Application of Dragon's Blood A in the Preparation of Anti-Helicobacter pylori Drugs

Abstract

This invention discloses the application of dracosanol A in the preparation of anti-Helicobacter pylori drugs, belonging to the pharmaceutical field. Draconisin A exhibits excellent antibacterial activity against standard sensitive Helicobacter pylori, clinically sensitive, and drug-resistant Helicobacter pylori, with a minimum inhibitory concentration (MIC) range of 4–16 μg / mL. It inhibits biofilm formation in Helicobacter pylori and can destroy mature Helicobacter pylori biofilms, showing stronger efficacy than metronidazole. Draconisin A can kill amoxicillin-induced and naturally induced Helicobacter pylori cocci. Draconisin A also exhibits strong bactericidal activity under acidic conditions, with a bactericidal effect superior to metronidazole. Draconisin A has a specific anti-Helicobacter pylori effect and can have synergistic or additive effects with commonly used clinical anti-Helicobacter pylori antibiotics and omeprazole. Draconisin A is less likely to induce drug resistance during anti-Helicobacter pylori treatment and has a strong killing effect on Helicobacter pylori colonizing the mouse stomach; its combined effect with omeprazole is comparable to standard triple therapy.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and provides pharmaceutical uses for dragon's blood extract A. Background Technology

[0002] As early as 1983, Barry Marshall discovered Helicobacter pylori (Hp). He found spiral or curved bacilli in gastric antral mucosal specimens from 58 patients and isolated and identified this pathogen as a Gram-negative microaerophilic bacterium. In subsequent studies, Hp was also shown to be associated with gastritis, gastric ulcers, duodenal ulcers, gastric mucosa-associated lymphoid tissue lymphoma (MALT), and gastric cancer. In 1994, the WHO (World Health Organization), through long-term epidemiological statistics and mammalian experiments, demonstrated the carcinogenicity of Hp and classified Helicobacter pylori as a Group 1 carcinogen, highlighting the severity and harmfulness of this pathogen to humans. After infection with *Helicobacter pylori* (Hp), it is mainly distributed in the stomach, duodenum, and other parts of the human body. In the stomach, *Hp* urease hydrolyzes urea to produce ammonia gas, forming an "ammonia cloud" that protects the bacteria from the erosion of gastric acid. The bacteria also secrete catalase and superoxide dismutase to evade the killing effect of neutrophils. After *Hp* colonizes and grows on the gastric mucosal epithelial cells, it releases virulence factors such as cytotoxin CagA and vacuole toxin VacA, which damage the epithelial cells, leading to acute and chronic gastritis. If the disease progresses and evolves further, it can lead to chronic atrophic gastritis, gastric intestinal metaplasia, dysplasia, and eventually gastric cancer.

[0003] Currently, the World Health Organization recommends triple or quadruple therapy for eradicating *Helicobacter pylori* (Hp) infection. The former involves a proton pump inhibitor (such as omeprazole) plus two antibiotics (choose two from clarithromycin, amoxicillin, levofloxacin, and metronidazole), while the latter adds a bismuth preparation (such as bismuth potassium citrate). However, with the widespread and prolonged use of antibiotics, Hp resistance is becoming increasingly severe, leading to lower eradication rates and a rise in "refractory cases." This resistance is particularly acute in regions with lower levels of economic development. Therefore, the development of novel anti-*Helicobacter pylori* compounds is of paramount importance in addressing this serious resistance situation. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing the application of dragon's blood A in the preparation of anti-Helicobacter pylori drugs.

[0005] The chemical formula of Dragon's Blood A is as follows. It is an effective active ingredient in the traditional Chinese medicine Dragon's Blood, which is used clinically. Dragon's Blood has pharmacological effects such as promoting blood circulation, anti-inflammation, antibacterial, analgesia, lowering blood lipids, and promoting epidermal repair.

[0006]

[0007] The application of dragon's blood A in the preparation of antibacterial drugs, wherein the bacteria is Helicobacter pylori, including clinically resistant Helicobacter pylori and susceptible Helicobacter pylori.

[0008] The application of the combination of dragon's blood A and proton pump inhibitor in the preparation of drugs against drug-resistant or sensitive Helicobacter pylori.

[0009] The aforementioned proton pump inhibitor is omeprazole.

[0010] Application of Dragon's Blood A in the preparation of drugs for treating acute and chronic gastritis and gastric and duodenal ulcers caused by drug-resistant or sensitive Helicobacter pylori infection.

