Citric acid and licochalcone B repairing composition and application

Abstract

The invention discloses a citric acid and licochalcone B repairing composition and application, and belongs to the technical field of medicine. According to the invention, the repair promoting potential of citric acid and the anti-inflammatory and anti-oxidation activity of licochalcone B are known, and the two natural active components with different action mechanisms are creatively compounded according to a specific proportion; therefore, the citric acid and licochalcone B repairing composition which has a synergistic effect on repairing gastric mucosa epithelial cell injury caused by HP infection is provided, the effect is better than that of a single component, safety and compliance are better, and a new technical approach is provided for solving the repairing problem of clinical gastric mucosa injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a citric acid and glycyrrhizin B repair composition and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Helicobacter pylori (HP) infection is a core pathological factor leading to various gastric diseases. It not only directly damages the gastric mucosal epithelium but also triggers persistent inflammation and oxidative stress, thereby disrupting the integrity of the mucosal barrier and its self-repair capabilities. While standard clinical treatments for this pathogen aim to eradicate the bacteria, they generally suffer from problems such as affecting gut microbiota balance, easily developing drug resistance, and having weak direct repair effects on mucosal cells. Therefore, developing alternative or adjuvant strategies that can directly and safely promote the repair of gastric mucosal damage has become a clear and urgent research direction in this field.

[0004] Against this backdrop, the search for active ingredients with mucosal protective potential from natural products has attracted considerable attention. Citric acid, as a natural intermediate in the body's energy metabolism, possesses good biocompatibility, and its involvement in cellular metabolic processes suggests it may have unique value in supporting tissue repair. On the other hand, chalcone B (LCB), derived from licorice, has been proven to have significant anti-inflammatory and antioxidant properties, providing a theoretical basis for alleviating Helicobacter pylori-related indirect damage. However, current research on these two components, particularly their scientific combination for targeted protection of damaged gastric mucosal cells and the exploration of their synergistic effects, remains lacking, failing to fully realize the potential value of their combined application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a citric acid and glycyrrhizin B repair composition and its application. The citric acid and glycyrrhizin B repair composition provided by the present invention can synergistically protect human gastric mucosal cells (GES-1 cells) damaged by Helicobacter pylori, significantly improve cell viability, reduce cell morphological damage, and has excellent synergistic protective effects.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a citric acid and glycyrrhizin B repair composition comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is (0~10):1, and the amount of citric acid is not 0.

[0007] Licorice chalcone B is an active chalcone compound extracted from the natural drug licorice. It possesses various biological activities, including clear anti-inflammatory, antioxidant, and cell-protective effects, and is theoretically suitable for alleviating HP-induced damage. Citric acid is an organic acid naturally found in citrus fruits. Physiologically, it promotes digestion, participates in energy metabolism, and enhances the body's absorption of minerals. In the pharmaceutical field, due to its good biocompatibility, it is used to aid in the recovery of gastrointestinal mucosal ulcers.

[0008] The inventors have creatively discovered that when citric acid and glycyrrhizin B are combined in a specific ratio, their protective effect on GES-1 cells in vitro is significantly enhanced, exhibiting a remarkable synergistic effect. This synergistic effect can alleviate Helicobacter pylori (HP) damage to gastric epithelial cells, improve cell viability, and enhance cell morphological integrity, indicating a positive synergistic relationship between the two in anti-HP activity and cell protection. Further experimental results show that when the mass ratio of citric acid to glycyrrhizin B is controlled within the range of 0-10:1 (with citric acid dosage not being 0), it can effectively repair Helicobacter pylori damage, with a drug synergy coefficient (CI value) of less than 1, demonstrating a significant synergistic effect.

[0009] Therefore, the combination based on the synergistic mechanism of citric acid and glycyrrhizin B is expected to provide a new solution for the prevention and treatment of Helicobacter pylori infection and related gastric diseases as a safe, effective, and low-drug-resistance novel drug or health product.

