Preparation method of phyllanthus extract and application thereof in anti-helicobacter pylori medicine

Abstract

The present application belongs to the technical field of natural product development and bioactivity application, and particularly relates to a preparation method of Phyllanthus niruri extract and application thereof in anti-Helicobacter pylori drugs. The Phyllanthus niruri medicinal material is crushed, soaked in purified water, extracted by water bath reflux, concentrated by rotary evaporation, and freeze-dried to obtain the Phyllanthus niruri water extract. The Phyllanthus niruri water extract has obvious anti-Helicobacter pylori activity. The MIC of the Phyllanthus niruri water extract dissolved in brain heart infusion for different strains is as low as 160-320 μg / mL, and the MBC is as low as 320-640 μg / mL. The Phyllanthus niruri water extract is mixed with different antibiotics in different proportions to test the minimum combined inhibitory concentration. It is found that the combined inhibitory index value of the Phyllanthus niruri water extract and clarithromycin for strain ATCC700392 is 0.75, which is an additive effect. Therefore, the Phyllanthus niruri water extract and clarithromycin can be combined to be used in anti-Helicobacter pylori drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural product development and bioactive application technology, specifically relating to a method for preparing Phyllanthus urinaria extract and its application in anti-Helicobacter pylori drugs. Background Technology

[0002] Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped bacterium that primarily colonizes the gastric mucosa and is closely associated with various gastric diseases. The antibiotic combination therapy commonly used in Western medicine to treat Helicobacter pylori infection has shown high rates of drug resistance globally. Furthermore, while antibiotics kill the bacteria, they can also disrupt the balance of intestinal flora, causing adverse reactions such as diarrhea and constipation.

[0003] In recent years, the search for non-antibiotic therapies has become a hot topic in medical research. Against this backdrop, the application of single-herb Chinese medicines, food-medicine homologous substances, health foods, and new resource foods in the treatment of Helicobacter pylori infection has received increasing attention. These natural substances exert antibacterial, anti-inflammatory, and immunomodulatory effects through multiple mechanisms, exhibiting low drug resistance and fewer adverse reactions, providing a natural and safe alternative for combating Helicobacter pylori. Multiple studies have shown that the efficacy of integrated traditional Chinese and Western medicine treatment is superior to Western medicine treatment alone. Chinese medicines and food-medicine homologous substances are of great significance in anti-Helicobacter pylori research. They generally have low toxicity and side effects, are suitable for long-term use, and exert their effects through multiple mechanisms such as regulating the body's immunity, inhibiting bacterial growth, and reducing inflammation. They are less likely to induce bacterial resistance and are suitable for treating drug-resistant strains.

[0004] Currently, research on traditional Chinese medicine (TCM) in combating Helicobacter pylori infection largely focuses on the clinical application and mechanism exploration of TCM compound formulas, demonstrating significant clinical advantages. However, research on single-herb TCM in combating Helicobacter pylori is relatively scarce, with insufficient depth and breadth of research. In conclusion, screening single-herb TCM with anti-Helicobacter pylori activity is of great significance.

[0005] Phyllanthus urinaria L., a plant belonging to the genus Phyllanthus in the family Euphorbiaceae, is named for its pearl-like fruit that grows beneath the leaves. It is also known as "Pearl Grass" or "Yin-Yang Grass." In traditional medicine, the dried whole plant of Phyllanthus urinaria has extensive applications, possessing various medicinal properties such as clearing heat and improving eyesight, promoting diuresis and reducing swelling, and detoxifying and eliminating stagnation. It is commonly used to treat diarrhea, jaundice, edema, urinary tract infections, red eyes, and dysentery. Phyllanthus urinaria mostly grows in fields, along roadsides, and on hillsides, primarily distributed in several southern provinces of my country, and is one of the commonly used folk medicines in the south. Modern research shows that Phyllanthus urinaria contains various chemical components, including lignans, flavonoids, phenolic acids, tannins, terpenes, and steroids. These components possess antiviral, antitumor, antibacterial, antioxidant, and hepatoprotective pharmacological effects. In recent years, with the deepening research on the chemical components and pharmacological activities of Phyllanthus urinaria, its application prospects in modern medicine have received widespread attention. However, no research has been reported on Phyllanthus urinaria's effectiveness against Helicobacter pylori.

[0006] The 2012 paper by RANILLA LG, APOSTOLIDIS E, SHETTY K. et al., entitled "Antimicrobial Activity of an Amazon Medicinal Plant (Chancapiedra)(Phyllanthus niruri L.) against Helicobacter pylori and Lactic Acid Bacteria," disclosed the antimicrobial activity of water extracts of Amazonian Phyllanthus niruri L. against Helicobacter pylori. However, Phyllanthus niruri L. and Phyllanthus urinaria L. are two different and independent species in the genus Phyllanthus of the family Euphorbiaceae, and they differ fundamentally in terms of origin, morphological characteristics, and chemical composition.