[0011] The drug for treating Helicobacter pylori infection consists of dragon's blood A and omeprazole.

[0012] Beneficial Effects: Dragon's Blood A can be used to prepare antibacterial and anti-infective drugs. The Dragon's Blood A demonstrated in this invention has excellent bactericidal effects on both planktonic and mature biofilm-forming Helicobacter pylori, and also has a bactericidal effect on amoxicillin-induced and naturally occurring cocci. It can be used to treat acute and chronic gastritis, gastric and duodenal ulcers, and other gastric diseases caused by drug-resistant or sensitive Helicobacter pylori infection, with minimal side effects. It also maintains the homeostasis of the intestinal flora and can effectively alleviate the problem of Helicobacter pylori drug resistance. Attached Figure Description

[0013] Figure 1 In vitro induction detection of Helicobacter pylori G27 strain's resistance to draconin A.

[0014] Figure 2 The bactericidal effect of draconin A on Helicobacter pylori G27 cocci induced under two conditions. A, Evaluation of the bactericidal effect of draconin A on amoxicillin-induced cocci under 4×MIC and 16×MIC conditions; B, Evaluation of the bactericidal effect of draconin A on naturally cultured cocci under 4×MIC and 16×MIC conditions; C, Observation of the bactericidal effect of draconin A on cocci under scanning electron microscopy; D, Morphology of cocci induced by amoxicillin and naturally induced cocci after treatment at 1 hour and 12 hours, respectively; Note: LrA, draconin A; MTZ, metronidazole; AMX, amoxicillin; CLA, clarithromycin; LEV, levofloxacin.

[0015] Figure 3Inhibitory effect of draconin A on biofilm formation of Helicobacter pylori G27 strain and its destructive effect on mature biofilm. A, Crystal violet staining method was used to detect the inhibitory effect of 1 / 4×MIC, 1 / 2×MIC, and 1×MIC draconin A on biofilm formation of Helicobacter pylori G27 strain; B, Alarm Blue staining method was used to detect the destructive effect of 1 / 4×MIC, 1 / 2×MIC, and 1×MIC draconin A on mature biofilm of Helicobacter pylori G27 strain. Note: LrA, draconin A; MTZ, metronidazole, *, P<0.05; **, P<0.01; ***, P<0.001; C, Alarm Blue staining method was used to detect the inhibitory effect of draconin A on biofilm formation of G27 strain; D, SYTO9-PI double staining and laser confocal microscopy were used to detect the killing effect of draconin A on mature biofilm of G27 strain.

[0016] Figure 4 Detection of the anti-Helicobacter pylori activity of LrA under acidic conditions. The bactericidal activity of 1 / 4×MIC, 1 / 2×MIC, and 1×MIC LrA against Helicobacter pylori G27 strain was detected under treatment conditions of pH 2.5 and containing 10 mM Urea. The bactericidal activity of the control drug metronidazole under acidic conditions was also detected under the same MIC multiplier treatment conditions. Note: LrA, LrA; MTZ, metronidazole. *, P<0.05; **, P<0.01; ***, P<0.001.

[0017] Figure 5 The bactericidal effect of draconin A on NSH57 and BHKS00388 strains in mice. A, Flowchart of establishing an acute gastritis animal model and drug treatment in mice infected with Helicobacter pylori; B, Detection results of the colonization of Helicobacter pylori NSH57 strain in the gastric mucosa of mice after treatment in different treatment groups; C, Detection results of the colonization of Helicobacter pylori BHKS00388 in the gastric mucosa of mice after treatment in different treatment groups. The study included a solvent control group (CMC, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80), a triple therapy group (OPZ + AC), and a combination therapy group of omeprazole and draconin A (OPZ + LrA). *, P<0.05; **, P<0.01; ***, P<0.001; D, Repair effect of draconin A on gastric mucosal inflammation in mice infected with drug-resistant Helicobacter pylori BHKS00388. Gastric mucosal tissues were stained with HE and TUNEL, magnified 400 times.