[0010] In some embodiments of the present invention, the mass ratio of citric acid to glycyrrhizin B is (2.5~10):1. Within this range, citric acid and glycyrrhizin B exhibit a very significant synergistic effect.

[0011] In some embodiments of the present invention, the mass ratio of citric acid to glycyrrhizin B is 2.5:1, 5:1, 6.25:1, 7.5:1, or 10:1. At this mass ratio, the synergistic effect of citric acid and glycyrrhizin B is more significant.

[0012] In some embodiments of the present invention, the citric acid and glycyrrhizin B repair composition can be prepared by the following method: Glycyrrhizin B is dissolved in DMSO to form a first mother liquor; citric acid is dissolved in water to form a second mother liquor; then the first mother liquor and the second mother liquor are mixed in proportion, stirred and dissolved evenly, and concentrated under reduced pressure or directly freeze-dried to obtain the citric acid and glycyrrhizin B repair composition.

[0013] The citric acid and glycyrrhizin B repair composition provided by this invention can synergistically improve the survival rate of GES-1 cells, effectively reduce cell damage caused by Helicobacter pylori infection, and exhibit excellent synergistic protective effects.

[0014] A second aspect of the present invention provides the use of the citric acid and glycyrrhizin B repair composition described in the first aspect in the preparation of a product for treating human gastrointestinal mucosal epithelial cell damage.

[0015] In some embodiments of the present invention, the damage to human gastrointestinal mucosal epithelial cells includes any one or more of the damage to human gastrointestinal mucosal epithelial cells caused by Helicobacter pylori infection and damage to human gastrointestinal mucosal epithelial cells caused by gastrointestinal diseases.

[0016] In some embodiments of the present invention, the Helicobacter pylori infection includes any one or more of acute HP infection and chronic HP infection.

[0017] In some embodiments of the present invention, the gastrointestinal diseases include any one or more of chronic superficial gastritis, atrophic gastritis, gastric ulcer, gastric mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), and precancerous lesions of the stomach.

[0018] In some embodiments of the present invention, the repair composition works by synergistically protecting GES-1 cells damaged by *Helicobacter pylori* infection, and its mechanism of action includes: (1) Reduce the cytotoxic damage of Helicobacter pylori to GES-1 cells; (2) Improve the morphological and structural integrity of infected cells; (3) Improve the survival rate of GES-1 cells, thereby protecting gastric epithelial cells and improving the gastric mucosal barrier function.

[0019] A third aspect of the present invention provides a product for repairing damage to human gastrointestinal mucosal epithelial cells, comprising a pharmaceutically acceptable carrier or excipient and the citric acid and glycyrrhizin B repair composition described in the first aspect.

[0020] In some embodiments of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, any one or more of lactose, starch, microcrystalline cellulose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, syrup, peanut oil, olive oil, and water.

[0021] In some embodiments of the present invention, the excipients include, but are not limited to, any one or more of the following: preservatives, colorants, diluents, granulators, emulsifiers, lubricants, binders, disintegrants, buffer solutions, flavoring agents, surfactants, thickeners, fillers, absorption promoters, and surfactants.

[0022] In some embodiments of the present invention, the dosage form of the drug is selected from any one or more of powder, pill, capsule, granule, tablet or oral liquid.

[0023] In some embodiments of the invention, tablets containing the restorative compositions of the invention can be prepared by compression or molding, optionally using one or more excipients or adjuvants. The active ingredient can be compressed in a free-flowing form (e.g., powder or granules) in a suitable machine, optionally mixed with binders, lubricants, inert diluents, surfactants, or dispersants. Molded tablets can be molded in a suitable machine, i.e., a mixture of powdered compounds moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of the active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of the active ingredient. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to about 5 g of the active drug, mixed with a suitable and convenient carrier material, which may comprise about 5% to 95% of the total composition. Unit dosage forms typically contain approximately 1 mg to approximately 2 g of active ingredient, usually in doses of 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0024] In some embodiments of the present invention, the product is a solution in which the total concentration of citric acid and glycyrrhizin B is 0.145-0.55 g / L.