[0007] First, Phyllanthus urinaria L., the dried whole herb of a plant in the genus Phyllanthus of the family Euphorbiaceae, is widely distributed in China (including multiple producing areas such as Jiangxi, Yunnan, and Zhejiang). This invention selects only samples from one producing area for research, which conforms to the inclusion specifications of "Chinese Materia Medica", "National Compendium of Chinese Herbal Medicine" and the standards of Chinese medicinal materials in multiple provinces. Its species characteristics are applicable to Phyllanthus urinaria from various producing areas in China. On the other hand, Phyllanthus niruri L., also known as "bitter Phyllanthus urinaria", is another independent species in the genus Phyllanthus of the family Euphorbiaceae. It is produced in the Amazon River Basin (Ecuador and Peru) and is included in the United States Pharmacopeia and the Indian Pharmacopoeia. It belongs to a different medicinal system from Phyllanthus urinaria used in traditional Chinese medicine.

[0008] Secondly, Phyllanthus urinaria L. has hairy stems, winged longitudinal ridges on its branches, and hairy leaf margins. Its leaves are simple, alternate, and arranged in two rows. Male flowers have 6 sepals and 6 glands on the floral disc. The capsule surface has tuberculate protuberances (scaly protuberances), and the pedicel is very short. Seeds have transverse grooves on the surface, and its thousand-seed weight is lower than that of Phyllanthus urinaria. In contrast, Phyllanthus niruri L. has cylindrical, slender branches, is entirely hairless, slightly curved or spreading, and its leaves have no hairy margins, a blunt or non-pointed apex, a rounded base, and are hairless on both sides. Its petioles are less than 1 mm long. Male flowers have 5 sepals (rarely 4), usually unequal in size, and 5 glands on the floral disc. The capsule diameter is 1–1.5 mm, smooth and hairless, with a short pedicel. Seeds have longitudinal grooves on the surface, and its thousand-seed weight is 193.18 mg, significantly higher than that of Phyllanthus urinaria. In addition, the main phenolic components of the Amazonian-originating *Phyllanthus urinaria* water extract are ellagic acid and hydroxycinnamic acid derivatives, with gallic acid detected only in samples from Ecuador. The total phenolic content is 65-73 mg / g dw, and the antioxidant activity (DPPH method) is approximately 89%. No flavonoids were detected, suggesting that its anti-Helicobacter pylori activity may be related to ellagitannins (such as corilagin and geraniol). It also contains lignans (nidulanin, nidulanin), alkaloids, and other components. However, experimental analysis showed that the chemical composition of *Phyllanthus urinaria* L. differs significantly from that of *Phyllanthus urinaria*, as detailed below: Phenolic components: mainly flavonoids (quercetin, rutin, catechin), phenolic acids (gallic acid) and tannins, with a total phenol content of 82~95 mg / g dw, which is higher than that of pearl grass.

[0009] Characteristic components: The flavonoids (quercetin, rutin, etc.) contained in Phyllanthus urinaria were not detected in Gynostemma pentaphyllum. Furthermore, Phyllanthus urinaria and Gynostemma pentaphyllum differ in their tannins and lignans; Phyllanthus urinaria contains lignans such as Phyllanthus urinaria succinate and Phyllanthus urinaria trifolia, while Gynostemma pentaphyllum contains lignans such as Phyllanthus urinaria succinate and Phyllanthus urinaria trifolia. Thin-layer chromatography experiments confirmed differences in the fluorescent spots of their petroleum ether extracts. After spraying with vanillin-concentrated sulfuric acid reagent, Phyllanthus urinaria showed a distinct pinkish-red spot at the corresponding location, while Gynostemma pentaphyllum did not exhibit this characteristic, further confirming the essential differences in their chemical compositions.

