Product for treating helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or with dysplasia and for preventing occurrence of gastric cancer, and treatment method therefor

Abstract

Provided are a product for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or with dysplasia, for reversing the pathology of gastric precancerous lesions, and for preventing the occurrence of gastric cancer, and a treatment method therefor. The product is a lotus leaf extract, and representative active ingredients thereof include, but are not limited to, isoquercetin, quercetin-3-O-glucuronide, hypericin, and nuciferine. The use comprises at least one of treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or with dysplasia, reversing gastric precancerous lesions, and preventing the occurrence of gastric cancer. In vitro experiments show that the lotus leaf extract can inhibit malignant proliferation of gastric precancerous lesion epithelial cells and reduce the active oxygen level thereof. In vivo experiments show that the lotus leaf extract can effectively improve the pathology of the Helicobacter pylori-negative gastric precancerous lesions and prevent the occurrence of gastric cancer in animal models. Clinical trials have shown that the lotus leaf extract can effectively treat the pathological conditions and clinical symptoms in patients with the Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or with dysplasia.

Description

A product and its treatment method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia and for preventing gastric cancer. Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a product and its treatment method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, reversing precancerous lesions of the stomach, and preventing the occurrence of stomach cancer. Background Technology

[0002] Stomach cancer has a high incidence rate in my country. According to data from the National Cancer Center of China's "2017 Latest Status and Trends of Cancer in China," stomach cancer ranks second in cancer incidence in my country, accounting for approximately 43.9% of new stomach cancer cases globally each year. The occurrence of stomach cancer is closely related to the malignant progression of gastritis. According to the classic Correa model, the development of stomach cancer involves a series of pathological states, including atrophic gastritis, intestinal metaplasia, and dysplasia (the latter two being referred to as precancerous lesions). [1] Taking effective intervention and reversal measures for these pathological conditions is the key to the early prevention and control of gastric cancer.

[0003] Treating precancerous lesions and preventing the progression of gastritis to gastric cancer remains a challenge in gastric cancer prevention and treatment, especially for patients negative for Helicobacter pylori (HP). Clinically, approximately 40% of patients are HP-positive. Studies have shown that eradicating HP is beneficial for gastric cancer prevention. A study published in the *New England Journal of Medicine*, involving 1676 participants, found that HP treatment reduced the risk of cancer development in participants with a family history of gastric cancer. [2] However, a meta-analysis published in the *Journal of Gastroenterology* found no significant reduction in gastric cancer incidence during a 10-year follow-up after *Helicobacter pylori* eradication, consistent with the findings of a randomized controlled clinical trial published in the *Journal of the American Medical Association*, which also found that *Helicobacter pylori* eradication did not lead to a significant reduction in gastric cancer incidence. [3,4] Expert consensus documents such as the "Expert Consensus on the Management Strategies of Precancerous State and Lesions of Gastric Mucosa in China (2020)" also indicate that the reversal effect of *Helicobacter pylori* eradication on precancerous lesions of gastric cancer still requires further research. [5] Celecoxib has been found to have a role in reversing precancerous lesions of the stomach, but it has no significant effect on patients who have undergone H. pylori eradication. [6] Current intervention strategies for Helicobacter pylori-negative atrophic gastritis mainly include lifestyle modifications, gastric mucosal protectants, and nonsteroidal anti-inflammatory drugs (NSAIDs). There are currently no intervention drugs that improve pathology or reverse the transformation of gastritis into cancer. Therefore, preventing Helicobacter pylori-negative or Helicobacter pylori-eradicated atrophic gastritis patients from progressing to gastric cancer remains a major challenge, urgently requiring the discovery of new drugs. [5-7] .

[0004] Lotus leaves are the leaves of the lotus (Nelumbo nuficera Gaertn.), a plant in the Nymphaeaceae family. They are also known as lotus leaves or lotus root leaves and are cultivated in both northern and southern China. The Compendium of Materia Medica and the Essentials of Materia Medica record that lotus leaves have the effects of "generating vital energy and benefiting the spleen and stomach" and "lightly dispersing, raising yang, and dispersing blood stasis".

[0005] The inventor's previous patent ZL201910849037.4 disclosed the "application of lotus leaf alkaloids and lotus leaf extracts as drugs for treating atrophic gastritis and / or blocking the transformation of gastritis into cancer," but it did not clarify the difference in efficacy for atrophic gastritis and precancerous lesions of gastric cancer in Helicobacter pylori-negative and Helicobacter pylori-positive cases, nor did it identify the relevant active ingredient group. This patent newly discovers the precise clinical localization of lotus leaf extract in Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, and the reversal of precancerous lesions of gastric cancer, and reveals the representative active ingredient group of lotus leaf extract that exerts this effect.

[0006] Summary of the Invention

[0007] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention discloses a treatment method and active ingredient group for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, reversing precancerous lesions of gastric cancer, and preventing gastric cancer. It clarifies the clinical indications and intervention mechanism, and defines the treatment method and clinical research evidence.

[0008] This invention provides a product and treatment method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, reversing precancerous lesions of gastric cancer, and preventing the occurrence of gastric cancer. The product is lotus leaf extract. The medicinal and edible herb involved in this invention is lotus leaf extract, which has a stable preparation process, mature quality standards, good safety, and low cost.

[0009] In a first aspect, the present invention provides a lotus leaf extract product. According to embodiments of the invention, it is characterized by comprising, but not limited to, the following substances as representative active ingredients: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid.

