A combination drug against helicobacter pylori

Abstract

The application discloses a combined medicine against helicobacter pylori, and aims to provide a combined medicine against helicobacter pylori, which has a significant inhibiting effect on a standard strain SS1 of helicobacter pylori, can effectively destroy a biofilm structure of the standard strain SS1 of helicobacter pylori, and has a synergistic bacteriostatic effect. The technical scheme is that an extract of fructus amomi essential oil is combined with antibiotics commonly used in clinical treatment of helicobacter pylori infection. Experiments show that the technology provided by the application has a synergistic effect with metronidazole, levofloxacin, clarithromycin and amoxicillin. The technology has an effect of reducing the dosage of the antibiotics used in clinical application, has potential application value for improving the drug resistance status of the antibiotics, and belongs to the technical field of medicines.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a combined drug for resisting Helicobacter pylori. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori H. pylori infection has been identified as a major cause of chronic gastritis, peptic ulcers, and gastric cancer. The current mainstay of treatment is quadruple therapy (a proton pump inhibitor, a bismuth agent, and two antibiotics), but this approach has significant drawbacks: first, antibiotic resistance is a significant concern, leading to a decrease in eradication rates; second, frequent side effects such as diarrhea, abdominal pain, allergic reactions, and intestinal flora disturbances reduce patient compliance; and finally, the risk of relapse is high, and repeated use can exacerbate drug resistance. Therefore, developing drugs with low resistance, minimal side effects, and the ability to reduce relapses is crucial.

[0003] Amomum villosum ( Amomum villosum ) is a classic Chinese herbal medicine for treating spleen and stomach disorders and pregnancy symptoms, focusing on "invigorating the spleen and regulating the middle," with the combined effects of eliminating dampness, warming the middle, and stabilizing the fetus. Its volatile oil components (such as bornyl acetate, camphor, and limonene) have been shown to exhibit broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria. However, existing studies have not confirmed the significant efficacy of Amomum villosum volatile oil or its extracts in combating H. pylori infection. In particular, systematic validation of its biofilm-inhibiting capacity and synergistic mechanisms is lacking, limiting its clinical application.

[0004] The existing Amomum villosum extraction process mainly uses water extraction and alcohol extraction. The alcohol extraction method easily dissolves inactive impurities such as polysaccharides and proteins, reduces the purity of volatile oils, and interferes with the efficacy of active ingredients. Traditional water extraction has low efficiency and limits the efficient acquisition and application of Amomum villosum's anti-Hp active ingredients. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a combined drug that has a significant inhibitory effect on H. pylori, can effectively destroy its biofilm structure and produce a synergistic antibacterial effect.

[0006] In view of the above problems, the purpose of the present invention is to provide a combined drug that has a significant inhibitory effect on H. pylori, can effectively destroy its biofilm structure and produce a synergistic antibacterial effect.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The invention discloses a combined medicine for resisting Helicobacter pylori, which is composed of an extract of Amomum villosum essential oil and an antibiotic.

[0009] The antibiotic is one of metronidazole, levofloxacin, clarithromycin or amoxicillin.

[0010] The mass ratio of Amomum villosum essential oil extract to metronidazole is 1:2×10 -3 composition; or the mass ratio of levofloxacin is 1:9.8×10 -4 composition; or the mass ratio of amoxicillin is 1:6×10 -5 composition; or the mass ratio of clarithromycin is 1:3.8×10 -6 composition.

[0011] The above-mentioned anti-Helicobacter pylori combined drug and the Amomum villosum essential oil extract are prepared by the following method:

[0012] 1) Grinding the Amomum villosum fruit and mixing it with pure water at a material-liquid ratio of 1:8 to obtain an Amomum villosum liquid;

[0013] 2) extracting the Amomum villosum solution of step 1) by steam distillation at 100-120° C. for 4 hours to obtain a crude Amomum villosum extract;

[0014] 3) collecting the upper oil phase of the Amomum villosum crude extract obtained in step 2) through an oil-water separator, and removing residual water in the oil phase by a physical method to obtain a pure Amomum villosum essential oil extract.