[0018] Figure 6Effects of Dragon's Blood A Treatment on the Diversity and Composition of Fecal Microbiota in Mice. A, Alpha-diversity analysis of the gut microbiota in the three drug treatment groups, calculated from 16S rRNA gene sequencing data using Chao1 (left) and Shannon (right) indices; B, PCA analysis based on OTU abundance, where the numbers in parentheses represent the contribution of major components to sample differences, with one point representing each sample and a confidence interval set at 0.99; C, Relative abundance of bacteria in each group of samples at the phylum level, determined from sequencing data. Data were clustered along the X-axis based on the number of samples in each group; D, Mean abundance analysis of 16S rRNA in each group of samples at the genus level. Detailed Implementation

[0019] The following embodiments are intended to enable those skilled in the art to fully understand the present invention, but do not limit the invention in any way.

[0020] Example 1: Determination of the in vitro anti-Helicobacter pylori activity of Dragon's Blood Extract A

[0021] The activity of dragon's blood extract A against Helicobacter pylori was determined by measuring its minimum inhibitory concentration (MIC).

[0022] (1) Materials

[0023] ①Chemicals: Dragon's blood essence A, B, C, D, as well as Loureiriol, sword leaf dragon's blood essence A, and sword leaf dragon's blood essence C were purchased from ChemFaces. Metronidazole, amoxicillin, clarithromycin, and levofloxacin were purchased from Aladdin.

[0024] ② Strains: Helicobacter pylori standard strains 26695 and G27; other clinical strains were isolated and identified from gastric mucosal samples of clinical patients by the First Affiliated Hospital and Yifu Hospital of Nanjing Medical University.

[0025] ③ Culture media and main reagents: Brain heart infusion broth (BHI), Columbia medium, selective antibiotics (vancomycin, polymyxin B, trimethoprim), fetal bovine serum (FCS), and 100% dimethyl sulfoxide (DMSO).

[0026] ④ Main instruments: BINDER CB160 three-gas incubator, ultraviolet spectrophotometer, constant temperature shaker (Thermo), centrifuge, electronic balance, etc.

[0027] ⑤ Consumables: EP tubes, centrifuge tubes, tip heads, etc.

[0028] (2) Methods: The minimum inhibitory concentration (MIC, 100 μL system) of draconin A against Helicobacter pylori was determined by liquid dilution method.

[0029] ① Prepare stock solutions of 6.4 mg / mL dracosanol A, B, C, D, Loureiriol, dracosanol A, dracosanol C, and 12.8 mg / mL metronidazole (MTZ), all in 100% DMSO.

[0030] ② Preparation of reserve bacterial suspension: Take Helicobacter pylori growing in the logarithmic phase on a solid plate and prepare a bacterial suspension using BHI (containing 10% FCS). Incubate the suspension in a tri-gas incubator until the logarithmic phase is reached, and adjust the concentration OD. 600 The value was 0.2 (the bacterial concentration was approximately 1 × 10⁻⁶). 8 (CFU / mL), diluted 10 times, the bacterial count is approximately 1×10⁻⁶. 7 CFU / mL, for later use.

[0031] ③ Preparation of 96-well plates: Add 178 μL of BHI culture medium (containing 10% FCS) to the first well, then add 2 μL of dracosanol A, B, C, D and Loureiriol, dracosanol A, and dracosanol C stock solutions, and dilute to the 8th well using the two-fold dilution method; add metronidazole to the other row of wells in the same way.

[0032] ④ Add bacterial solution: Take 10 μL of the prepared bacterial solution and add it to each well (the bacterial concentration per well is approximately 1.0 × 10⁻⁶). 6 Within the range of CFU / mL, the concentrations of dracosanol A, B, C, D, and Loureiriol, dracosanol A, and dracosanol C were 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. In another row, 100 μL of BHI culture medium (containing 10% FCS) was added to each well as a sterile control group; in yet another row, 90 μL of BHI culture medium (containing 10% FCS) and 10 μL of the prepared bacterial culture were added to each well as a positive control group. All wells were then incubated in a tri-gas incubator.

[0033] ⑤ Result Interpretation: Interpret the results after 48 or 72 hours of incubation. The minimum drug concentration that completely inhibits bacterial growth in the wells is defined as the MIC. The experiment is only meaningful when there is significant bacterial growth in the positive control wells (i.e., without drug) and no bacterial growth in the sterile control group. Repeat the experiment three times.

[0034] (3) Results

[0035] The results are shown in Table 1.

[0036] Table 1. Minimum inhibitory concentrations (μg / mL) of Dragon's Blood A and its series of compounds against Helicobacter pylori.