[0010] In a second aspect, the present invention provides the use of lotus leaf extract in the preparation of pharmaceuticals. According to embodiments of the present invention, the lotus leaf extract comprises, but is not limited to, the following substances as representative active ingredients: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid. The pharmaceuticals are used to treat Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia and / or to treat Helicobacter pylori-negative precancerous lesions of the stomach and / or to prevent the occurrence of stomach cancer.

[0011] In a third aspect, the present invention provides a method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned lotus leaf extract product.

[0012] In a fourth aspect, the present invention provides a method for preventing gastric cancer. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned lotus leaf extract product.

[0013] This invention establishes the production process and quality standards, and further explores the application of lotus leaf extract in treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, reversing precancerous lesions of gastric cancer, and preventing gastric cancer. The inventors identified the active ingredient group of lotus leaf extract that reverses Helicobacter pylori-negative precancerous lesions of gastric cancer through a series of analyses and experiments, verified the intervention mechanism and efficacy through in vitro and in vivo experiments, and determined its treatment method and clinical efficacy through rigorous real-world studies and multicenter randomized double-blind placebo-controlled trials.

[0014] The dosage form of the medicine or food is selected from tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations, powders, beverages, etc.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 shows the experimental results of lotus leaf extract inhibiting the malignant proliferation of Helicobacter pylori-negative gastric precancerous lesion epithelial cells in Example 2 of the present invention. Among them, (A) is the result of the change in the proportion of Helicobacter pylori-negative gastric precancerous lesion epithelial cells under different concentrations of lotus leaf extract intervention, and (B) is the result of the IC50 value of lotus leaf extract inhibiting the malignant proliferation of cells.

[0018] Figure 2 shows the effect of lotus leaf extracts prepared by different extraction processes in Example 3 of the present invention on inhibiting the malignant proliferation of Helicobacter pylori-negative gastric precancerous epithelial cells. Among them, (A) is the result of the change of relative cell content under different concentrations of lotus leaf extract treated by 20% ethanol extraction process, (B) is the result of the change of relative cell content under different concentrations of lotus leaf extract treated by 80% ethanol extraction process, (C) is the result of the change of relative cell content under different concentrations of lotus leaf extract treated by water extraction and alcohol precipitation supernatant, and (D) is the result of the change of relative cell content under different concentrations of lotus leaf extract treated by water extraction and alcohol precipitation.

[0019] Figure 3 shows the results of the study on the effect of lotus leaf extract on the level of reactive oxygen species (ROS) in gastric precancerous lesions in Example 4 of the present invention. In this figure, (A) is the percentage effect of the model group / lotus leaf extract group on gastric precancerous lesions (B525-A+ cells), and (B) is the ROS cell ratio of the model group / lotus leaf extract group.

[0020] Figure 4 shows the results of the efficacy study of lotus leaf extract on Tff1-KO Helicobacter pylori-negative gastric precancerous lesion model mice in Example 5 of the present invention. Among them, (A) is the proportion of mice in each group of model group / lotus leaf extract group / celecoxib group that progressed to severe dysplasia, and (B) is the proportion of mice in each group of model group / lotus leaf extract group / celecoxib group that progressed to gastric cancer.

[0021] Figure 5 shows the proportion of cellular atypical proliferation grades in mice in each group (model group, lotus leaf extract group, and celecoxib group) in Example 5 of this invention. Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0026] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0027] product

[0028] This invention provides a product for treating Helicobacter pylori-negative chronic atrophic gastritis and / or precancerous lesions of the stomach, and for preventing the occurrence of stomach cancer. The product is a lotus leaf extract. The medicinal and edible herb involved in this invention is a lotus leaf extract, whose representative active ingredients include, but are not limited to: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid, and it also contains other excipients.

[0029] The dosage form of the medicine or food is selected from tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations, powders, beverages, etc.

[0030] use

[0031] This invention proposes the use of lotus leaf extract in the preparation of pharmaceuticals. According to embodiments of the invention, the lotus leaf extract contains, but is not limited to, the following substances as representative active ingredients: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid. The pharmaceuticals are used to treat Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia and / or to treat Helicobacter pylori-negative gastric precancerous lesions and / or to prevent gastric cancer. Specifically, the inventors identified the group of active ingredients in lotus leaf extract that reverse precancerous gastric lesions through a series of analyses and experiments, verified the intervention mechanism and effect through in vitro and in vivo experiments, compared the effects of different preparation processes on precancerous gastric lesions, and determined the treatment method and clinical efficacy through rigorous real-world studies and multicenter randomized double-blind placebo-controlled clinical trials.

[0032] Disease treatment methods

[0033] This invention provides a method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned lotus leaf extract product.

[0034] The present invention also proposes a method for preventing gastric cancer. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned lotus leaf extract product.

[0035] In this article, the term “pharmaceuticalally acceptable amount” is the amount of substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., the amount of substance with a reasonable benefit / risk ratio.

[0036] In this document, the term "application" is equivalent to "administration," referring to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The lotus leaf extract product of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the lotus leaf extract product of the present invention is administered orally.

[0037] In this document, the term "treatment" refers to the use of a drug or product to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any use of medicine or product to treat, cure, relieve, improve, reduce, or inhibit a disease in an individual, including but not limited to administering medicines containing lotus leaf extract products as described herein to individuals in need.

[0038] In carrying out and completing this invention, the inventors conducted a series of experimental studies and clinical trials. The results of these experiments are given in the following description of the embodiments. These experimental results demonstrate that lotus leaf extract, at safe doses, can significantly treat Helicobacter pylori-negative atrophic gastritis with intestinal metaplasia and / or dysplasia, reverse precancerous lesions of the stomach, and prevent the occurrence of stomach cancer. In the above experiments, and in other work related to this invention, the inventors have made the following findings:

[0039] (1) Study on the active ingredients of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis with intestinal metaplasia and / or dysplasia, and reversing precancerous lesions of the stomach.