[0015] in:

[0016] Step 1) The powder obtained after crushing the Amomum villosum fruit needs to be passed through a 40-mesh sieve;

[0017] The physical method of removing the residual water in the oil phase in step 3) is specifically to remove the residual water in the oil phase by drying with anhydrous sodium sulfate.

[0018] Preferably, the active ingredients of the above-mentioned anti-Helicobacter pylori combined drug Amomum villosum essential oil extract include camphor, bornyl acetate, limonene, camphene and bornol.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The essential oil extracted by the technical solution provided by the present invention has a significant inhibitory effect on Hp, with a minimum inhibitory concentration (MIC) of 256µg / mL, and can effectively destroy its biofilm structure (inhibition rate >80%).

[0021] The Amomum villosum essential oil extract provided by the present invention shows synergistic effect (FIC≤1) when used in combination with four antibiotics. The present invention provides a basis for developing new natural anti-Hp drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the GC-MS detection spectrum of Amomum villosum essential oil extract;

[0023] Figure 2 The results of three kinds of Amomum villosum extracts against Hp;

[0024] Figure 3 This is the result graph of the absorbance value of the anti-Hp activity of the essential oil extract of Amomum villosum;

[0025] Figure 4 This is the time-concentration curve of the anti-Hp effect of Amomum villosum essential oil extract;

[0026] Figure 5 This is the result of the inhibition of Hp biofilm by the extract of Amomum villosum essential oil;

[0027] Figure 6 This is the absorbance value result of the Amomum villosum essential oil extract inhibiting Hp biofilm. DETAILED DESCRIPTION

[0028] The present invention is further explained below with reference to the examples and accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] Example 1

[0030] This embodiment provides a combined drug for treating Helicobacter pylori, comprising an extract of Amomum villosum essential oil and metronidazole in a mass ratio of 1:2×10 -3 composition.

[0031] Example 2

[0032] The present embodiment provides a combined drug for treating Helicobacter pylori, which is composed of an extract of Amomum villosum essential oil and levofloxacin in a mass ratio of 1:9.8×10 -4 composition.

[0033] Example 3

[0034] This embodiment provides a combined drug for treating Helicobacter pylori, comprising an extract of Amomum villosum essential oil and amoxicillin in a mass ratio of 1:6×10 -5 composition.

[0035] Example 4

[0036] This embodiment provides a combined drug for treating Helicobacter pylori, comprising an extract of Amomum villosum essential oil and clarithromycin in a mass ratio of 1:3.8×10 -6 composition.

[0037] The Amomum villosum essential oil extracts used in Examples 1-4 were prepared by the following steps:

[0038] S1. Wash Amomum villosum with pure water, dry naturally at room temperature, and grind through a 40-mesh sieve to obtain Amomum villosum fine powder;

[0039] S2. Amomum villosum powder was mixed with pure water at a material-liquid ratio of 1 (g):8 (mL), and heated to boiling state (100-120°C) in an electric heating mantle for 4 hours. The upper oily extract was collected by an oil-water separator;

[0040] S3. Add anhydrous sodium sulfate to the oily extract obtained in step S2 and dry it to obtain a pure Amomum villosum essential oil extract.

[0041] The prepared Amomum villosum essential oil extract was sent to the Analysis and Testing Center of Guangdong University of Technology for GC-MS detection of the Amomum villosum essential oil extract components. The GC-MS spectrum is shown in FIG. Figure 1 The specific ingredients are shown in Table 1 below.