[0037]

[0038]

[0039] Note: LrA, Draconis A; LrB, Draconis B; LrC, Draconis C; LrD, Draconis D; Con A, Draconis A; Con C, Draconis C; MTZ, Metronidazole.

[0040] As shown in the table above, the MIC range of Dragon's Blood A against 30 strains of Helicobacter pylori (including 2 standard strains and 28 clinically resistant strains) is 4–16 μg / mL, which is better than the antibacterial effect of other series of compounds. This indicates that Dragon's Blood A has strong antibacterial activity against Helicobacter pylori in vitro and has good development prospects.

[0041] Example 2: Detection of the in vitro antibacterial spectrum of dragon's blood extract A

[0042] (1) Materials

[0043] ①Chemicals: Dragon's Blood A was purchased from Chem Faces, metronidazole from Aladdin, and ampicillin, amphotericin B, and vancomycin from MCE.

[0044] ② Strains: Helicobacter pylori standard strains 26695 and G27, as well as ATCC strains. Other clinical strains were isolated and identified from gastric mucosal samples of clinical patients by the First Affiliated Hospital of Nanjing Medical University and its affiliated Yifu Hospital.

[0045] ③ Culture media and main reagents: BB medium, LB medium, and the rest are as described above.

[0046] ④ Main instruments: 37℃ incubator, the rest are as described above.

[0047] ⑤ Consumables are as described above.

[0048] (2) Methods: The minimum inhibitory concentration (MIC, 100 μL system) of draconin A against Gram-negative bacteria, Gram-positive bacteria, and fungi was determined using the liquid dilution method. The detection method for Helicobacter pylori was the same as above. The final bacterial concentration for other non-Hp strains was approximately 1.0 × 10⁻⁶ per well. 5 CFU / mL, the final concentration of fungi was 1.0 × 10⁻⁶. 2 CFU / mL

[0049] (3) Results

[0050] The results are shown in Table 2.

[0051] Table 2. Antibacterial spectrum of Dragon's Blood A (μg / mL)

[0052]

[0053] As shown in Table 2, Dragon's Blood A has a specific antibacterial effect against Helicobacter pylori, but no antibacterial effect against other Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0054] Example 3: In vitro checkerboard assay for drug susceptibility testing of Dragon's Blood Extract A with commonly used clinical antibiotics and omeprazole.

[0055] (1) Materials

[0056] ①Chemicals: Dragon's Blood A was purchased from Chem Faces, Levofloxacin, Metronidazole, Clarithromycin, Amoxicillin, Tetracycline, and Furazolidone were purchased from Aladdin, and Omeprazole was purchased from MCE Bioreactors.

[0057] ② Strains: Helicobacter pylori standard strains 26695 and G27, and other clinical strains were isolated and identified from gastric mucosal samples of clinical patients by the First Affiliated Hospital of Nanjing Medical University and its affiliated Yifu Hospital.

[0058] ③ Culture media and main reagents: BB medium, LB medium, and the rest are as described above.

[0059] ④ Main instruments: 37℃ incubator, three-gas incubator, the rest are as described above.

[0060] ⑤ Consumables are as described above.

[0061] (2) Methods: The combined antibacterial effects of dracosanol A with levofloxacin, metronidazole, clarithromycin, amoxicillin, tetracycline, furazolidone, and omeprazole were detected by combined drug sensitivity testing. The specific procedures are as follows:

[0062] ① Preparation of compound a plate: Dilute compound a to 400 times MIC, take 50 μl to well B3 of a 96-well plate, add 25 μl of DMSO to wells B4 to B12, take 25 μl of the drug solution from well B3 to well B4, mix by pipetting, take another 25 μl of the drug solution to the next well, mix by pipetting, and then dilute by a 2-fold serial number to well B12 for later use;

[0063] ② Preparation of compound b plate: First, spread 90 μl of BHI + 10% FCS liquid medium on the entire 96-well plate. Then, spread 89 μl of BHI + 10% FCS liquid medium on wells C2 to C12. Add 1 μl of compound b stock solution to make the final concentration of compound b 4 times the MIC. Serially dilute downwards. Row A is set as negative control wells with no bacteria and only 100 μl of liquid medium. Column 1 is the positive control wells with no drug and only 90 μl of liquid medium and 10 μl of bacterial solution.