[0010] Amino acid composition: The methionine content in Phyllanthus urinaria is significantly higher than that in Phyllanthus urinaria, while the total content of other amino acids in Phyllanthus urinaria is slightly higher than that in Phyllanthus urinaria. There are obvious differences in the types and proportions of amino acids, which further confirms the difference in their material basis. Summary of the Invention

[0011] To address the problems existing in the prior art, this invention provides a method for preparing Phyllanthus urinaria extract and its application in anti-Helicobacter pylori drugs. The Phyllanthus urinaria water extract obtained by this invention through a specific extraction process exhibits significant anti-Helicobacter pylori activity. The MIC (microinhibitory concentration) of the Phyllanthus urinaria water extract dissolved in brain and heart extract against different strains is as low as 160-320 μg / mL, and the MBC (microinhibitory concentration) is as low as 320-640 μg / mL. Further testing of the minimum combined inhibitory concentration (MIC) by mixing the Phyllanthus urinaria water extract with different antibiotics in different proportions revealed that the combined inhibitory index (MIC) of the Phyllanthus urinaria water extract and clarithromycin against strain ATCC700392 is 0.75, indicating an additive effect. Therefore, the Phyllanthus urinaria water extract and clarithromycin can be combined for use in anti-Helicobacter pylori drugs.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity, comprising the following steps: After pulverizing Phyllanthus urinaria, the extract was soaked in purified water and then subjected to water bath reflux extraction. After extraction, the extract was filtered under reduced pressure, and the residue was subjected to multiple reflux extractions and filtrations. The filtrates were combined, concentrated by rotary evaporation, and freeze-dried to obtain Phyllanthus urinaria water extract freeze-dried powder.

[0013] Furthermore, the amount of ethanol solution added each time is 10-15 times the amount of Phyllanthus urinaria medicinal material.

[0014] Furthermore, the soaking time is 30-60 minutes.

[0015] Furthermore, the reflux extraction temperature is 80-90℃, and the time is 1-1.5h.

[0016] Furthermore, the number of times the multiple reflux extraction and filtering are performed is 1-3 times.

[0017] Furthermore, the preparation method also includes the step of dissolving the lyophilized powder of Phyllanthus urinaria water extract in brain and heart extract.

[0018] Furthermore, the preparation method of the brain and heart extract is as follows: add 100mL / 3.7g of double-distilled water to the brain and heart extract powder, shake well, autoclave, and cool to room temperature.

[0019] A second aspect of the present invention provides a Phyllanthus urinaria extract with anti-Helicobacter pylori activity prepared by the above-described preparation method.

[0020] A third aspect of the present invention provides the application of the above-mentioned Phyllanthus urinaria extract in the preparation of an anti-Helicobacter pylori drug, wherein the key active components 5-demethoxyniranthin, Methyl brevifolincarboxylate, and Urinatetralin in the Phyllanthus urinaria extract bind to key targets of Helicobacter pylori SRC, HSP90AA1, and PTPN11, thereby affecting the progression of Hp infection by influencing the PI3K-Akt signaling pathway and the Prostate cancer pathway.

[0021] A fourth aspect of the present invention provides a combination composition for combating Helicobacter pylori, comprising the above-mentioned Phyllanthus urinaria extract and clarithromycin.

[0022] Furthermore, the mass ratio of Phyllanthus urinaria extract to clarithromycin is 320-160:0.001-0.0005.

[0023] Compared with the prior art, the beneficial effects of the present invention are: This invention involves pulverizing Phyllanthus urinaria (a type of medicinal herb), soaking it in purified water, extracting it under reflux in a water bath, concentrating it by rotary evaporation, and then freeze-drying it to obtain an aqueous extract of Phyllanthus urinaria. This aqueous extract of Phyllanthus urinaria exhibits significant anti-Helicobacter pylori activity. The MIC (microinhibitory concentration) of the aqueous extract of Phyllanthus urinaria dissolved in brain and heart extract against different strains is as low as 160-320 μg / mL, and the MBC (microinhibitory concentration) is as low as 320-640 μg / mL. Further testing of the minimum inhibitory concentration (MIC) of the aqueous extract of Phyllanthus urinaria with different antibiotics in varying proportions revealed that the combined inhibitory index (MIC) of the aqueous extract of Phyllanthus urinaria and clarithromycin against strain ATCC700392 is 0.75, indicating an additive effect. Therefore, the aqueous extract of Phyllanthus urinaria and clarithromycin can be combined for use in anti-Helicobacter pylori drugs. Attached Figure Description

[0024] Figure 1 The time-inhibition curve of Phyllanthus urinaria water extract against Helicobacter pylori strain ATCC700392 is shown.

[0025] Figure 2 The results show the effects of different concentrations of Phyllanthus urinaria aqueous extract on the motility of Helicobacter pylori.

[0026] Figure 3 Scanning electron micrographs of Helicobacter pylori in the growth control group (left) and the drug-treated group (right).

[0027] Figure 4 Venn diagram of the common target of Phyllanthus urinaria and Hp.

[0028] Figure 5 This is a network diagram of "drug-ingredient-potential target".

[0029] Figure 6 This is a network diagram of potential target PPIs.

[0030] Figure 7 The image shows the results of the GO enrichment analysis.

