Extraction methods and applications of total extracts and compositions of *Lycoperdon perlatum*.

Abstract

This invention relates to the extraction method and application of total extracts and compositions of *Helicobacter pylori* (Northern Green Dragon Leaf) medicinal materials, belonging to the field of traditional Chinese medicine extraction and chemical separation. To address the problems of high dependence, addiction, and significant adverse reactions of commonly used clinical anti-Helicobacter pylori drugs, the extraction method is as follows: *Helicobacter pylori* is weighed, soaked in ethanol (volume concentration), extracted, and the extracts are combined, the solvent is recovered, and the mixture is freeze-dried to obtain the total extract of *Helicobacter pylori*. The total extract is dissolved in water, and eluted with 20% ethanol using a chromatographic column to obtain the composition of *Helicobacter pylori*. The total extract and composition of *Helicobacter pylori* are used as active ingredients in drugs for treating *Helicobacter pylori*-positive gastric ulcers. Both exhibit significant antibacterial effects, low dependence and addiction, and no adverse reactions.

Description

Technical Field

[0001] This invention relates to a method for extracting a traditional Chinese medicine composition and its application, belonging to the field of traditional Chinese medicine extraction and chemical separation. Background Technology

[0002] Gastric ulcer (GU) is a common digestive system disease worldwide. Its pathological essence is localized tissue loss of the gastric mucosa after exposure to various aggressive factors, with the defect reaching the muscularis mucosae. Epidemiological surveys show that the annual incidence of gastric ulcers in adults is approximately 10%-20%, severely impacting patients' quality of life. Helicobacter pylori is a microaerophilic Gram-negative bacillus that specifically colonizes the gastric mucosal epithelium. The detection rate of Helicobacter pylori is extremely high in patients with peptic ulcers. Studies show that the infection rate of Helicobacter pylori in duodenal ulcer patients is over 90%, while the infection rate in gastric ulcer patients reaches 70%-90%. Currently, quadruple therapy based on proton pump inhibitors (PPIs) is a commonly used clinical treatment for gastric ulcers. However, due to the irrational use of antibiotics, drug resistance in patients is constantly increasing, leading to a decline in the overall effectiveness of quadruple therapy. Therefore, finding alternative drugs with low resistance, stable and sustained efficacy, and good antibacterial effects is a problem that needs to be solved at this stage.

[0003] Traditional Chinese medicine (TCM) has advantages in regulating multiple targets and pathways in the fight against Helicobacter pylori, with low risk of drug resistance, few adverse reactions, and the ability to holistically regulate and improve gastric mucosal function. It shows great promise in the clinical treatment of Helicobacter pylori-positive cases. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems of high dependence, addiction and significant adverse reactions of commonly used clinical anti-Helicobacter pylori drugs, and to provide an extraction method for a composition of *Helicobacter pylori* and its application.

[0005] The extraction method for the total extract of *Leymus chinensis* is as follows:

[0006] Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

[0007] The extraction method of the Northern Qinglong Garlic Composition is as follows:

[0008] 1. Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract the extracts three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

[0009] 2. Dissolve 100g of total extract of *Leymus chinensis* (BQLY-Z) obtained in step 1 in water, and elute with 20% ethanol using a macroporous resin D101 column to obtain 20% ethanol eluent of *Leymus chinensis* ethanol extract (BQLY-20), which is the *Leymus chinensis* composition.

[0010] The total extract of *Heliotropium indicum* is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

[0011] The North Qinglongyi composition is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

[0012] The total extract of *Ilex cornuta* and the composition of *Ilex cornuta* (20% ethanol eluent of *Ilex cornuta* ethanol extract) of the present invention have significant antibacterial effects.