[0040] First, production process standards were established to clarify the preparation process. Then, the inventors constructed a method based on "network pharmacology prediction + multivariate experimental verification" to analyze the potential intervention targets and active ingredients of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis and reversing precancerous gastric lesions. First, network pharmacology methods were used to analyze the potential intervention modules of major components of lotus leaf extract at various stages of Helicobacter pylori-negative atrophic gastritis and precancerous gastric lesions from a biological functional perspective. Then, a cell-molecule-compound multilayer biological network was constructed to identify potential intervention targets and active ingredients. Finally, the binding kinetics and binding affinity between lotus leaf extract components and target proteins were characterized using thermoproteomics and biomembrane interference techniques, and the potential active ingredients were experimentally verified from multiple perspectives.

[0041] (2) Lotus leaf extract inhibits the malignant proliferation of epithelial cells in precancerous gastric lesions.

[0042] Using the human gastric epithelial cell GES-1 precancerous lesion model stimulated by the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as the subject, the half-maximal inhibitory concentration (IC50) of lotus leaf extract on MNNG-stimulated GES-1 gastric precancerous lesion model cells was determined to be 537.8 μg / mL. The results indicate that lotus leaf extract can inhibit the proliferation of gastric precancerous lesion-related cells and block the transformation process of gastritis into cancer.

[0043] (3) Lotus leaf extract reduces reactive oxygen species levels in precancerous gastric cells.

[0044] Using the MNNG-stimulated human gastric epithelial cell GES-1 precancerous lesion model as the subject, the proportion of cells with high reactive oxygen species in the GES-1 gastric precancerous lesion model cells under MNNG stimulation was reduced by 23%.

[0045] (4) Lotus leaf extract effectively reversed the pathological changes of precancerous gastric lesions in mice.

[0046] Using a Tff1 knockout (Tff1-Knockout, Tff1-KO) mouse model that has been shown in multiple studies to spontaneously generate various stages of gastric cancer, such as atrophic gastritis, intestinal metaplasia, and dysplasia, as the subject, after successful modeling, mice were given celecoxib and lotus leaf extract by gavage. Pathological results showed that lotus leaf extract could effectively reverse the pathological changes of precancerous lesions in the mice.

[0047] (5) Real-world studies and multicenter randomized double-blind placebo-controlled trials of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis with intestinal metaplasia and / or dysplasia and reversing the pathology of precancerous gastric lesions.

[0048] To investigate the clinical efficacy of lotus leaf extract, a real-world efficacy study of 138 patients and a multicenter, randomized, double-blind, placebo-controlled trial of 200 patients were conducted. In the real-world study, patients who took lotus leaf extract for an average of 8 weeks (range 2-24 weeks) were assigned to the treatment group, while those who did not take lotus leaf extract were assigned to the control group. Pathological improvement in the treatment and control groups was analyzed. The results showed that the treatment group had significantly better pathological improvement in the precancerous lesion stage than the control group (P<0.01), and also significantly better pathological improvement in the atrophic stage (P<0.05). Notably, subgroup analysis based on Helicobacter pylori infection status revealed that the improvement rate in the Helicobacter pylori-negative group reached 37.0%, significantly higher than that in the Helicobacter pylori-positive group (Chi-Square test, P<0.05), suggesting that lotus leaf extract may be the first drug to block or reverse the inflammatory-cancer transformation process in Helicobacter pylori-negative patients. This randomized, double-blind trial recruited patients with Helicobacter pylori-negative atrophic gastritis accompanied by intestinal metaplasia and / or dysplasia. The primary outcome measure was the pathological improvement rate after 6 months of treatment. Results showed no significant difference in pathological distribution between the lotus leaf extract group and the placebo group at baseline. At the 6-month follow-up, the pathological improvement rates in patients with Helicobacter pylori-negative precancerous lesions were 46.2% and 13.2% in the lotus leaf extract group and the placebo group, respectively, showing a statistically significant difference (P<0.01). This also provided clinical evidence for symptom improvement and data support for clarifying clinical localization and further research.

[0049] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0050] Example 1: Study on the pathological active components of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis and / or reversing precancerous lesions of the stomach.

[0051] First, a process for preparing lotus leaf extract was established based on the quality standards published in the inventor's patent "A method for determining the content of effective components in lotus leaves and their preparations" (patent application number: 202410950437.5).

[0052] (1) Analyzing the mechanism of lotus leaf extract in intervening in precancerous lesions of gastric cancer based on network pharmacology methods

[0053] The UNIQ system (ZL201910902205.1), independently developed by the inventors, was used to predict and calculate the target spectrum of components contained in lotus leaf extract. The top 100 targets were selected as the candidate target set for each compound based on the algorithm's accuracy. The reliability of the predicted target sets for each compound was verified using literature mining methods. The recall rate of each component's predicted target set in literature reports was calculated.

[0054] Next, an enrichment analysis of the overall target was performed. Based on the gastritis-cancer transformation mechanism obtained from previous studies, the enrichment results were screened to identify potential pathways and biological processes by which lotus leaf extract intervenes in gastritis-cancer transformation. The pathway enrichment results are shown in Table 1. The analysis revealed that lotus leaf extract plays an interventional role in biological functions such as inflammation, immunity, lipid metabolism, oxidative phosphorylation, and cell proliferation in gastritis-cancer transformation.