[0042] Table 1: GC-MS analysis of components of Amomum villosum essential oil extract

[0043] Serial number Retention time / min Retention Index name Molecular formula Relative content (%) 1 4.504 938 α-pinene [C 10 H 16 ]]> 1.400 2 4.730 969 Camphene [C 10 H 16 ]]> 7.351 3 5.274 985 β-Myrcene C 10 H 16 ]]> 4.502 4 5.898 1018 Limonene C 10 H 16 ]]> 10.367 5 6.989 1081 Linalool C 10 H 18 O]]> 2.067 6 7.865 1145 Camphor <![CDATA[C 10 H 16 O]]> 39.697 7 8.124 1191 Borneol C 10 H 18 O]]> 6.460 8 9.916 1267 Bornyl acetate C 12 H 20 O2]]> 16.459 9 11.780 1420 α-Santalen C 15 H 24 ]]> 0.742 10 11.855 1432 β-Caryophyllene <![CDATA[C 15 H 24 ]]> 1.607 11 12.170 1449 (+)-Epi-β-Santalen <![CDATA[C 15 H 24 ]]> 0.575 12 12.313 1465 α-Caryophyllene <![CDATA[C 15 H 24 ]]> 0.345 13 12.554 1524 α-Longifolinene <![CDATA[C 15 H 24 ]]> 0.506 14 12.943 1547 β-Bisabolene <![CDATA[C 15 H 24 ]]> 1.212 15 13.186 1554 Cardenadiene <![CDATA[C 15 H 24 ]]> 1.084 16 13.603 1565 Nerolidol <![CDATA[C 15 H 26 O]]> 0.470 17 14.716 1622 T-cadinol <![CDATA[C 15 H 26 O]]> 0.738 18 14.887 1639 α-Biasol <![CDATA[C 15 H 26 O]]> 1.025 19 15.006 1668 β-Bisabolol <![CDATA[C 15 H 26 O]]> 1.206 20 15.069 1674 trans-sesquisabinene hydrate <![CDATA[C 15 H 26 O]]> 0.444 21 15.366 1681 α-Santalol <![CDATA[C 15 H 24 O]]> 0.485

[0044] Total content / % 98.741 Monoterpenes / % 23.620 Oxygenated monoterpenes / % 48.223 Sesquiterpenes / % 6.071 Oxygenated sesquiterpenes / % 4.368 other / % 16.459

[0045] Comparative Example 1

[0046] This comparative example provides an aqueous extract of Amomum villosum, the preparation method of which comprises the following steps:

[0047] S1. Wash Amomum villosum with pure water, dry naturally at room temperature, and grind through a 40-mesh sieve to obtain Amomum villosum fine powder;

[0048] S2. Amomum villosum powder (g) and pure water (mL) were mixed at a material-liquid ratio of 1:8, and then placed in an electric heating mantle for heating and reflux. The mixture was kept at a boiling state (100-120°C) for 4 hours, and the filtrate was collected.

[0049] S3. The filtrate obtained in step S2 is filtered using a rotary evaporator to obtain a pure Amomum villosum water extract.

[0050] Comparative Example 2

[0051] The present embodiment provides an alcohol extract of Amomum villosum, and the preparation method thereof comprises the following steps:

[0052] S1. Wash Amomum villosum with pure water, dry naturally at room temperature, grind with a grinder, and pass through a 40-mesh sieve to obtain Amomum villosum fine powder;

[0053] S2. Amomum villosum powder (g) and anhydrous ethanol (mL) were mixed at a material-liquid ratio of 1:8, and then placed in an electric heating mantle for heating and reflux, maintaining a boiling state (70-80°C) for 4 hours, and the filtrate was collected;

[0054] S3. The filtrate obtained in step S2 is filtered using a rotary evaporator to obtain a pure Amomum villosum alcohol extract.

[0055] In order to verify the effectiveness of the technical solution provided by this application, the following is an experimental test to prove the technical solution provided by this application:

[0056] 1. The in vitro anti-Hp activities of the Amomum villosum essential oil extracts provided in Examples 1-4, the Amomum villosum water extract prepared in Comparative Example 1, and the Amomum villosum alcohol extract prepared in Comparative Example 2 were tested.

[0057] 1. Experimental subjects: Helicobacter pylori standard strain SS1 (HpSS1, donated by Professor Chen Ye of Southern Medical University, stored in a -80°C refrigerator), Amomum villosum essential oil extracts of Examples 1-4, and extracts of Comparative Examples 1-2.