[0064] ③ Preparation of combined drug sensitivity plates: Using a multi-pipette, 1 μl of compound a from the compound a plate is added to the corresponding compound b plate. That is, 1 μl of compound a from wells B3 to B12 is added to wells B3 to B12, C3 to C12, D3 to D12, E3 to E12, F3 to F12, G3 to G12, and H3 to H12 of the b plate.

[0065] ④ Inoculation of bacterial suspension: The Hp standard strain G27 was cultured to the logarithmic growth phase and adjusted to OD600 = 0.015 (approximately 1 × 10⁷ CFU / ml) in BHI + 10% FCS liquid medium. 10 μl of the bacterial suspension was inoculated into each well of a combined drug sensitivity plate. The positive control wells were inoculated with bacteria, while the negative control wells were not. The plates were incubated in a three-gas incubator for 2-3 days, and the results were observed.

[0066] ⑤ Result Interpretation: After 48–72 hours of incubation, if the bacteria in the positive control wells grow well, the results can be interpreted. Column 2 allows observation and recording of the MIC of compound a alone, and row B allows observation and recording of the MIC value of compound b alone. When combined use of drugs to inhibit bacterial growth is observed in the wells, the drug concentrations of compounds a and b are recorded, and the FICI value is calculated. FICI = MIC a United / MIC a Individual + MIC b United / MIC b (Single) If FICI ≤ 0.5, it indicates that the two compounds have a synergistic effect; if the FICI value is between 0.5 and 1, it indicates that the two compounds have an additive effect; if the FICI value is between 1 and 4, it indicates that the two compounds have no interaction; if FICI > 4, it indicates that the two compounds have an antagonistic effect. The combined drug sensitivity test was performed in three independent replicates.

[0067] (3) Results

[0068] The results are shown in Table 3.

[0069] Table 3. Combined antibacterial effects of dracoside A, commonly used clinical antibiotics, and PPIs against 10 strains of Helicobacter pylori.

[0070]

[0071] Note: LrA is Dragon's Blood A; LEV is Levofloxacin; MTZ is Metronidazole; CLR is Clarithromycin; AMX is Amoxicillin; TET is Tetracycline; FZD is Furazolidone; PPI is Omeprazole

[0072] As shown in the table above, when used in combination with commonly used clinical antibiotics, dracosanol A exhibits a greater additive effect, while when used in combination with omeprazole, it shows a synergistic effect in 60% of bacterial strains. Therefore, dracosanol A has the potential to synergistically treat Helicobacter pylori infection with commonly used clinical antibiotics or omeprazole, and has good development prospects.

[0073] Example 4: In vitro resistance test of dragon's blood extract A to Helicobacter pylori

[0074] (1) Materials

[0075] Materials as in Example 1.

[0076] (2) Method

[0077] Inoculate Helicobacter pylori G27 strain from a fresh solid plate into 5 mL of BHI (containing 10% FCS), and adjust the bacterial concentration to OD. 600 The initial concentration was approximately 0.2, then dracoside A was added to a final concentration of 4 μg / mL (1 / 2 × MIC). The mixture was incubated in a tri-gas incubator for 48 hours, and bacterial growth was observed. The bacterial culture was then passaged, and the above method was repeated to induce drug resistance. If growth was good, the dracoside A concentration was doubled; if growth was slow or absent, the existing dracoside A concentration was maintained. Drug resistance induction was performed for a total of 60 days, with MIC measured every 4 days. The control group consisted of metronidazole, also with an initial concentration of 1 / 2 × MIC.

[0078] See results Figure 1 .Depend on Figure 1 As shown, no resistance to draconin A was observed in Helicobacter pylori G27 during successive passages, while Helicobacter pylori G27 developed resistance to metronidazole after the first 5 passages, resulting in a 4-fold increase in MIC. These results demonstrate that draconin A has a very low tendency to induce resistance in Helicobacter pylori.

[0079] Example 5: In vitro antibacterial effect of dragon's blood extract A on Helicobacter pylori.

[0080] (1) Materials

[0081] Materials as in Example 1.

[0082] (2) Method: The antibacterial activity of dracoside A against Helicobacter pylori was detected by plate colony counting method.

[0083] ① Amoxicillin-induced cocci: Helicobacter pylori G27 strain was inoculated from fresh solid plates into Brucella broth (containing 2% FCS), and the bacterial concentration was adjusted to OD. 600=Approximately 0.4, then amoxicillin was added to a final concentration of 0.063 μg / mL, and the mixture was incubated in a tri-gas incubator for 24 hours. Gram staining and microscopic observation confirmed that all G27 strains had transformed into spherical bacteria, while in the control experiment (without amoxicillin), the G27 strain was spiral-shaped and remained spherical after 12 hours of incubation. Figure 2 D).