[0055] Table 1: Pathway Enrichment Results

[0056] (2) Prediction of active ingredients in lotus leaf extract based on network targets

[0057] We further constructed a multi-level "component-molecule-cell" network for the intervention of lotus leaf extract in precancerous gastric lesions. Previous studies identified three cell types most associated with early gastric cancer cells: stem-related MSCs (intestinal-like stem cells), metabolism-related enterocytes, and proliferation-related PCs (proliferating cells), revealing their crucial roles in the transformation of gastritis into cancer. Therefore, we compared the predicted targets of lotus leaf extract with the characteristic genes of MSCs, enterocytes, and PCs, constructing a component-molecule-cell network. This network includes compounds, genes, cells, and the relationships between them. The network displays cellular characteristic genes predicted to be targets of at least one compound or adjacent to the predicted targets of at least one compound on the PPI. Compounds are categorized according to their chemical structure. Within each category, compounds predicted to directly and indirectly (via PPI) target more genes are represented in the network. A compound category is associated with a cell type if compounds in that category are predicted to target more than five characteristic genes of that cell type. The connections between genes represent their direct links in the protein-protein interaction network from the STRING database (V11.5).

[0058] Based on network pharmacology analysis, it was found that alkaloids, flavonoids, and organic acids in lotus leaves have synergistic regulatory effects on immunity, inflammation, dryness, oxidative stress, and lipid metabolism. Coumarins played a significant regulatory role in immunity, lipid metabolism, and oxidative stress, while lotus leaf polysaccharides had a significant regulatory effect on immune inflammation, energy metabolism, and oxidative stress.

[0059] Finally, based on component analysis and network pharmacology analysis, we further analyzed which components in lotus leaf extract might intervene in precancerous lesions of the stomach. First, we screened for compounds that could intervene in characteristic genes of at least two of the three cell types: MSCs, Enterocytes, and PCs. Then, we performed functional analysis on the predicted target profile of each compound, focusing on its effects on cell proliferation (including cell cycle, mitosis, etc.) and metabolic processes (including glucose metabolism, lipid metabolism, oxidative phosphorylation, etc.). We found that among the alkaline components, lotus leaf alkaloids intervene in cell proliferation through HDAC2 and YWHAQ, and in metabolic processes through APOB and ALDOA; N-norophylline intervenes in cell proliferation through HDAC2, CDKN2A, and YWHAQ, and in metabolic processes through APOB and ALDOA; among the flavonoids, quercetin-3-O-β-D-glucuronide intervenes in cell proliferation through CDK1, CDC20, and BUB3, and in metabolic processes through OAT, IDH2, and MTTP; isoquercitrin intervenes in cell proliferation through PCNA, CDK1, and CCND2, and in metabolic processes through FABP1 and OAT; hyperoside and myricetin intervene in cell proliferation through PCNA, PLK1, and HDAC2, and in metabolic processes through LDHA, LDHB, IMPDH2, and APOA1. Furthermore, all these components can directly or indirectly interfere with key molecules in the p53 and TNF signaling pathways.

[0060] (4) Validation of the interaction between active ingredients and targets of lotus leaf extract

[0061] Experimental materials: lotus leaf alkaloid, isoquercitrin, quercetin-3-O-glucuronide, rutin, hyperoside, NQO1-His, PRC1-His, PRDX2-His.

[0062] Experimental Methods: Proteins were diluted to 30–50 μg / mL and then immobilized on a Ni-NTA sensor. The sensor was placed in buffer solutions containing different concentrations of the test compounds, and the interaction between the protein and the test compounds was detected using a ForteBio Octet Red96e molecular interaction analyzer. The binding signals were identified and analyzed using OctetHT V10.0 software, and KD (affinity) and R² (estimated goodness of fit, close to 1 indicating a good fit) were calculated.

[0063] Experimental Results: Based on the network pharmacology analysis results, targets such as PRC1, NQO1, and β-catenin, which play important roles in the biological module related to gastritis-cancer transformation, were selected for further study. Bio-Layer Interferometry (BLI) was used to characterize the binding kinetics and affinity between lotus leaf components and target proteins. The study revealed interactions between lotus leaf extract compounds such as lotus leaf alkaloids and rutin binding to PRC1, hyperoside and rutin binding to NQO1, and isoquercitrin and quercetin-3-O-glucuronide binding to β-catenin. KD and R2 values ​​are shown in Table 2.

[0064] Table 2: Results of interactions between components and targets in lotus leaf extract

[0065] The above results indicate that, by combining network pharmacology analysis, blood component analysis, and component-target interaction verification analysis, isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloids were screened as the main representative active ingredients for intervening in the transformation of gastritis into cancer, and can be used as indicators for the design of the preparation process and quality control of lotus leaf extract.

[0066] Example 2: Experiment on the inhibition of malignant proliferation of Helicobacter pylori-negative gastric precancerous epithelial cells by lotus leaf extract

[0067] Experimental materials: GES-1 cells, MNNG, lotus leaf extract, CCK-8 reagent.

[0068] Experimental methods: GES-1 cells induced by MNNG were seeded in 96-well plates and treated with an equal volume of water or different concentrations of lotus leaf extract for 24 hours. Cell proliferation rate was determined using CCK-8 reagent. After incubation at 37℃ and 5% CO2 for 2 hours, absorbance at 450 nm was measured using a microplate reader. The IC50 was calculated by fitting the curve.

[0069] The experimental results of the inhibition of malignant proliferation of Helicobacter pylori-negative gastric precancerous lesions by lotus leaf extract are shown in Figure 1.