[0013] The combination of the total extract of *Helicobacter pylori* and *Helicobacter pylori* of this invention resulted in a reduction in gastric ulcer area, a decrease in gastric tissue damage, a recovery in pepsin content, and a reduction in the expression of inflammatory factors. This demonstrates that the extract of *Helicobacter pylori* can not only directly kill *Helicobacter pylori*, but also repair gastric inflammation, with low dependence and addiction potential and no adverse reactions. Attached Figure Description

[0014] Figure 1 These are images of gastric tissue ulcers and HE staining from Experiment 2. In the image, NC represents the normal control group, MODLE represents the model control group, BQLY-Z represents the total extract of *Ilex cornuta*, BQLY-20 represents the 20% ethanol eluent of *Ilex cornuta* ethanol extract, and Amoxicillin represents the positive drug amoxicillin group.

[0015] Figure 2 This is a graph showing the ulceration surface and statistics in Experiment 2;

[0016] Figure 3 This is a statistical chart of stomach capacity from Experiment 2;

[0017] Figure 4 This is a graph showing the pepsin content in Experiment 2;

[0018] Figure 5 This is a graph showing the LPS content in the stomach tissue of mice in Experiment 2.

[0019] Figure 6 This is a graph showing the TNF-α content in the stomach tissue of mice in Experiment 2. Detailed Implementation

[0020] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0021] Specific Implementation Method 1: The extraction method of the total extract of *Pterocarya stenoptera* in this implementation method is as follows:

[0022] Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

[0023] Specific Implementation Method Two: The extraction method of the Northern Azure Dragon Skin Composition in this implementation method is as follows:

[0024] 1. Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract the extracts three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

[0025] 2. Dissolve 100g of total extract of *Leymus chinensis* (BQLY-Z) obtained in step 1 in water, and elute with 20% ethanol using a macroporous resin D101 column to obtain 20% ethanol eluent of *Leymus chinensis* ethanol extract (BQLY-20), which is the *Leymus chinensis* composition.

[0026] Specific Implementation Method 3: The total extract of *Hemiberlesia lingua* described in Specific Implementation Method 1 is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

[0027] Specific Implementation Method Four: The Northern Qinglongyi Composition described in Specific Implementation Method Two is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

[0028] The following experiments were used to verify the effectiveness of the invention:

[0029] Experiment 1:

[0030] Antibacterial effect experiment of total extract and 20% ethanol eluent of ethanol extract of *Leymus chinensis*:

[0031] I. Materials and Methods

[0032] 200 mg / mL of deionized water, 50% ethanol, and extracts of *Helicobacter pylori* (total extract and 20% ethanol eluent of *Helicobacter pylori* ethanol extract) were serially diluted with their respective extraction solvents. The extracts were cultured in sterile solid medium at 60°C to achieve final concentrations of 100, 50, 25, 12.50, 6.25, 3.13, 1.57, 0.79, and 0.40 mg / mL, respectively. After solidification, *Helicobacter pylori* suspension (NCTC11673) was added for incubation. The MIC (minimum inhibitory concentration) was the concentration at which no test bacteria grew. The blank control was the corresponding extraction solvent. Each treatment group was repeated in triplicate.

[0033] Antibacterial rate Y = [1 - (OD drug + bacterial solution - OD drug background) / (OD growth control - OD culture medium blank)] * 100%

[0034] II. Experimental Results

[0035] The 20% ethanol eluent of the ethanol extract of *Leymus chinensis* has a significant antibacterial effect.

[0036] Table 1. Drug MIC Statistics

[0037]

[0038] Experiment 2:

[0039] I. Materials and Methods

[0040] 1. Use SPF-grade male C57BL / 6 mice, 7 weeks old, weighing 18-22g, 36 mice in total. The room temperature was 22±2°C, the humidity was 55±5%, and the day and night were alternated for 12 hours. The mice had free access to food, water and feed.

[0041] 2. Animal grouping

[0042] Thirty-six SPF-grade male C57BL / 6 mice were randomly divided into five groups: control group (n=9), model group (n=9), model + BQLY-Z group (n=6), model + BQLY-20 group (n=6), and model + amoxicillin group (n=6).

