sea cucumber n-butanol extract.

[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) chromatography column. Then, water and 70% ethanol were used as mobile phases for elution, and the 70% ethanol eluent was collected.

[0078] Take 70% ethanol eluent and remove the solvent by rotary evaporation to obtain total sea cucumber saponins.

[0079] The sea cucumber saponins were detected using an Agilent 1260 Infinity II liquid chromatography system (Agilent Technologies (China) Co., Ltd.). The saponins were identified as sea cucumber saponins of the sea cucumber alkyl type, with the general structural formula shown below. Figure 1 A, The liquid chromatography peak chromatogram of total saponins from sea cucumber is shown below. Figure 2 A.

[0080] 1.1.2 Extraction of Holothurin A, a saponin monomer from sea cucumber

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

[0082] Collect the eluent of chloroform:methanol:water (7:2.5:0.2) and remove the solvent by rotary evaporation.

[0083] The analysis was performed using an Agilent HPLC system (Agilent 1260 Infinity II, Agilent Technologies (China) Co., Ltd.). The result was identified as holothurin A (HA), a saponin monomer from sea cucumbers. The general structural formula is shown below. Figure 1 B, see liquid chromatography peak chromatogram. Figure 2 B.

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

[0085] The total saponin extract of sea cucumber obtained by vacuum concentration 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 packing, Fuji Chemical Co., Ltd., Japan), and then eluted with 50% methanol as the mobile phase.

[0086] Collect the eluent and remove the solvent by rotary evaporation.

[0087] The analysis was performed using an Agilent HPLC system (Agilent 1260 Infinity II, Agilent Technologies (China) Co., Ltd.), and the result was identified as sea cucumber saponin monomer Echinoside A (EA). The general structural formula is shown below. Figure 1 C, see liquid chromatography peak chromatogram. Figure 2 C.

[0088] 1.1.4 Preparation of desaccharified sea cucumber saponins

[0089] Sea cucumber saponin monomer HA was used as a substrate and mixed with Novozyme pectinase (Pectinex Ultra SP-L, Novozymes Biosciences) and sodium acetate buffer solution (pH=4.6) at a ratio of 25:1:12.5 (w / v / v). The mixture was incubated at 40℃ with shaking at 130 rpm for 72 h. The reaction solution was then centrifuged at 12000 rpm for 10 min, and the precipitate was dried in a 65℃ oven to obtain the desaccharified product HA-2 of sea cucumber saponin HA. The general structural formula of the desaccharified product HA-2 of sea cucumber saponin is shown below. Figure 1 D.

[0090] Sea cucumber saponin monomer EA was used as a substrate and 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). The mixture was incubated at 40℃ with shaking at 130 rpm for 72 h. The reaction solution was then centrifuged at 12000 rpm for 10 min, and the precipitate was dried in a 65℃ oven to obtain the desaccharified product EA-2 of sea cucumber saponin EA. The general structural formula of the desaccharified product EA-2 of sea cucumber saponin is shown below. Figure 1 E.

[0091] 0.5 g of sea cucumber saponin monomer HA was dissolved in 500 mL of 2 mol / L trifluoroacetic acid aqueous solution, sealed, and hydrolyzed at 110 °C for 3 hours. The hydrolysate was neutralized with NaOH solution, and extracted four times with an equal volume of n-butanol. The n-butanol layer was collected, and the solvent was removed by rotary evaporation to obtain the detetrasaccharide product of sea cucumber saponin HA—the aglycone HA-4 of sea cucumber saponin monomer HA. The general structural formula of sea cucumber saponin aglycone HA-4 is shown below. Figure 1 F.

[0092] 0.5 g of sea cucumber saponin monomer EA was dissolved in 500 mL of 2 mol / L trifluoroacetic acid aqueous solution, sealed, and hydrolyzed at 110 °C for 3 hours. The hydrolysate was neutralized with NaOH solution, and extracted four times with an equal volume of n-butanol. The n-butanol layer was collected, and the solvent was removed by rotary evaporation to obtain the detetrasaccharide product of sea cucumber saponin EA—the aglycone EA-4 of sea cucumber saponin monomer EA. The general structural formula of sea cucumber saponin aglycone EA-4 is shown below. Figure 1 G.

[0093] 1.1.5 Preparation of Sea Cucumber Saponin Desulfurization Products

[0094] Sea cucumber saponin desulfurization products were prepared by a mild acid hydrolysis method.

[0095] Take 1g of sea cucumber saponin monomer HA, add 200mL of 0.2mol / L sulfuric acid aqueous solution, dissolve thoroughly, and hydrolyze in a water bath shaker at 80℃ for 22 hours. Neutralize the hydrolysate with NaOH solution, and extract four times with an equal volume of n-butanol. Take the n-butanol layer solution, and remove the solvent by rotary evaporation to obtain the desulfurized product of sea cucumber saponin HA—desulfurized saponin HA-dS. The general structural formula of desulfurized saponin HA-dS of sea cucumber saponin monomer HA is shown below. Figure 1 H.