[0070] Experimental results: Lotus leaf extract inhibited the proliferation of precancerous gastric cells in a dose-dependent manner, IC50 = 537.8 μg / mL.

[0071] Example 3: Effects of lotus leaf extracts prepared by different extraction processes on the proliferation of precancerous gastric cancer cells.

[0072] Experimental materials: GES-1 cells, MNNG, lotus leaf extract, CCK-8 reagent.

[0073] Experimental Methods: 50 mg of lotus leaf extract prepared using different processes was weighed and added to 5 mL of enzyme-free sterile water. The mixture was heated in a 60°C water bath with vortexing and sonication to aid dissolution, and then filtered through a 220 nm filter. MNNG-induced GES-1 cells (MNNG-GES-1) were seeded in 96-well plates. Different volumes of lotus leaf extract solution were added to the experimental groups to concentrations of 100, 200, 400, 800, and 1600 μg / mL, respectively. After 24 h of treatment with lotus leaf extract, cell counts were detected using the CCK-8 assay, and the IC50 was calculated.

[0074] The effects of lotus leaf extracts prepared by different extraction processes on inhibiting the proliferation of precancerous lesion cells in the gastric cancer model are shown in Figure 2.

[0075] Experimental results: Lotus leaf extracts prepared by different extraction processes all inhibited the proliferation of MNNG-GES-1 cells, and the inhibitory effect was concentration-dependent. The calculated IC50 values ​​for each group are shown in Table 3. Combined with the results of Example 2, the IC50 of the lotus leaf extract obtained by the water extraction process was significantly lower than that of the lotus leaf extracts obtained by the 20% ethanol extraction, 80% ethanol extraction, water extraction with alcohol precipitation, and water extraction with alcohol precipitation supernatant.

[0076] Table 3: Comparison of IC50 of lotus leaf extracts from different extraction processes

[0077] Example 4: Lotus leaf extract reduces reactive oxygen species (ROS) levels in precancerous gastric cells.

[0078] Experimental materials: GES-1 cells, MNNG, lotus leaf extract, Hochest dye, DCFH-DA fluorescent probe.

[0079] Experimental methods:

[0080] (1) Cell culture and treatment: GES-1 cells induced by 20 μM MNNG were seeded in 6-well plates and cultured for 24 h. The lotus leaf extract group was treated with 600 ng / mL lotus leaf extract for 10 h, while the model group was not treated.

[0081] (2) Probe incubation: Prepare working solution (1% Hochest and 0.05% DCFH-DA added to 1640 medium), wash cells once with 1640 medium, add 1 mL of staining working solution and incubate for 20 min, discard the working solution, wash cells three times with 1640 medium, collect cells and perform flow cytometry detection.

[0082] The results of the study on the effect of lotus leaf extract on the level of reactive oxygen species (ROS) in precancerous gastric cells are shown in Figure 3.

[0083] Experimental results: Compared with the model group, lotus leaf extract intervention reduced the proportion of high ROS precancerous lesion cells (B525-A+ cells) in the stomach from 78.8% to 60.5%, a decrease of 23%.

[0084] Example 5: Study on the efficacy of lotus leaf extract on a mouse model of Tff1-KO Helicobacter pylori-negative precancerous gastric lesions

[0085] Experimental methods:

[0086] (1) Tff1-KO Helicobacter pylori-negative mice aged 12 months were randomly divided into three groups: G1 (model group), G2 (lotus leaf extract group), and G3 (celecoxib group). There was no significant difference in the average weight of mice in each group.

[0087] (2) The dosage was determined by referring to previous rat drug administration experiments, selecting the medium-dose group with the best effect, and using the experimental animal dosage conversion formula.

[0088] (3) The administration time was the same as that of rats, which was 1 month. After another month, the rats were sacrificed.

[0089] (4) The evaluation indicators mainly consider the changes in mouse weight and pathological results after intervention.

[0090] (5) Based on the pathological interpretation results, samples from which no gastric antrum was obtained, as well as samples with poor sampling quality, were removed. The number of mice in each group was as follows: G1 (26 mice), G2 (16 mice), G3 (17 mice).

[0091] The results of the study on the efficacy of lotus leaf extract on Tff1-KO Helicobacter pylori-negative gastric precancerous lesion model mice are shown in Figure 4. The results of the study on the proportion of cellular dysplasia grades in each group of mice (model group, lotus leaf extract group, and celecoxib group) are shown in Figure 5.

[0092] Experimental results: The lotus leaf extract group had a lower grade of dysplasia than other groups. The incidence of carcinogenesis and severe dysplasia was 0%, while the incidence of carcinogenesis and severe dysplasia in the model group was 38.1% and 11.5%, respectively; and the incidence in the positive control group was 33.3% and 11.8%, respectively. These results suggest that the lotus leaf extract group is significantly more effective than the model group and the positive control group in treating Helicobacter pylori-negative precancerous lesions of the stomach and in preventing the occurrence of stomach cancer.

[0093] Example 6: A real-world study and multicenter randomized double-blind placebo-controlled trial of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis and reversing the pathology of Helicobacter pylori-negative gastric precancerous lesions.

[0094] Clinical validation of the efficacy and safety of lotus leaf extract in treating Helicobacter pylori-negative atrophic gastritis and reversing the pathological changes of Helicobacter pylori-negative gastric precancerous lesions was conducted in a real-world efficacy study of 138 patients and a multicenter randomized double-blind placebo-controlled trial of 200 patients.