[0025] In some embodiments of the present invention, the concentration of glycyrrhizin B in the solution is 0.02-0.05 g / L, and the concentration of citric acid is 0.125-0.5 g / L.

[0026] In some embodiments of the present invention, the preparation method of the solution includes dissolving glycyrrhizin B in a cosolvent (dimethyl sulfoxide (DMSO)), then mixing it evenly with citric acid dissolved in a pharmaceutically acceptable solvent (e.g., water) and diluting it to obtain the solution.

[0027] The preparation process can be carried out at room temperature. After mixing, the solution is filtered through a 0.22 μm filter membrane for sterilization to obtain a clear solution. The resulting solution can be used directly in in vitro experiments or further prepared into a pharmaceutical composition for oral administration.

[0028] The beneficial effects of this invention are as follows: This invention provides a repair composition consisting of citric acid and glycyrrhizin B. By combining citric acid and glycyrrhizin B in a specific ratio, a significant synergistic effect was unexpectedly discovered in protecting gastric mucosal epithelial cells damaged by Helicobacter pylori. This composition is not a simple additive effect of their activities, but rather more effectively improves cell survival, inhibits the release of inflammatory factors, and enhances antioxidant enzyme activity, thereby achieving synergistic protection at multiple targets and pathways. This synergistic effect has been clearly confirmed in cell models by calculating the combination index (CI<1). Compared to existing clinical therapies that focus on antibacterial activity while having indirect repair effects, the composition of this invention can directly and efficiently promote the repair of gastric mucosal cells and the reconstruction of barrier function. Furthermore, both components are derived from natural sources and have high biocompatibility, providing a novel solution for developing a safe product focused on mucosal repair for the prevention and treatment of Helicobacter pylori-related gastric diseases.

[0029] Specifically, this particular combination significantly reduced the damage of *Helicobacter pylori* (HP) to gastric epithelial cells (such as GES-1 cells), effectively enhanced the viability of infected cells, and improved the integrity of cell morphology, demonstrating a good protective function for gastric mucosal cells. This synergistic protective effect is closely related to the inhibition of harmful factors associated with *Helicobacter pylori* infection.

[0030] Most importantly, this synergistic effect is strictly ratio-dependent. When the ratio deviates from this optimized range (e.g., greater than 10:1), the synergistic index (CI) will be greater than 1, and the synergistic effect will be significantly weakened or disappear. This highlights the technical advantages and non-obviousness of the specific ratio of this invention. Meanwhile, both citric acid and glycyrrhizin B have good biocompatibility, and their combination provides a new approach for developing anti-HP-related products focusing on gastric mucosal repair. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 The results of the determination of the repair composition provided in Example 5 of the present invention in repairing GES-1 cell damage are shown; wherein, LCB is glycyrrhizin B and CA is citric acid.

[0033] Figure 2 The experimental results of the repair composition provided in Example 5 of this invention improving the survival rate of GES-1 cells are shown below; where a is the effect diagram of the control group, b is the effect diagram of the model group, c is the effect diagram of 0.50 g / L citric acid, d is the effect diagram of 0.50 g / L citric acid, and e is the effect diagram of 0.50 g / L citric acid + 0.05 g / L glycyrrhizin B. Detailed Implementation

[0034] This invention provides a citric acid and glycyrrhizin chalcone B repair composition and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention. To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] The solutions in Examples 1-5 and Comparative Examples 1 and 2 were prepared by the following method: A measured amount of glycyrrhizin chalcone B was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) to form a mother liquor. A measured amount of citric acid (CA) raw material powder was dissolved in a measured amount of ultrapure water to form a mother liquor. After mixing the two mother liquors in a predetermined ratio, the mixture was diluted with ultrapure water to the target concentration and then filtered through a 0.22 μm filter membrane for sterilization to obtain a clear solution. The obtained solution can be used directly in in vitro GES-1 cell protection experiments or further used to prepare oral pharmaceutical compositions.

[0036] Example 1 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 6.25:1.