[0096] Take 1g of sea cucumber saponin monomer EA, add 200mL of 0.2mol / L sulfuric acid aqueous solution, dissolve thoroughly, and hydrolyze in a water bath shaker at 80℃ for 22 hours. Neutralize the hydrolysate with NaOH solution, and extract four times with an equal volume of n-butanol. Take the n-butanol layer solution, and remove the solvent by rotary evaporation to obtain the desulfurized product of sea cucumber saponin EA—desulfurized saponin EA-dS. The general structural formula of desulfurized saponin EA-dS of sea cucumber saponin monomer EA is shown below. Figure 1 I.

[0097] Example 2: In vitro antibacterial activity of sea cucumber saponins and their derivatives against Helicobacter pylori

[0098] 2.1 Culture of Helicobacter pylori

[0099] The Sydney strain 1 (SS1) of Helicobacter pylori, preserved in glycerol tubes, was taken out of the -80℃ freezer and rapidly thawed in a 37℃ 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 have grown on the plate, pick a single colony with a sterile inoculation loop and resuspend it in Brucella 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. 8 The sample contained *Helicobacter pylori* culture at CFU / mL. The concentrations of the test samples in wells A1–A10 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. Well A11 served as a sterile control, containing no bacterial culture, no test sample, and only 200 μL of Brucella broth. Each well was used in triplicate.

[0107] The 96-well plate was placed in a microaerophilic environment (7% oxygen, 10% carbon dioxide, 83% nitrogen) and incubated at 37°C for 48 hours. The lowest concentration at which no turbidity was observed was the minimum inhibitory concentration (MIC). Suspensions from each well were aspirated and evenly spread onto Columbia blood agar plates, and incubated again at 37°C for 48 hours. Colony counting was performed, and the lowest concentration at which no colony growth was observed was the minimum bactericidal concentration (MBC). The experimental results are shown in Table 1.

[0108] Table 1. Results of determination of minimum inhibitory concentration and minimum bactericidal concentration of sea cucumber saponins and their derivatives against Helicobacter pylori

[0109]

[0110] As shown in Table 1, the two sea cucumber saponin monomers HA and EA have better antibacterial and bactericidal effects on Helicobacter pylori than ginsenoside monomer Rd.

[0111] Compared with sea cucumber saponin monomer HA, the antibacterial and bactericidal effects of HA-2 (derived from the removal of two sugar groups) decreased, and the antibacterial and bactericidal effects of HA-4 (derived from the removal of four sugar groups) further decreased. Compared with sea cucumber saponin monomer EA, the antibacterial and bactericidal effects of EA-2 (derived from the removal of two sugar groups) decreased, and the antibacterial and bactericidal effects of EA-4 (derived from the removal of four sugar groups) further decreased. These results indicate that the antibacterial and bactericidal effects of sea cucumber saponins are positively correlated with sugar chain length; the shorter the sugar chain, the weaker the antibacterial and bactericidal effects.

[0112] Compared with sea cucumber saponin monomers HA and EA, the antibacterial and bactericidal effects of the desulfurization products HA-dS and EA-dS of sea cucumber saponin monomers HA and EA were significantly reduced, indicating that the antibacterial and bactericidal abilities of sea cucumber saponin monomers are dependent on the sulfate groups.

[0113] 2.3 Inhibition zone test of Helicobacter pylori

[0114] The diameter of the inhibition zone is a core indicator for quantifying the antibacterial effect of antimicrobial drugs in antimicrobial experiments. Its significance lies not only in judging bacterial sensitivity, but also in providing a key basis for screening antimicrobial substances.

[0115] Take 100 μL of a concentration of 1×10 8A CFU / mL 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 a 25 μg / mL test sample (including HA-2 (after removing two sugar groups from HA), EA-2 (after removing two sugar groups from EA), HA-4 (after removing four sugar groups from HA), EA-4 (after removing four sugar groups from EA), HA-dS (desulfurized product of HA), EA-dS (desulfurized product of 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.

[0116] Table 2. Evaluation criteria for antibacterial effect

[0117]

[0118] Table 3. Results of inhibition zone diameter determination of sea cucumber saponins and their derivatives against Helicobacter pylori

[0119]

[0120] 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.

[0121] Example 3: In vivo study on the ability of sea cucumber saponins and their derivatives to colonize and clear Helicobacter pylori in the stomach.

[0122] 3.1 Animal Experiments

[0123] Eighty-four SPF-grade male BALB / c mice (7 weeks old, weighing 30.0±1.0g) were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd. The animal housing environment was maintained at 21℃~23℃, humidity at 45%~55%, and a 12h light / 12h dark cycle. During the experiment, the mice had free access to AIN-93G standard complete nutritional feed and water. After 7 days of acclimatization, the mice were randomly divided into 12 groups of 7 mice each. The experimental period was 5 weeks. The specific procedures for grouping and drug administration in the animal experiment are shown in Table 4.