[0084] ② Naturally induced spherical bacteria: Helicobacter pylori G27 strain was inoculated from fresh solid plates into Brucella broth (containing 2% FCS), and the bacterial concentration was adjusted to OD. 600 = Approximately 0.1, incubated in a three-gas incubator for 96 hours, Gram staining and microscopic observation confirmed that all G27 strains transformed into spherical bacteria, and remained spherical after 12 hours of incubation. Figure 2 D).

[0085] ③ Drug treatment: Centrifuge spherical strains, discard the supernatant, and add Brucella broth (containing 2% FCS) to dilute to a final bacterial concentration of approximately OD. 600 =0.5, then add amoxicillin, metronidazole, clarithromycin, levofloxacin and 4×MIC / 16×MIC dragon's blood A to a final concentration of 64×MIC, and incubate in a three-gas incubator.

[0086] ④ Plate counting: Take 100 μL of bacterial culture at 1 h and 12 h, add 10 μL of AlarmBlue detection reagent directly to each 100 μL system, and incubate in a three-gas incubator for 4 h in the dark. Detect the fluorescence intensity using an ELISA reader with excitation light at 530 nm and emission light at 590 nm.

[0087] (3) Results

[0088] See results Figure 2 .Depend on Figure 2 As shown, Dragon's Blood A exhibits a strong bactericidal effect against Helicobacter pylori cocci in vitro. Under treatment conditions of 4×MIC and 16×MIC, it can gradually kill cocci induced by both conditions over time. In contrast, commonly used clinical antibiotics such as amoxicillin, metronidazole, clarithromycin, and levofloxacin are also difficult to kill cocci under 64×MIC conditions. The data indicate that Dragon's Blood A has a stronger and better bactericidal effect against Helicobacter pylori cocci and has good development prospects.

[0089] Example 6: The bactericidal effect of dragon's blood extract A on Helicobacter pylori biofilm.

[0090] (1) Materials

[0091] Crystal violet was purchased from Aladdin Company. The detection reagents included two fluorescent dyes, SYTO9 and propidium iodide (PI). The remaining materials for Alarm Blue were the same as those in Example 1.

[0092] (2) Methods: Crystal violet staining, fluorescence confocalization and plate colony counting were used to detect the anti-biofilm activity of draconin A against Helicobacter pylori.

[0093] ① Inhibitory effect of dragon's blood A on Helicobacter pylori biofilm formation: Helicobacter pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, and diluted with the above fresh culture medium to OD. 600 The concentration was 0.15, and different concentrations of dragon's blood A, metronidazole (as a positive control), or an equal amount of DMSO (as a solvent control) were added to it. Then, it was spread on a 96-well plate and incubated in a three-gas incubator for 3 days. The relative amount of biofilm was then detected by crystal violet staining.

[0094] ② The destructive effect of dragon's blood A on established mature biofilms: Helicobacter pylori G27 strain was cultured overnight in Brucella broth with 10% FBS added, and then diluted with the above fresh culture medium to OD. 600 The concentration was 0.15, and the culture medium was then spread onto 96-well plates and incubated in a tri-gas incubator for 3 days to form a biofilm. The culture medium was then discarded, and the plates were washed twice with PBS. Different concentrations of draconin A, MTZ (as a positive control), or an equal amount of DMSO (as a solvent control) were added to fresh Brucella broth and added to 96-well plates for further incubation for 24 hours. The relative amount of biofilm was detected using crystal violet staining.

[0095] ③ Effect of Dragon's Blood A on Bacterial Activity in Biofilms Detected by Laser Confocal Microscopy. Bacterial activity within the biofilm was assessed using the Live / Dead BacLight Bacterial Viability Kit (Invitrogen, USA), which consists of two fluorescent dyes, SYTO9 and propidium iodide (PI). Helicobacter pylori G27 biofilms were prepared as described above and treated with different concentrations of dragon's blood A, MTZ (as a positive control), or an equal volume of DMSO (as a solvent control). After incubation in a tri-gas incubator for 24 hours, unattached biofilms were washed three times with PBS and then stained with the two fluorescent dyes in the dark at room temperature for 30 minutes. After rinsing, images were observed using a confocal laser scanning microscope (LSM710; Carl Zeiss, Germany), with additional fields of view examined randomly. The fluorescent dyes SYTO9 and PI distinguished between live cells (green cells) and dead cells (red cells).