[0037] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.02 g / L, the concentration of citric acid is 0.125 g / L, and the total concentration is 0.145 g / L.

[0038] Example 2 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 2.5:1.

[0039] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.05 g / L, the concentration of citric acid is 0.125 g / L, and the total concentration is 0.145 g / L.

[0040] Example 3 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 5:1.

[0041] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.05 g / L, the concentration of citric acid is 0.25 g / L, and the total concentration is 0.3 g / L.

[0042] Example 4 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 7.5:1.

[0043] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.05 g / L, the concentration of citric acid is 0.375 g / L, and the total concentration is 0.425 g / L.

[0044] Example 5 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 10:1.

[0045] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.05 g / L, the concentration of citric acid is 0.5 g / L, and the total concentration is 0.55 g / L.

[0046] Comparative Example 1 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 12.5:1.

[0047] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.02 g / L, the concentration of citric acid is 0.25 g / L, and the total concentration is 0.27 g / L.

[0048] Comparative Example 2 A citric acid and glycyrrhizin B repair composition, comprising citric acid and glycyrrhizin B, wherein the mass ratio of citric acid to glycyrrhizin B is 18.75:1.

[0049] This citric acid and glycyrrhizin B repair composition can be formulated into a solution for repairing damage to human gastrointestinal mucosal epithelial cells (i.e., a mixed aqueous solution of citric acid and glycyrrhizin B). The concentration of glycyrrhizin B is 0.02 g / L, the concentration of citric acid is 0.375 g / L, and the total concentration is 0.395 g / L.

[0050] Experimental example: Improvement effect of each group: The improvement effect (E) was calculated using formulas (1) and (2).

[0051] E A =(C A -C M ) / (C0-C m (1) E AB =(C AB -C M ) / (C0-C m (2) Where C0 represents the cell viability of the blank control group, C m C represents the cell survival rate of the model group. A C represents the cell survival rate of the single-drug group. AB E represents cell viability in the combined drug administration group. A E represents the protective effect of a single drug. AB This represents the protective effect of the combination of citric acid and glycyrrhizin B.

[0052] The following experiments validated the synergistic effect: the expected summation and drug synergistic coefficient (CI) were calculated using formulas (3) and (4).

[0053] Expected summation effect = E A +E B -E A ×E B (3) CI=(E A +E B -E A ×E B ) / E AB (4) Among them, E A E represents the protective effect of citric acid. B Representing the protective effect of glycyrrhizin B, E AB This represents the protective effect of the combination of citric acid and glycyrrhizin B. If CI is less than 1, i.e., E... AB The effect was greater than the expected summation, demonstrating a synergistic effect between citric acid and glycyrrhizin B.

[0054] Cell viability (MTT) assay GES-1 cells were seeded in 96-well plates at a density of 5000 cells per well, and RPMI-1640 medium containing 10% fetal bovine serum was added. The plates were incubated at 37°C and 5% CO2 for 24 h. After cell attachment, different test solutions (100 μL per well) were added to each well, along with 1×10⁻⁶ ppm of the solution. 6 One Helicobacter pylori bacterium was co-cultured for 24 h. After culturing, MTT solution (final concentration 0.05 mg / mL) was added to each well, and incubation continued for 4 h. The culture medium was then discarded, and DMSO was added to dissolve the formazan crystals. The absorbance (OD) was measured at 570 nm using a microplate reader. 570 The relative cell viability of the uninfected group was set to 1, and the relative cell viability of the other experimental groups was normalized to 1. The specific concentrations of each group are shown in Table 1.

[0055] Table 1 Concentration of each group

[0056] The test results are shown in Table 2 below (n=3, mean ± standard deviation, relative to the blank control group as 1).

[0057] Table 2 Test Results

[0058] As shown in Table 2, citric acid and glycyrrhizin B, within a certain concentration range, have a synergistic protective effect on GES-1 cells infected with Helicobacter pylori.