[0031] Figure 8 The graph shows the results of the KEGG pathway analysis.

[0032] Figure 9 This is a docking diagram of the key active ingredient and the core target molecule. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0035] 1. Preparation of bacterial culture medium (1) Preparation of blood agar plates: Pour 15.6g of Columbia agar base into a 500mL Erlenmeyer flask, add 380mL of distilled water, shake to mix, and autoclave at 121℃ for 20min. After sterilization, cool in an oven to 55℃, add 5% sterile defibrinated sheep blood (i.e., 20mL), shake in one direction to mix, and pour into agar plates while still hot. The prepared blood agar plates should be left to stand overnight in a biosafety cabinet to allow moisture to evaporate, and should be used within 2 weeks.

[0036] (2) Preparation of brain and heart extract (HBI): Weigh 14.8g of brain and heart extract powder into a 500mL Erlenmeyer flask, add 400mL of double-distilled water, shake well, and place in a 121℃ autoclave for 20min. After sterilization, cool to room temperature and store in a 4℃ refrigerator. Use within 2 weeks.

[0037] 2. Bacterial resuscitation, passage, and cryopreservation (1) Bacterial resuscitation: Take the frozen bacterial strain out of the ultra-low temperature freezer (-80℃) and allow it to thaw naturally at room temperature. Take 100µL of bacterial solution and spread it evenly on the blood agar plate using an L-shaped spreader in a single direction. Place the plate in a three-gas incubator and incubate upside down at 37℃ under microaerophilic conditions (5% O2, 10% CO2, 85% N2) for 72 hours. The resuscitation is complete when the bacterial strain covers the surface of the blood agar plate and the bacterial growth has a frosted appearance.

[0038] (2) Subculturing of bacteria: When the bacterial strain has covered the surface of the blood agar plate and the bacterial growth is frosted, it can be subculturing. Use a moist sterile cotton swab soaked in BHI medium containing 20% ​​fetal bovine serum (FBS) to scrape and collect the bacterial growth on the blood agar plate. Suspend it in sterile 20% FBS-BHI to prepare a bacterial suspension. Then take 100µL of the bacterial suspension and spread it evenly on a new blood agar plate. Place it in a tri-gas incubator and invert it for 3 days. When the bacterial growth covers the surface of the blood agar plate and the bacterial growth is frosted, the subculturing is complete.

[0039] (3) Cryopreservation of bacteria: Select blood agar plates that have been cultured for 48-72 hours and are growing vigorously. Collect bacterial growth with a moistened sterile cotton swab, suspend it in 20% FBS-BHI, and then mix it with 50% sterile glycerol in a 1:1 ratio to prepare a bacterial suspension containing 10% FBS and 25% glycerol. Take 1 mL of the bacterial suspension and place it in a 2 mL cryovial. Store it in an ultra-low temperature freezer for 1-2 years. It needs to be thawed and stored regularly each year.

[0040] Example 1: Extraction and dissolution of Phyllanthus urinaria extract (1) Take Phyllanthus urinaria from Jiangxi Province, batch number 2405012, pulverize it, weigh 10g into a 500mL round-bottom flask, add 12 times the amount of purified water and soak for 30min, reflux extract in a 90℃ water bath for 1h, filter under reduced pressure, add 12 times the amount of purified water to the residue for a second extraction, extract a total of three times, combine the three filtrates, concentrate by rotary evaporation at 75℃, freeze-dry the obtained extract to obtain freeze-dried powder of the herbal water extract, and weigh it. The freeze-dried powder is packed into EP tubes, placed in a desiccator for drying and storage for later use.

[0041] (2) Weigh 20.48 mg of lyophilized powder into a 5 mL centrifuge tube, add 4 mL of brain heart extract in a clean bench, vortex mix and sonicate for 30 min, place in a centrifuge at 10000 rpm, at room temperature, centrifuge for 5 min, take the supernatant and filter through a 0.22 µm aqueous phase filter membrane to prepare a mother liquor with a concentration of 5120 µg / mL.