[0096] ④ Effect of Alarm Blue on the activity of draconin A in biofilms. Biofilms of Helicobacter pylori G27 cells were prepared and treated as described above. After treatment under microaerophilic conditions for 24 hours, 10 μl of Alarm Blue reagent was added directly to each 100 μl system. The system was then incubated in a tri-gas incubator for 4 hours in the dark. The cells were then photographed and statistically analyzed to observe the bacterial activity in the biofilm through color changes.

[0097] (3) Results

[0098] The results are as follows Figure 3 .Depend on Figure 3 As shown in Figure A, dragon's blood element A can inhibit the formation of Helicobacter pylori biofilm, by... Figure 3 As shown in Figure B, dragon's blood extract A has the ability to disrupt the mature biofilm of Helicobacter pylori, and its anti-biofilm activity is stronger than that of metronidazole. Figure 3 As shown in C and 3D, Dragon's Blood A can kill Helicobacter pylori in biofilms, and its activity is stronger than that of metronidazole.

[0099] Example 7: Bactericidal effect of Dragon's Blood A on Helicobacter pylori under acidic conditions

[0100] (1) Materials

[0101] The BHI medium was adjusted to pH 2.5 with dilute hydrochloric acid, and 1M Urea buffer was used. The other materials were the same as those in Example 1.

[0102] (2) Method: The bactericidal effect of dragon's blood A on Helicobacter pylori under acidic conditions was detected by CFU counting method.

[0103] ① Incubate Helicobacter pylori G27 strain overnight in BHI medium supplemented with 10% FCS. The first group was diluted to OD using neutral BHI + 10% FCS. 600 The concentration was 0.3. The samples were treated with DMSO, 1 / 4×MIC MTZ and LrA, 1 / 2×MIC MTZ and LrA, and 1×MIC MTZ and LrA for 30 minutes respectively. 100 μl of each sample was taken out and serially diluted with neutral BHI + 10% FCS, spread on Columbia blood agar medium, and incubated in a tri-gas incubator.

[0104] ② The second group was diluted to OD using BHI + 10% FCS + 10mM Urea buffer at pH 2.5. 600 The concentration was 0.3. The samples were treated with DMSO, 1 / 4×MIC MTZ and LrA, 1 / 2×MIC MTZ and LrA, and 1×MIC MTZ and LrA for 30 minutes each. 100 μl of each sample was taken and serially diluted with neutral BHI + 10% FCS. The samples were then spread on Columbia blood agar medium and incubated in a tri-gas incubator. CFU was counted after 4 days.

[0105] (3) Results

[0106] The results are as follows Figure 4 . Figure 4 It is evident that Dragon's Blood A exhibits bactericidal activity against Helicobacter pylori G27 at pH 2.5 under conditions of 1 / 4×MIC, 1 / 2×MIC, and 1×MIC, and its bactericidal effect is superior to metronidazole under each concentration gradient condition.

[0107] Example 8: Detection of the killing effect of dragon's blood extract A on Helicobacter pylori NSH57 strain colonized in mouse stomach

[0108] (1) Materials

[0109] The strain was Helicobacter pylori mouse-domesticated strain NSH57, and the mice were SPF-grade 6-week-old female C57BL / 6 mice, with other materials as in Example 1.

[0110] (2) Method

[0111] ① In vivo model of Helicobacter pylori NSH57 infection in mice: Refer to the article (Huang Y, Hang X, Jiang X, Zeng L, Jia J, Xie Y, Li F, Bi H. In Vitro and In Vivo Activities of ZincLinolenate, a Selective Antibacterial Agent against Helicobacter pylori. Antimicrob Agents Chemother[J]. 2019;63(6):e00004-19). 10% of mouse gastric tissue was used to detect Helicobacter pylori colonization, with a colonization amount ranging from 1×10 5 A CFU / g level or higher indicates successful colonization; all test results show Helicobacter pylori colonization, indicating successful model construction.

[0112] ② Grouping: The successfully modeled infected group was divided into 3 groups: omeprazole plus draconin A (28 mg / kg) group, omeprazole plus amoxicillin (14 mg / kg) and clarithromycin (7 mg / kg) group (standard triple therapy group), and solvent control group, with 8 mice in each group; the dosage of omeprazole in all groups was 138.2 mg / kg, and 8 mice not infected with Helicobacter pylori served as the negative control group.