[0124] Table 4. Animal experimental grouping and administration methods

[0125]

[0126] During weeks 1-2, each mouse in the normal control group and model group was administered 100 μL of physiological saline by gavage; each mouse in the experimental groups was administered 100 μL of the corresponding saponin or saponin derivative by gavage, with the gavage volume calculated at 1 mg / kg body weight. The positive control group was administered 100 μL of quadruple therapy by gavage, with the following dosages: esomeprazole (purchased from Shandong New Era Pharmaceutical Co., Ltd.) 3.03 μg / g body weight, amoxicillin (purchased from Shandong Lukang Pharmaceutical Co., Ltd.) 151.67 μg / g body weight, clarithromycin (purchased from Jiangsu Hengrui Medicine Co., Ltd.) 75.83 μg / g body weight, and colloidal bismuth pectin (purchased from Anhui Yongshengtang Pharmaceutical Co., Ltd.) 45.5 μg / g body weight.

[0127] Starting from week 3, each group continued to be administered physiological saline, saponins or saponin derivatives, and quadruple therapy by gavage, while simultaneously receiving Helicobacter pylori bacterial suspension by gavage. The total volume of sample and bacterial suspension administered to each mouse by gavage was 100 μL, and the Helicobacter pylori inoculation amount was approximately 1 × 10⁻⁶. 8 CFU was administered via gavage every two days for a total of seven times. Patients were kept fasting for 12 hours before each gavage and for four hours afterward.

[0128] Starting from week 5, continue to administer the corresponding solution via gavage, following the method used in weeks 1 and 2, to expel as many Helicobacter pylori bacteria as possible that have not colonized the stomach.

[0129] On the last day after gavage, mice were kept NPO (fasting) but allowed free access to water for 12 hours. After anesthesia with isoflurane, the mice were enucleated to collect blood, euthanized by cervical dislocation, and their gastric tissue and gastric juice were collected.

[0130] 3.2 Determination of urease activity:

[0131] The colonization of Helicobacter pylori in the stomach of mice in each group was determined using a urease (UE) activity assay kit (Solarbio, catalog number: BC4110, Beijing Solarbio Technology Co., Ltd.), and the Helicobacter pylori clearance rate was calculated. The entire procedure was performed according to the kit instructions. 0.1 g of mouse stomach tissue was added to 1 mL of extraction buffer, homogenized on ice, and centrifuged at 12000 g for 15 min at 4 °C. 100 μL of the supernatant was taken, and 200 μL of reagent one and 400 μL of reagent two were added, mixed thoroughly, and reacted at 37 °C for 1 h. 400 μL of the above reaction mixture was taken, and 80 μL of reagent three and 60 μL of reagent four were added, mixed thoroughly, and allowed to stand at room temperature for 20 min. Then, 460 μL of distilled water was added, mixed thoroughly, and the absorbance at 630 nm was measured using a visible spectrophotometer. The results are shown in the table below. Figure 3 OD 630nm The higher the value, the higher the urease activity of Helicobacter pylori, the greater the number of bacteria colonizing the stomach, and the lower the clearance rate.

[0132] Figure 3 The results showed that sea cucumber saponin monomer HA exhibited the strongest ability to clear Helicobacter pylori from the stomach, superior to sea cucumber saponin monomer EA and ginsenoside monomer Rd, which had the strongest antibacterial activity, and comparable to the effect of quadruple therapy antibiotics. Both sea cucumber saponin monomers HA and EA showed that the shorter the sugar chain, the weaker the antibacterial effect; that is, the order of antibacterial effect was: saponin monomer > desaccharified saponin > saponin aglycone. Furthermore, the antibacterial effect of saponin desulfurization products decreased significantly, i.e., saponin monomer > desulfurized saponin. These results indicate that the sugar chains and sulfate groups in the sea cucumber saponin structure play an important role in enhancing the ability to clear Helicobacter pylori from the gastric mucosa.

[0133] In summary, the sea cucumber saponins and their derivatives provided by this invention can inhibit Helicobacter pylori colonization, promote the clearance of Helicobacter pylori from the stomach, and inhibit the activity of Helicobacter pylori. In vitro antibacterial experiments show that the antibacterial effect of sea cucumber saponins is directly proportional to the effective concentration and is superior to that of ginsenoside monomer Rd; the antibacterial ability of sea cucumber saponins depends on the presence of sugar chains and sulfate groups, and the shorter the sugar chain, the weaker the antibacterial effect. In vivo animal experiments show that sea cucumber saponins and their derivatives can effectively reduce the number of Helicobacter pylori colonies in the stomach of mice.

Claims

1. The application of sea cucumber saponin monomers and / or sea cucumber saponin derivatives in the preparation of anti-Helicobacter pylori products, wherein the sea cucumber saponin monomers are sea cucumber saponin monomers Holothurin A and / or sea cucumber saponin monomers Echinoside A; and the sea cucumber saponin derivatives are dedisaccharide products of sea cucumber saponin monomers Holothurin A and / or desulfurization products of sea cucumber saponin monomers Echinoside A.

2. The application as described in claim 1, characterized in that: The Helicobacter pylori mentioned is the Helicobacter pylori found in the stomach of humans or other animals.

3. The application as described in claim 1 or 2, characterized in that: The dosage of sea cucumber saponin monomers or sea cucumber saponin derivatives is 1–10 mg / kg body weight.

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