[0042] Test Example 1: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Test The stock solution from Example 1 was finally diluted to concentrations of 2560 µg / mL, 1280 µg / mL, 640 µg / mL, 320 µg / mL, 160 µg / mL, and 80 µg / mL using a two-fold dilution method in 96-well plates. 50 µL of the drug solution was added to each well, with three replicates for each concentration gradient. Negative control wells (containing only the drug, without bacterial inoculation), growth control wells (containing only the bacterial inoculation, without the drug), and positive control wells (clarithromycin) were also included. The bacterial strain cultured for 72 hours was scraped onto 20% FBS-BHI using a moistened sterile swab. The concentration was determined based on OD... 600Adjust the turbidity of the bacterial suspension to 1 McF, dilute it 10-fold with 20% FBS-BHI, and inoculate 50 µL of bacterial suspension per well into the above-mentioned drug-containing 96-well plates (the final turbidity of the bacterial suspension in the 96-well plate is approximately 1 × 10⁻⁶). 6 (CFU / mL). The 96-well plates were placed in a tri-gas incubator and incubated at 37°C under microaerophilic conditions with shaking at 150 rpm for 3 days. The results were then observed. Visually inspected from bottom to top under light, the growth control showed significant turbidity. The drug concentration at which the bacterial turbidity was significantly reduced compared to the control group was the MIC (repeated 3 times). The test results are shown in Table 1 below.

[0043] Table 1 It can be seen that the MIC of Phyllanthus urinaria aqueous extract dissolved in brain heart extract is as low as 160-320 μg / mL and the MBC is as low as 320-640 μg / mL for different strains.

[0044] Test Example 2: Plotting Time-Antibacterial Curve The stock solution from Example 1 was diluted twice to prepare drug solutions with concentration gradients of 2 MIC, MIC, 1 / 2 MIC, and 1 / 4 MIC. 1 mL of each solution was added to each well of a 6-well plate. A growth control group consisting of drug-free BHI was set up. Using strain ATCC700392 as the research object, the strain was collected and the bacterial concentration was adjusted according to the method in Test Example 1. After diluting 10-fold with 20% FBS-BHI, 1 mL of bacterial solution was added to each well, and the plates were incubated in a tri-gas incubator with shaking at 150 rpm. At 0, 8, 12, 24, 28, 36, 48, 60, and 72 h, 100 µL of culture medium was added to a 96-well plate to measure the OD. 600 Value. With time as the x-axis, OD 600 The values ​​are plotted on the ordinate as the time-antibacterial curve of the herbal extract, and OD is used to plot the curve. 600 A higher value indicates a greater number of bacteria and a weaker antibacterial effect. Test results are as follows: Figure 1 As shown in the figure, the antibacterial effect of the Phyllanthus urinaria water extract is concentration-dependent. The *Helicobacter pylori* (Hp) growth rate is faster during the culture period of 38–60 h, with the bacterial count reaching its peak at 60 h. Compared with the control group, the drug concentration at the MIC effectively inhibited Hp growth.

[0045] Test Example 3: Anti-Helicobacter pylori Motility Test Preparation of semi-solid culture medium: Add 3.7g BHI, 0.3g agar, and 93mL double-distilled water, shake to mix, and autoclave at 121℃ for 20min. After sterilization, cool in an oven to 55℃. Then, dispense into 50mL centrifuge tubes, adding 200μL, 100μL, 50μL, and 25μL of 0.16 g / mL stock solution (diluted from the stock solution in Example 1) and 200μL BHI to each tube, respectively, to prepare drug-containing culture media with final concentrations of 640μg / mL, 320μg / mL, 160μg / mL, and 80μg / mL, as well as a negative control. Add 3.5mL FBS and shake well. Measure 25mL and invert each plate. The semi-solid motility culture medium needs to be left for 2-3 days to allow sufficient moisture to evaporate before starting the experiment.

[0046] Preparation of bacterial culture medium: ATCC700392 strain was collected according to the method in Test Example 1, and the bacterial concentration was adjusted to 4 McF (approximately 1.2 × 10⁻⁶). 9 (CFU / mL) Immerse a disposable inoculation needle in the serum broth containing resuspended bacteria for approximately 3 seconds, then remove and vertically insert it into the culture medium for 3 seconds. Inoculate two wells per semi-solid motility plate, using three plates per concentration. The semi-solid motility plates do not need to be inverted. Incubate for five days in a tri-gas incubator at 37°C under microaerophilic conditions. After five days, photograph the semi-solid plates vertically in the same field of view as a ruler, and analyze the bacterial area using ImageJ.

[0047] Test results are as follows Figure 2 As shown in Table 2, compared with the control group, the aqueous extract of Phyllanthus urinaria significantly inhibited the motility of Helicobacter pylori at the MIC concentration.