[0095] 1. Real-world clinical trials of lotus leaf extract

[0096] The real-world study included 138 patients. Among them, the lotus leaf extract treatment group included 46 patients who underwent endoscopy at the China-Japan Friendship Hospital and Yijishan Hospital of Wannan Medical College in Anhui Province between May 2019 and December 2022. The baseline mean age was 57.35 years; this group included 17 males and 29 females; and included 25 cases of chronic atrophic gastritis, 16 cases of intestinal metaplasia, and 5 cases of low-grade dysplasia. The average duration of lotus leaf extract treatment was 8 weeks. The control group was matched using the propensity score method. Age, sex, and *Helicobacter pylori* infection were selected as covariates. Propensity scores were calculated, and a logistic regression model was used to calculate the comprehensive propensity score for each covariate. Samples were then matched according to the score levels, and the final control group was determined after evaluating the matching results. This trial included 92 patients in the control group who did not receive lotus leaf extract, with a mean baseline age of 58.12 years; 49 were male and 47 were female. The control group included 48 patients with atrophy, 34 with intestinal metaplasia, and 10 with low-grade intraepithelial neoplasia at baseline. No significant differences were found between the treatment and control groups in these clinical indicators, ensuring comparability of the intervention effects of lotus leaf extract.

[0097] Based on the patients' endpoint pathological outcomes, the pathological conditions of different groups of patients at different stages were statistically analyzed to assess the effect of lotus leaf extract on improving the pathology of patients with precancerous gastric lesions. Pathological progression was defined as the difference between the endpoint pathological score and the baseline pathological score. A score less than 0 indicated improvement; a score of 0 indicated maintenance; and a score greater than 0 indicated deterioration. At the baseline stage of intestinal metaplasia and low-grade intraepithelial neoplasia, the pathological improvement rate in the treatment group was 38.0%, while that in the control group was 22.7%. The Chi-Square test showed that the pathological improvement in the treatment group was significantly better than that in the control group at the precancerous lesion stage (P<0.01). At the atrophic gastritis stage, the pathological improvement rate in the treatment group was 28%, while that in the control group was 18.8%. The Chi-Square test also showed that the pathological improvement in the treatment group was significantly better than that in the control group at the atrophic stage (P<0.05).

[0098] The results of the subgroup analysis of Helicobacter pylori in patients enrolled in the real-world study of the lotus leaf extract treatment group are shown in Table 4.

[0099] Subgroup analysis of the lotus leaf extract treatment group based on Helicobacter pylori infection status revealed that the improvement rate in the Helicobacter pylori-negative group was 37.0%, significantly higher than that in the Helicobacter pylori-positive group (Chi-Square test, P<0.05), suggesting that lotus leaf extract may become the first drug to block or reverse the inflammatory-cancer transformation process in Helicobacter pylori-negative patients.

[0100] Table 4: Subgroup analysis of Helicobacter pylori in patients enrolled in the real-world study of the lotus leaf extract treatment group

[0101] 2. Randomized, double-blind, placebo-controlled clinical trial of lotus leaf extract

[0102] This study employs a multicenter, randomized, double-blind, placebo-controlled parallel clinical trial design to investigate the intervention efficacy in patients with Helicobacter pylori-negative intestinal metaplasia and dysplasia.

[0103] Study population

[0104] (1) Selection criteria

[0105] 1) Age between 18 and 70 years old

[0106] 2) Endoscopic and pathological diagnoses show atrophic gastritis with intestinal metaplasia or dysplasia.

[0107] (2) Exclusion criteria

[0108] 1) Has a history of gastric ulcers, gastric polyps, gastroesophageal reflux disease, gastric surgery, or other digestive system diseases such as chronic liver disease;

[0109] 2) Those who have used glucocorticoids, nonsteroidal anti-inflammatory drugs, or similar medications within the past three months;

[0110] 3) Individuals with a confirmed diagnosis of serious primary diseases of the cardiovascular, respiratory, digestive, or endocrine systems, whose conditions have not been stabilized;

[0111] 4) Patients with severe liver or kidney dysfunction (ALT or AST values ​​greater than twice the upper limit of normal; serum creatinine values ​​greater than twice the upper limit of normal);

[0112] 5) Individuals with known allergies to lotus leaf-based foods or medications;

[0113] 6) Pregnant or expectant women, and breastfeeding women;

[0114] 7) Individuals with mental illnesses or other diseases that affect clinical assessment or make it difficult for them to cooperate with clinical investigations;

[0115] 8) Individuals suffering from cancer or other life-threatening serious illnesses with an expected survival time of less than 12 months;

[0116] 9) Those who have participated in other drug clinical trials within the past three months.

[0117] Sample size and randomization method

[0118] This study aims to enroll 200 participants using a block randomization method, with a 1:1 ratio of experimental to control groups (100 participants in each group). Dedicated statisticians will generate random numbers and use these numbers to code the investigational drug and placebo.

[0119] test drug

[0120] The test drug in this study was a lotus leaf extract preparation.

[0121] Treatment group: Based on the consensus of TCM experts on chronic atrophic gastritis, pharmacopoeia standards and other TCM intervention studies, the dosage regimen was 10g / bag, 1 bag / day, for a course of 6 months, taken orally.

[0122] Control group: In addition to routine treatment, a placebo was given as a control drug, 10g per day. The placebo was similar in appearance, color and size to lotus leaf powder solid beverage.

[0123] Concomitant Medications: During the study period, under the guidance of a professional clinician and referring to the "Chinese Consensus on Chronic Gastritis (2017, Shanghai)," PPIs, H2 receptor antagonists, gastric mucosal protectants, and other medications may be used as appropriate based on the patient's condition. The use of traditional Chinese medicine preparations (including decoctions and proprietary Chinese medicines) related to the treatment of gastric diseases is prohibited. Concomitant medication use during the study period should be accurately recorded in the CRF form.