[0043] After 7 days of acclimatization, mice were administered 0.2 mL of NCTC11673 H. pylori bacterial suspension (approximately 1 × 10⁹ CFU / mL) by gavage. All animals were fasted for 12 hours before gavage, and then given H. pylori suspension once a day for 5 days, for a total of 3 times. The negative control group consisted of 9 mice, which were given sterile saline instead of H. pylori suspension. Eight weeks after gavage, 3 mice from the control group and 3 mice from the model group were randomly sacrificed, and gastric mucosa was collected to assess H. pylori infection and inflammation, thus excluding mice with natural H. pylori infection.

[0044] Thirty-six male SPF-grade C57BL / 6 mice (20g each) were prepared and divided into a blank control group, a model control group, a BQLY-Z group, a BQLY-20 group, and an amoxicillin group. After numbering, all mice were fed with 25 mg / mL ampicillin aqueous solution for 4 days to kill any bacteria in the mice's stomach that might affect the experiment.

[0045] The frozen bacterial culture was activated at a ratio of 1:100 in Brucella broth (manufacturer: Qingdao Haibo, model: HB0241) + 10% FBS (Sijiqing, model: 13011-8611), and then transferred to fresh culture medium and cultured again to mid-log phase for bacterial activation. The bacterial count was adjusted to 1×10⁹ with PBS solution (phosphate buffered PBS) for later use.

[0046] Mice were fasted for 12 hours to reduce the interference of gastric contents with infection. A 20G gavage syringe was used. Each mouse was gavaged with 0.2 mL of NCTC11673 H. pylori bacterial solution daily, and fed and watered again 4 hours after gavage. The gavage was repeated every other day for a total of 5 times. The control group mice were gavaged with an equal volume of PBS solution.

[0047] After randomization, the daily dose required per mouse was calculated using the body surface area conversion method, referring to the clinical dosage of amoxicillin for a 60kg adult (dose per mouse (mg) = total adult dose (mg) × (mouse body surface area / adult body surface area). Based on the recommended daily dose of amoxicillin for adults (1500mg), it was determined that each group should receive 6mg of the drug per mouse in this experiment. The dosage of raw Qinglongyi (30g / person / day) and the extraction rate of the raw drug (BQLY-Z extraction rate 14.6%, using BQLY-Z) were also considered. 100g of sample was eluted to obtain BQLY-20: 4.45g; the oral gavage volume for mice (300μL / 30g) yielded the following drug concentrations: BQLY-Z: 85.30mg / mL, BQLY-20: 3.80mg / mL. Ultrapure water was used to prepare the drug solutions. The prepared drug solution concentrations were: BQLY-Z: 85.30mg / mL, BQLY-20: 3.80mg / mL, and amoxicillin: 2.49mg / mL. Each group was administered 0.2mL of the corresponding drug solution by gavage. The blank control group and model group were administered 0.2mL of distilled water by gavage daily for 28 days. The treatment was conducted in an SPF environment at 24℃ with a 12h light-dark cycle.

[0048] After anesthetizing mice with isoflurane, the stomach of the mice was removed, and the stomach contents were aspirated using a 20G gavage needle. After centrifugation at 5000g for 3 minutes, the supernatant was collected to measure the gastric fluid volume.

[0049] After the above operations, gently rinse with PBS solution to remove impurities from the stomach surface. The mouse stomach tissue was longitudinally sectioned and its size recorded before photographing. ImageJ software was opened and approximate calibration was performed using a known size reference. The ulcer area was manually circled using the Freehand Selection Tool, and the ulcer area was obtained by Analyze → Measure.

[0050] Randomly selected portions of gastric tissue were placed in 4% paraformaldehyde solution and fixed at room temperature for 2 hours, then stored overnight at 4°C for sectioning. 0.5g of gastric tissue was added to 500μL of lysis buffer (the homogenizing medium provided in the pepsin assay kit) and homogenized. After thawing, the gastric antrum tissue was removed and the LPS and TNF-α levels were determined strictly according to the ELISA kit instructions. The absorbance values ​​of the samples obtained using a microplate reader (detection wavelength 450 nm) were used to calculate the concentrations of IL-6 and IL-8 in the samples using a standard curve.