[0048] Table 2 Group N <![CDATA[Area (mm 2 )]]> Control 3 8.33 ± 1.79 1 / 4 MIC 3 6.83 ± 2.41 1 / 2MIC 3 6.45 ± 1.68 MIC 3 1.91 ± 0.48 2MIC 3 0.91 ± 0.17 Test Example 4: Scanning Electron Microscopy Observation of the Effects of Phyllanthus urinaria Water Extract on Bacterial Cells ATCC700392 strain cultured for 48 hours was scraped from blood agar plates, and the turbidity was adjusted to 1 McF. The samples were then placed in a tri-gas incubator and cultured at 37°C under microaerophilic conditions with shaking at 130 rpm for 1 day until the logarithmic growth phase. 1 mL of the bacterial culture was added to 49 mL of 10% FBS-BHI containing the drug (obtained by dilution of the stock solution from Example 1). The final concentration of the drug was its MIC. A growth control group was set up with 1 mL of bacterial culture and 49 mL of 10% FBS-BHI without the drug, and a positive control group was set up with 1 mL of bacterial culture and 49 mL of 10% FBS-BHI containing clarithromycin. The final concentration of clarithromycin was its MIC. After 24 hours of shaking culture in a tri-gas incubator, the bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm at 26°C for 3 minutes. The supernatant was discarded, and the precipitate was washed 2-3 times with 1×PBS (pH 7.0), fixed with 2.5% pentylene glycol, and incubated overnight at 4°C. When preparing samples, the bacterial precipitate was washed three times with PBS, dehydrated with a series of 30%, 50%, 70%, 80%, and 95% ethanol, soaked and replaced with tert-butanol, freeze-dried at the critical point, mounted on a platform, sputter-coated with gold, and then sent for observation.

[0049] The changes in Hp cell morphology after culturing at the MIC concentration of Phyllanthus urinaria for 24 hours were observed using SEM. For example... Figure 3 As shown, the control group bacteria were spiral, curved, or rod-shaped, with a rough surface and intact surface membrane structure. In contrast, the bacteria cultured with the drug showed a broken surface membrane structure, exhibiting an adhesive, stringy appearance. The bacterial length was slightly shortened, and the surface became rougher and granular. This indicates that Phyllanthus urinaria may inhibit Hp by disrupting the integrity of the bacterial biofilm and inhibiting biofilm synthesis. At this time, Hp was in the logarithmic growth phase, and the bacterial surface became rough to evade drug attack, so the inhibitory effect was not very obvious. If the drug concentration and the drug-bacterial co-culture time are increased, the bacterial rupture effect may be more obvious.

[0050] Example 2: Combination of Phyllanthus urinaria aqueous extract and clarithromycin and combined antibacterial test The mother liquor in Example 1 was prepared into drug solutions with concentration gradients of 4MIC, 2MIC, MIC, 1 / 2MIC, and 1 / 4MIC by the two-fold dilution method. Different concentrations of antibiotics (amoxicillin 0.8 - 0.0125 μg / mL, clarithromycin 0.32 - 0.002 μg / mL, metronidazole 12.8 - 0.8 μg / mL, levofloxacin 3.2 - 0.2 μg / mL) were prepared in the same way. The strains were collected and the concentration of the bacterial suspension was adjusted according to the method in Test Example 1. According to the microbroth checkerboard dilution method, the bacterial suspension: drug solution: antibiotic = 2:1:1, and the total volume of each well was 100 μL, which was inoculated into a 96-well plate. It was placed in a triple-gas incubator and cultured with shaking at 37°C, in a microaerophilic environment, and 130 rpm for 72 h. The well with obvious clarification observed by the naked eye against light was taken as the minimum combined inhibitory concentration, and the combined inhibitory index (FICI) was calculated according to the formula. When FICI ≤ 0.5, there was a synergistic effect; 0.5 < FICI ≤ 1, there was an additive effect; 1 < FICI ≤ 4, there was no relevant effect; FICI > 4, there was an antagonistic effect. Each group was conducted with 3 parallel tests.

[0051] The test results are shown in Table 3 below. It can be seen that the combined inhibitory index (FICI) values of Phyllanthus niruri Linn. and levofloxacin, metronidazole, and amoxicillin are between 1 and 4, showing no relevant effect. The combined inhibitory index (FICI) value of Phyllanthus niruri Linn. and clarithromycin is 0.75, showing an additive effect. Phyllanthus niruri Linn. has no antagonistic effect in vitro with the 4 antibiotics, providing preliminary evidence for the combined use of the water extract of Phyllanthus niruri Linn. and various clinical treatment regimens for Hp infection.

[0052] Table 3 Test Example 5: Exploring the mechanism of action of Phyllanthus niruri Linn. in the treatment of Helicobacter pylori infection based on network pharmacology Obtaining the active ingredients of the drug and the corresponding target genes: Enter the keyword "Phyllanthus niruri Linn." in the HERB database to search for the active ingredients, screen according to the Lipinsk five principles, download and save the screened ingredients, and import them into the SwissTargetPrediction database (P > 0) to obtain the gene names of individual main ingredients.