[0124] Endpoint Indicators

[0125] (1) Key endpoint indicators

[0126] Referring to the "Technical Guidelines for Clinical Efficacy Evaluation of New Traditional Chinese Medicine Drugs for Chronic Gastritis" published in 2022 and related expert interpretations, gastric tissue samples were taken at baseline, 6 months of follow-up, and 12 months of follow-up. The Correa pathological scoring system was used to score the samples and calculate the improvement rate. Two experienced pathologists scored each microscopic tissue sample according to the Sydney criteria for acute gastritis, chronic gastritis, atrophy, intestinal metaplasia, and dysplasia, with scores of none (0), mild (1), moderate (2), and severe (3).

[0127] (2) Secondary endpoint indicators

[0128] Improvement in Correa pathology score at 6 months.

[0129] Symptom improvement rate: The treatment method was based on the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines". The symptoms of chronic gastritis and cold-heat syndrome were scored according to the frequency and severity of the symptoms. None (0), mild (1), moderate (2), severe (3).

[0130] Analyze datasets

[0131] This trial has been registered with the Chinese Clinical Trial Register (ChiCTR), a primary registry on the World Health Organization's international clinical trial registration platform, under registration number ChiCTR2000039549. All statistical analyses followed the intention-to-treat principle. During the follow-up period, 21 patients dropped out; 6 patients voluntarily withdrew their informed consent, and 15 patients were lost to follow-up due to failure to complete outcome marker testing caused by factors such as the COVID-19 pandemic. Baseline clinical characteristics and intergroup analyses were performed on 179 eligible patients. There were no significant differences in baseline gender, age, ethnicity, or pathological diagnosis between the lotus leaf extract and placebo groups, ensuring comparability of outcome markers between the two groups. To clarify the optimal clinical application of lotus leaf extract, subgroup analysis was conducted based on Helicobacter pylori infection status, comparing the efficacy in 118 Helicobacter pylori-negative patients.

[0132] Security Analysis

[0133] Safety assessment indicators in this study included both laboratory tests and adverse events. Laboratory tests included: complete blood count (CBC): red blood cell count, hemoglobin, white blood cell count, neutrophil percentage, lymphocyte percentage, platelet count, etc.; blood biochemistry tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, etc.; and urinalysis: red blood cells (high power field), white blood cells (high power field), protein, glucose, bacteria (high power field), occult blood, etc. Adverse events included serious adverse events (liver function impairment, kidney function impairment, etc.) and other adverse events. In the first interim analysis, all laboratory test indicators of enrolled patients were within the normal clinical reference range, with an abnormality rate of 0. The incidence of all adverse events during the 6-month follow-up in both groups was 0, and there was no significant correlation with demographic or other baseline characteristics.

[0134] Therapeutic effect analysis

[0135] Outcome analysis was performed on 118 Helicobacter pylori-negative patients. For primary outcome indicators, the pathological condition (intestinal metaplasia and dysplasia) was recorded at enrollment. After 6 months of continuous medication, a follow-up endoscopy was performed, and the pathological condition was statistically analyzed and compared between the two endoscopic examinations. Outcome indicators were defined as follows: Improvement: The pathological condition at 6 months was less severe than the baseline pathological score, such as intestinal metaplasia changing to atrophic gastritis; Maintenance: The pathological condition at 6 months was consistent with the baseline pathological condition, such as intestinal metaplasia remaining unchanged; Progression: The pathological condition at 6 months was more severe than the baseline pathological condition, such as intestinal metaplasia changing to dysplasia. The degree of improvement in pathological condition among different groups was determined, and the pathological changes between groups were statistically analyzed based on the baseline pathological condition.

[0136] Table 5: Pathological outcomes of different patient groups in RCT trials

[0137] The pathological improvement rate of each patient in the Helicobacter pylori-negative group after 6 months of follow-up was calculated. Results showed no significant difference in pathological distribution between the two groups at baseline. At the 6-month follow-up point, the improvement rates of intestinal metaplasia in the lotus leaf extract treatment group and the placebo group were 41.5% (22 / 53) and 15.5% (7 / 46), respectively, with the lotus leaf extract group significantly superior to the placebo group (Chi-Square test, P<0.01). However, the improvement rates of dysplasia in the two groups were 66.7% (8 / 12) and 0 (0 / 7), respectively, with the lotus leaf extract group significantly superior to the placebo group (Chi-Square test, P<0.01). In the precancerous lesion group with a combination of intestinal metaplasia and dysplasia, the pathological improvement rates were 46.2% (30 / 65) and 13.2% (7 / 53), respectively, with the lotus leaf extract group showing significantly better improvement (Chi-Square test, P<0.01). Therefore, the lotus leaf extract of the present invention has significant clinical efficacy in treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia or dysplasia, providing data support for further research.