[0051] II. Experimental Results

[0052] The experiment showed that compared with the control group, the model group mice had increased ulcer area, more severe gastric tissue damage, decreased pepsin content, and increased expression of inflammatory factors, indicating successful model establishment. In contrast, the drug intervention group mice, compared with the model group, showed reduced ulcer area, lessened gastric tissue damage, restored pepsin content, and decreased expression of inflammatory factors. This demonstrates that *Helicobacter pylori* extract can not only directly kill *Helicobacter pylori* but also repair gastric inflammation.

[0053] Experiment 3:

[0054] Liquid chromatography-mass spectrometry (LC-MS) operation methods and spectral interpretation results:

[0055] Dissolve 0.1 g of BQLY-20 in 10 mL of methanol. Inject the sample using the following chromatographic and mass spectrometric conditions.

[0056] 1. Chromatographic conditions

[0057] Watres ACQUITY™ UPLC system; ACQUITY™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm); ACQUITY™ UPLC BEH C18 pre-column (5.0 mm × 2.1 mm, 1.7 μm); column temperature 35 °C; mobile phase 0.1% formic acid aqueous solution (A) - 0.1% formic acid acetonitrile (B), gradient elution (0 min, 5% B; 5 min, 10% B; 15 min, 35% B; 25 min, 70% B; 30 min, 100% B; 32 min, 100% B); flow rate 0.3 mL / min -1 The injection volume was 2 μL; the autosampler temperature was 4 °C.

[0058] 2. Mass spectrometry conditions

[0059] Electrospray ionization was performed in both positive and negative ion modes. The positive and negative ion source voltages were 5500V and -4500V, respectively, with an ion source temperature of 550℃. The nebulizing gas was N2, with both the nebulizing gas and auxiliary gas pressures at 379.2 kPa and the curtain gas pressure at 241.3 kPa. The pyrolysis voltage was ±80V, the collision energy was ±40 eV, and the collision energy spread was 20 eV. The TOF / MS scan range was 100–1500 m / z. IDA was set to scan the eight highest peaks with response values ​​exceeding 100 cps using secondary mass spectrometry. The Product Ion scan range was 50–1000 m / z, with dynamic background subtraction (DBS) enabled. Data acquisition was performed using Analyst TF 1.6 software, and data processing was performed using Peakview 2.0-Masterview 1.0.

[0060] The results of BQLY-20 liquid chromatography-mass spectrometry are as follows:

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Claims

1. A method for extracting the total extract of *Leymus chinensis*, characterized in that... The extraction method for the total extract of *Lycoris radiata* is as follows: Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

2. A method for extracting the composition of *Hemiberlesia lingua*, characterized in that... The extraction method of the Northern Qinglong Garlic Composition is as follows:

1. Weigh 20 kg of *Ilex cornuta* and soak it in 80% ethanol at a ratio of 1 kg:10 L for 7 days. Extract the extracts three times, combine the extracts, recover the solvent, and freeze-dry to obtain the total extract of *Ilex cornuta*.

2. Dissolve 100g of total extract of *Leymus chinensis* obtained in step 1 in water, and elute with 20% ethanol using a macroporous resin D101 column to obtain the 20% ethanol eluent of *Leymus chinensis* ethanol extract, which is the *Leymus chinensis* composition.

3. The application of the total extract of *Gynostemma pentaphyllum* as described in claim 1, characterized in that... The total extract of *Heliotropium indicum* is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

4. The application of the Northern Azure Dragon Garment Composition according to claim 1, characterized in that... The North Qinglongyi composition is used as the active ingredient in a drug for treating HP-positive gastric ulcers.

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