[0053] Obtaining disease genes: Search in the GeneCards and DisGeNET databases with the keyword "Helicobacter pylori", merge and remove duplicates after obtaining the results to obtain the relevant targets of Hp.

[0054] Identification of potential targets and construction of the "drug-component-potential target" network: The main component genes and disease genes were imported into Venny 2.1.0 and their intersection was taken. The resulting intersection genes are the potential targets of Phyllanthus urinaria for anti-Hp activity. The main active ingredient of the drug and the intersection genes were imported into Cytoscape 3.10.3 to create a "drug-component-potential target" network diagram, and nodes with higher degree values ​​were selected as the main active ingredients.

[0055] Construction of the protein-protein interaction (PPI) network: The obtained intersection genes were input into the STRING database, the species was set as "Homosapiens", and the PPI network was constructed. Finally, Cytoscape 3.10.3 software was used for visualization analysis, and nodes with higher degree values ​​were selected as core targets.

[0056] GO biological analysis and KEGG pathway analysis: The DAVID database was used to perform GO functional enrichment analysis and KEGG signaling pathway analysis on the intersecting genes. The results were sorted in ascending order of P-value. The top 10 results for biological process (BP), cellular component (CC), and molecular function (MF) were selected to create a GO functional enrichment bar chart. The top 10 results for KEGG signaling pathway enrichment were selected to create a Sankey bubble chart.

[0057] Molecular docking validation: Based on the degree values, the top three key active ingredients and core targets were screened for molecular docking analysis. The chemical structures of the main compounds and the protein structures of the core targets were obtained using the PubChem database, PDB database, and BatchVinaGUI software, and preprocessing was performed before molecular docking. The optimized ligands and receptors were then used for molecular docking via AutoDock Vina, and the docking results were visualized using PyMOL software.

[0058] After screening, a total of 8 active ingredients of Phyllanthus urinaria were obtained, including phenolic acids, flavonoids, tannins and other compounds, as shown in Table 4 below. A total of 247 effective target genes corresponding to the active ingredients were obtained from the SwissTargetPrediction database.

[0059] Table 4 Ingredient id Ingredient name Molecular Formula Molecular Weight HBIN011510 5-demethoxyniranthin <![CDATA[C 23 H 30 O6]]> 402.5 HBIN027030 gallic acid <![CDATA[C7H6O5]]> 170.12 HBIN031753 kaempferol <![CDATA[C 15 H 10 O6]]> 286.24 HBIN035118 methyl brevifolin carboxylate <![CDATA[C 14 H 10 O8]]> 306.22 HBIN039736 phyllodulcin <![CDATA[C 16 H 14 O5]]> 286.28 HBIN041495 quercetin <![CDATA[C 15 H 10 O7]]> 302.23 HBIN047585 urinaligran <![CDATA[C 22 H 24 O7]]> 400.4 HBIN047586 urinatetralin <![CDATA[C 22 H 24 O6]]> 384.4 A total of 2312 *H. pylori* genes were retrieved from the GeneCards and DisGeNET databases. The 94 intersection genes obtained after cross-referencing were considered potential gene targets for *Phyllostachys edulis* in treating *H. pylori* infection. Figure 4As shown. A "drug-ingredient-potential target" network was constructed by combining Phyllanthus urinaria and its corresponding active ingredients and potential targets, as shown in [the diagram]. Figure 5 The key active ingredients with high concentrations are 5-demethoxyniranthin, Methyl brevifolin carboxylate, and Urinatetralin. These ingredients have multiple target sites and are considered key active ingredients in Phyllanthus urinaria for treating Helicobacter pylori.

[0060] like Figure 6 As shown, the PPI network contains 45 nodes. Larger nodes with darker colors represent higher degree values ​​and greater roles in inter-node communication. There are 118 lines, connecting two related nodes. Based on degree values, the top three nodes were selected: SRC (steroid receptor coactivator), HSP90AA1 (heat shock protein 90α family class A member 1), and PTPN11 (protein tyrosine phosphatase non-receptor type 11). These targets are considered to play a key role in Phyllanthus urinaria's anti-Hp activity. SRC is a non-receptor tyrosine kinase encoded by the SRC proto-oncogene, which regulates various processes such as cell division, movement, adhesion, angiogenesis, and survival, playing a crucial role in maintaining normal physiological functions. It induces the transformation of various malignant cells and participates in tumor generation, growth, and metastasis. HSP90AA1 belongs to the heat shock protein family and is an important molecular chaperone protein involved in the regulation of intracellular protein folding, stability, and function, playing a vital role in cellular stress responses. PTPN11 is a tyrosine phosphatase that is widely involved in the dephosphorylation of human proteins. It is also involved in the regulation of multiple signaling pathways related to cell metabolism, anti-apoptosis, and immune regulation. Mutations in the PTPN11 gene are associated with the occurrence of various diseases. Abnormal regulation of PTPN11 has been found in various human diseases, including autosomal dominant genetic diseases and various tumors.