[0138] Furthermore, the symptom improvement of all 179 patients was evaluated after a 6-month follow-up. Referring to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines," typical symptoms of chronic gastritis and cold-heat syndrome were scored according to the frequency and severity of the symptoms. Four levels were assigned: none (0), mild (1), moderate (2), and severe (3). The statistical symptoms included: bloating or fullness in the hypochondrium, stomach pain, belching and acid reflux, loose stools, fatigue, bitter taste in the mouth, dry mouth, nausea and vomiting, tenderness upon palpation, localized pain, loss of appetite, chest tightness, constipation, shortness of breath and reluctance to speak, vomiting clear fluid, bland taste in the mouth, yellow urine, dry heaving, constipation, melena, and dull complexion. Comparing the symptom scores at the 6-month follow-up point with the baseline, it was found that at the 6-month follow-up point, the lotus leaf extract group showed significant improvement in scores for the five symptoms of bloating, loss of appetite, heartburn, dry mouth, and fatigue (* indicates P<0.01), and the improvement scores were better than those in the placebo group (Wilcoxon test). Among the five symptom groups, the improvement rates were as follows: bloating: 17.9% in the placebo group and 52.3% in the lotus leaf extract group; loss of appetite: 7.7% in the placebo group and 13.6% in the lotus leaf extract group; heartburn: 5.1% in the placebo group and 31.8% in the lotus leaf extract group; dry mouth: 2.6% in the placebo group and 20.5% in the lotus leaf extract group; fatigue: 15.4% in the placebo group and 20.5% in the lotus leaf extract group. The improvement rates in the lotus leaf extract group were significantly better than those in the placebo group in all groups (Chi-Square test, P<0.01).

[0139] Table 6: Improvement of symptom scores in patients during RCT trials

[0140] The experimental results of the above embodiments collectively demonstrate that the lotus leaf extract prepared using the production process and active ingredients proposed in this invention can effectively treat Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, reverse precancerous lesions of the stomach, and prevent the occurrence of stomach cancer.

[0141] The examples provided are merely illustrative and not intended to limit the implementation of the invention. Those skilled in the art will recognize that various variations and modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations, and obvious variations or modifications derived therefrom remain within the scope of this invention. It is understood that the above embodiments are exemplary and should not be construed as limiting the invention. Those skilled in the art can make variations, modifications, substitutions, and alterations to the above embodiments within the scope of this invention.

[0142] References

[0143] [1] Correa P, Haenszel W, Cuello C, et al. A model for gastric cancer epidemiology. Lancet. 1975; 2 (7924): 58-60.

[0144] [2] Choi IJ, Kim CG, Lee JY, et al. Family History of Gastric Cancer and Helicobacter pylori Treatment. N Engl J Med. 2020; 382(5): 427-436.

[0145] [3] Ford AC, Yuan Y, Moayyedi P. Long-Term Impact of Helicobacter pylori Eradication Therapy on Gastric Cancer Incidence and Mortality in Healthy Infected Individuals: AMeta-Analysis Beyond 10Years of Follow-Up. Gastroenterology. 2022; 163(3):754-756.e1.

[0146] [4]Wong BC, Lam SK, Wong WM, et al. Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of China: a randomized controlled trial. JAMA. 2004; 291(2):187-194.

[0147] [5]Wong BC, Zhang L, Ma JL, et al. Effects of selective COX-2inhibitor and Helicobacter pylori eradication on precancerous gastric lesions. Gut. 2012; 61(6):812-818.

[0148] [6] National Clinical Research Center for Digestive Diseases (Shanghai), National Alliance of Early Digestive Cancer Prevention and Treatment Centers, Helicobacter pylori Group of Gastroenterology Branch of Chinese Medical Association, et al. Expert consensus on management strategies for precancerous state and precancerous lesions of gastric mucosa in China (2020). Chinese Journal of Gastroenterology, 2020, 40(11):11.

[0149] [7]Dinis-Ribeiro,M.,Shah,S.,El-Serag,H.,Banks,M.,Uedo,N.,Tajiri,H.,Coelho,LG,Libanio,D.,Lah ner,E.,Rollan,A.,Fang,JY,Moreira,L.,Bornschein,J.,Malfertheiner,P.,Kuipers,EJ,&El-Omar,EMThe road to a world-unified approach to the management of patients with gastric intestinal metaplasia: a review of current guidelines. Gut, 2024; gutjnl-2024-333029.

Claims

1. A lotus leaf extract product, characterized in that, The lotus leaf extract contains, but is not limited to, the following substances as representative active ingredients: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid.

2. The product according to claim 1, characterized in that, The product described herein is used to treat Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia.

3. The product according to claim 1, characterized in that, The product described therein is used to treat Helicobacter pylori-negative precancerous lesions of the stomach.

4. The product according to claim 1, characterized in that, The product described therein is used to prevent stomach cancer.

5. The product according to any one of claims 1 to 4, characterized in that, The product is either a pharmaceutical or a food product. The dosage form of the pharmaceutical is selected from tablets, capsules, pills, injections, sustained-release preparations, and controlled-release preparations. The product form of the food product is selected from powders and beverages.

6. The product according to claim 5, characterized in that, The product further contains pharmaceutical or food-grade excipients.

7. The use of lotus leaf extract in the preparation of pharmaceuticals, characterized in that, The lotus leaf extract contains, but is not limited to, the following substances as representative active ingredients: isoquercitrin, quercetin-3-O-glucoside, hyperoside, and lotus leaf alkaloid. The drug is used to treat Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or with dysplasia and / or to treat Helicobacter pylori-negative gastric precancerous lesions and / or to prevent the occurrence of gastric cancer.

8. A method for treating Helicobacter pylori-negative chronic atrophic gastritis with intestinal metaplasia and / or dysplasia, characterized in that, The method comprises administering to a subject a pharmaceutically acceptable amount of the lotus leaf extract product according to any one of claims 1 to 6.

9. A method for preventing gastric cancer, characterized in that, The method comprises administering to a subject a pharmaceutically acceptable amount of the lotus leaf extract product according to any one of claims 1 to 6.

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