[0113] ③ Administration: Omeprazole was administered by gavage 30 minutes before other drugs. After administration, mice were fasted and deprived of water for 4 hours. The average weight of mice was calculated as 20g / mouse. The drug was administered once a day for 3 consecutive days. The solvent control group was given 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80 solution, with the same volume and frequency as above.

[0114] ④ Mouse treatment: 48 hours after the last administration, mice were euthanized, and stomach tissue was taken for Helicobacter pylori isolation, culture and identification, and colonization was calculated.

[0115] (3) Results

[0116] The results are as follows Figure 5 .Depend on Figure 5 As shown in B, the omeprazole plus dragon's blood A group had a similar killing effect on Helicobacter pylori in mice as the triple therapy group (omeprazole plus amoxicillin and clarithromycin).

[0117] Example 9: Detection of the killing effect of Dragon's Blood Extract A on multidrug-resistant Helicobacter pylori strain BHKS00388 colonized in the stomach of mice.

[0118] (1) Materials

[0119] The strain was Helicobacter pylori mouse-domesticated strain BHKS00388, and the mice were SPF-grade 6-week-old female C57BL / 6 mice, with other materials as in Example 1.

[0120] (2) Method

[0121] ①In vivo model of Helicobacter pylori BHKS00388 infection in mice: Refer to the article (Huang Y, Hang X, Jiang X, Zeng L, Jia J, Xie Y, Li F, Bi H. In Vitro and In Vivo Activities of Zinc Linolenate, a Selective Antibacterial Agent against Helicobacter pylori. Antimicrob Agents Chemother[J]. 2019;63(6):e00004-19). 10% of mouse gastric tissue was used to detect Helicobacter pylori colonization, with a colonization amount ranging from 1×10 5 A CFU / g level or higher indicates successful colonization; all test results show Helicobacter pylori colonization, indicating successful model construction.

[0122] ② Grouping: The successfully modeled infected mice were divided into three groups: omeprazole plus dermal extract A (28 mg / kg) group, omeprazole plus amoxicillin (14 mg / kg) and clarithromycin (14 mg / kg) group (standard triple therapy group), and solvent control group, with 6 mice in each group; the dosage of omeprazole in all groups was 64.1 mg / kg, and 6 mice not infected with Helicobacter pylori served as the negative control group.

[0123] ③ Administration: Omeprazole was administered by gavage 30 minutes before other drugs. After administration, patients were kept from eating or drinking for 4 hours. The administration was once a day for 4 consecutive days. The solvent control group was given 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80 solution, with the same volume and frequency as above.

[0124] ④ Mouse treatment: One day after the last administration, mouse feces were collected for 16S rRNA gene (V3-V4 region) sequencing analysis. Forty-eight hours after the last administration, the mice were euthanized, and stomach tissue was taken. Half of the tissue was used for Helicobacter pylori isolation, culture and identification, and colonization was calculated. The other half of the tissue was used for pathological section staining.

[0125] (3) Results

[0126] like Figure 5 As shown in Figure C, the omeprazole plus dermal extract A combination therapy showed comparable efficacy in killing drug-resistant Helicobacter pylori in mice as the triple therapy group. Figure 5 D. The dermal erythrorhizon A treatment group showed significantly better repair ability of gastric mucosal damage in mice than the triple therapy group, and also reduced the apoptosis effect on gastric mucosal cells. Figure 6 As shown, there were no significant differences in α- and β-diversity between the OPZ+LrA treatment group and the CMC-Na group, while the microbial community structure of the triple therapy group showed significant changes. Therefore, LrA treatment resulted in very little change in the diversity and composition of the mouse gut microbiome.

Claims

1. Application of Dragon's Blood A in the preparation of anti-Helicobacter pylori drugs.

2. Application of the combination of dragon's blood A and omeprazole in the preparation of drugs against drug-resistant or sensitive Helicobacter pylori.

3. Application of Dragon's Blood A in the preparation of drugs for treating acute and chronic gastritis, gastric and duodenal ulcers caused by drug-resistant or sensitive Helicobacter pylori infection.

4. A pharmaceutical composition for treating Helicobacter pylori infection, characterized in that... The components are dragon's blood A and omeprazole.

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