[0059] The results obtained by calculating formulas (3) and (4) show that the CI values ​​of each example (Example 1-Example 5) are all less than 1, indicating that there is a significant synergistic effect between citric acid and glycyrrhizin B, which can significantly improve the survival rate of GES-1 cells under Helicobacter pylori infection. The CI values ​​of Comparative Example 1 and Comparative Example 2 are higher than those of Examples 1-Example 5, indicating that when the mass ratio of citric acid to glycyrrhizin B deviates from the range (0~10):1 set by the present invention, the synergistic effect is significantly weakened or disappears.

[0060] Figure 2 Figure a shows the control group, where cells exhibit a typical phenotype of high density, well-developed shape, and intact structure. Figure 2 Figure b shows the results of the model group. Compared with the control group, the GES-1 cells in the model group showed a significant decrease in cell density, shrunken morphology, and impaired integrity due to Helicobacter pylori damage. Figure 2Figures c and d in the figure show the effect of the single-factor treatment group. Compared with the control group, the number of GES-1 cells in the single-factor treatment group was still smaller and the morphology was poor, indicating that its protective effect on GES-1 cells was weak. Figure 2 Figure e shows the treatment group of the anti-Helicobacter pylori composition of the present invention, in which the GES-1 cell density is close to that of the control group, the morphology is relaxed and the structural integrity is good, and there are significant phenotypic differences with the model group and the single-factor treatment group.

[0061] In summary, the citric acid-glycyrrhizin B compound composition provided by the present invention can produce significant synergistic protective effects within a specific ratio range, and has good anti-Helicobacter pylori activity and gastric epithelial cell protection effect.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A citric acid and glycyrrhizin B repair composition, characterized in that, It includes citric acid and glycyrrhizin B, with a mass ratio of citric acid to glycyrrhizin B of (0~10):1, and the amount of citric acid used is not 0.

2. The citric acid and glycyrrhizin B repair composition as claimed in claim 1, characterized in that, The mass ratio of citric acid to glycyrrhizin B is (2.5~10):

1.

3. The citric acid and glycyrrhizin B repair composition as claimed in claim 1, characterized in that, The mass ratio of citric acid to glycyrrhizin B is 2.5:1, 5:1, 6.25:1, 7.5:1 or 10:

1.

4. The use of the citric acid and glycyrrhizin B repair composition according to any one of claims 1-3 in the preparation of a product for treating human gastrointestinal mucosal epithelial cell damage.

5. The application as described in claim 4, characterized in that, The damage to human gastrointestinal mucosal epithelial cells includes any one or more of the damage to human gastrointestinal mucosal epithelial cells caused by Helicobacter pylori infection and damage to human gastrointestinal mucosal epithelial cells caused by gastrointestinal diseases; Preferably, the gastrointestinal diseases include any one or more of the following: chronic superficial gastritis, atrophic gastritis, gastric ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and precancerous lesions of the stomach.

6. A product for repairing damage to human gastrointestinal mucosal epithelial cells, characterized in that, Includes pharmaceutically acceptable carriers or excipients and the citric acid and glycyrrhizin B repair composition according to any one of claims 1-3.

7. The product as described in claim 6, characterized in that, The pharmaceutically acceptable carriers include any one or more of lactose, starch, microcrystalline cellulose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, syrup, peanut oil, olive oil, and water. Preferably, the excipients include any one or more of the following: preservatives, colorants, diluents, granulators, emulsifiers, lubricants, binders, disintegrants, buffer solutions, flavoring agents, surfactants, thickeners, fillers, absorption promoters, and surfactants.

8. The product as described in claim 6, characterized in that, The dosage form of the drug is selected from any one or more of the following: powder, pill, capsule, granule, tablet or oral liquid.

9. The product as described in claim 6, characterized in that, The product is a solution, and the total concentration of citric acid and glycyrrhizin B in the solution is 0.145-0.55 g / L.

10. The product as described in claim 9, characterized in that, In the solution, the concentration of glycyrrhizin B is 0.02-0.05 g / L, and the concentration of citric acid is 0.125-0.5 g / L.

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