[0061] The GO functional enrichment analysis yielded 448 functional entries, such as Figure 7As shown, BP analysis revealed significant enrichment effects on inflammatory response, signal transduction, epidermal growth factor receptor signaling pathway, and chromatin remodeling; CC analysis showed significant enrichment effects on cytoplasm, cytosol, nucleoplasm, and receptor complex; MF analysis showed significant enrichment effects on ATP binding, protein binding, histone H3Y41 kinase activity, and histone H2AXY142 kinase activity. KEGG pathway enrichment analysis yielded 128 signaling pathways, such as... Figure 8 As shown, Phyllanthus urinaria's treatment of Helicobacter pylori infection is closely related to the PI3K-Akt signaling pathway, prostate cancer, pathways in cancer, fluid shearstress and atherosclerosis, chemical carcinogenesis, and reactive oxygen species. Helicobacter pylori infection can induce gastric ulcers, and the PI3K-Akt signaling pathway is believed to play an important role in the pathogenesis and treatment of gastric ulcers. This signaling pathway occupies a central position in intracellular and extracellular signal transduction and plays a key regulatory role in physiological processes such as inflammation, oxidative stress, and apoptosis.

[0062] Three chemical components with high degree values ​​were selected and molecularly docked with three target sites with high degree values ​​to verify their binding. The results are shown in Table 5. The molecular docking binding energy is <-5.0 kcal / mol, indicating a stable binding characteristic between the components and the target sites, which has significant biological significance, and the docking results are worth further verification. Among them, Urinatetralin and HSP90AA1, SRC and Methyl brevifolincarboxylate and HSP90AA1 have relatively low binding energies, suggesting that HSP90AA1 and SRC may be important targets for Phyllanthus urinaria in the treatment of Helicobacter pylori infection. The visualization results are shown in Table 5. Figure 9As shown, Urinatetralin and HSP90AA1 form hydrogen bonds with ASN-106 and ASN-51 residues, Urinatetralin and SRC form hydrogen bonds with LYS-155 and ALA-148 residues, and Methylbrevifolincarboxylate and PTPN11 form hydrogen bonds with ASN-106 and ASN-51 residues.

[0063] Table 5 Active ingredient name HSP90AA1 PTPN11 SRC Urinatetralin -8.1 -5.2 -7.5 5-demethoxyniranthin -6.2 -4.6 -6.3 Methyl brevifolincarboxylate -7.1 -4.9 -6.4 The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity, characterized in that, Includes the following steps: After pulverizing Phyllanthus urinaria, the extract was soaked in purified water and then subjected to water bath reflux extraction. After extraction, the extract was filtered under reduced pressure, and the residue was subjected to multiple reflux extractions and filtrations. The filtrates were combined, concentrated by rotary evaporation, and freeze-dried to obtain Phyllanthus urinaria water extract freeze-dried powder.

2. The method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity according to claim 1, characterized in that, The amount of purified water added each time should be 10-15 times the weight of Phyllanthus urinaria medicinal material.

3. The method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity according to claim 1, characterized in that, The soaking time is 30-60 minutes.

4. The method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity according to claim 1, characterized in that, The reflux extraction temperature is 80-90℃, and the time is 1-1.5h.

5. The method for preparing Phyllanthus urinaria extract with anti-Helicobacter pylori activity according to claim 1, characterized in that, The preparation method also includes the step of dissolving the freeze-dried powder of Phyllanthus urinaria water extract in brain and heart extract.

6. A Phyllanthus urinaria extract with anti-Helicobacter pylori activity prepared by the preparation method according to any one of claims 1-5.

7. The use of Phyllanthus urinaria extract as described in claim 6 in the preparation of an anti-Helicobacter pylori drug, characterized in that, The key active ingredients in the Phyllanthus urinaria extract, 5-demethoxyniranthin, Methyl brevifolincarboxylate, and Urinatetralin, bind to key targets of Helicobacter pylori, SRC, HSP90AA1, and PTPN11, thereby affecting the progression of Hp infection by influencing the PI3K-Akt signaling pathway and the Prostate cancer pathway.

8. A combination composition against Helicobacter pylori, characterized in that, It contains the Phyllanthus urinaria extract as described in claim 6 and clarithromycin.

9. The combination composition against Helicobacter pylori according to claim 8, characterized in that, The mass ratio of Phyllanthus urinaria extract to clarithromycin is 320-160:0.001-0.0005.

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