[0058] 2. Experimental method: Brain heart infusion broth (BHI) powder was accurately weighed and mixed with pure water according to the manufacturer's instructions. The mixture was stirred until completely dissolved, sealed with a sealing film, and then sterilized by high pressure. After the sterilized BHI broth cooled, sterile fetal bovine serum (FBS) was added to the broth at a volume ratio of 1:10 (v / v) in a clean bench and mixed evenly as the final culture medium. The mixture was sealed and used for later use. At the same time, a 20 mg / mL stock solution of Amomum villosum extract was prepared with anhydrous ethanol as the solvent, sterilized by filtration through a 0.22 µm filter membrane, and then diluted with culture medium to 1024 µg / mL. Subsequently, 512 µg / mL, 256 µg / mL, 128 µg / mL, and 64 µg / mL extract solutions were obtained by two-fold dilution as experimental groups. A blank group (culture medium only), a negative control group (containing bacteria but no drug), a solvent control group (containing 5% ethanol), and a positive drug control group (containing 2 μg / mL metronidazole); HPSS1 strains within the third generation of culture were scraped using PBS buffer and diluted with culture medium. Mixtures of Amomum villosum extract and HpSS1 suspension were added to 96-well plates, respectively, to make the final concentrations of Amomum villosum extract 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL, and the final concentration of HpSS1 suspension 1×10 6 CFU / mL. After incubation in a 96-well plate at 37°C in a microaerobic environment for 3 days, the minimum inhibitory concentration (MIC) was determined visually, and the absorbance at 600 nm was measured using a microplate reader. Each experiment was repeated three times, and one-way analysis of variance was performed using GraphPad software.

[0059] The results are shown in Table 2. The MIC of the Amomum villosum essential oil extract provided by Examples 1-4 is lower than the MIC of the Amomum villosum water extract and Amomum villosum alcohol extract provided by Comparative Examples 1-2, indicating that the Amomum villosum essential oil extract prepared by this method contains more anti-HpSS1 active ingredients, thereby significantly enhancing its antibacterial activity; Figure 2Visual observation showed that the blank group and the positive group solutions were clear, while the negative group and the solvent control group were turbid, confirming that 5% ethanol had no inhibitory effect on HpSS1 and its antibacterial activity was entirely due to the Amomum villosum extract. Figure 2 It is clear that the antibacterial effect of Amomum villosum essential oil extract is better than that of Amomum villosum water extract and Amomum villosum alcohol extract, with a MIC value of 256 μg / mL; Figure 3 The results of microplate reader detection showed that 256 μg / mL of Amomum villosum essential oil extract had a significant inhibitory effect on HpSS1 (P** < 0.01). This result was consistent with the conclusion of naked eye observation, verifying the effectiveness of the experiment.

[0060] Table 2: MICs of Amomum villosum extracts against HpSS1 using different extraction methods

[0061]

[0062] 2. Draw a time-inhibition curve to dynamically monitor the antibacterial process of Amomum villosum essential oil extract on HpSS1

[0063] Test sample: the Amomum villosum essential oil extract of the present invention.

[0064] Determination method: In parallel experiments, a mixture of Amomum villosum essential oil extract and HpSS1 suspension was added to seven 96-well plates, respectively, to make the final concentrations of the extract 0 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL and 512 μg / mL, and the final concentration of HpSS1 suspension was 1×10 6 CFU / mL, cultured under microaerobic conditions at 37°C; a well plate was taken at 0, 12, 24, 36, 48, 60 and 72 h, and its absorbance at 600 nm was measured (all experimental group data were recorded after deducting the absorbance value of the blank group on the same plate), and the time-inhibition curve was drawn accordingly.

[0065] like Figure 4As shown, HpSS1 populations decreased and growth slowed in all groups before 24 hours, indicating a lag phase. The curve for the 0 µg / mL group indicates that HpSS1 entered the logarithmic phase at 24 hours. All concentrations of Amomum villosum essential oil extract significantly inhibited HpSS1 proliferation before 36 hours (experimental groups entered the logarithmic phase after 36 hours). The 128, 256, and 512 µg / mL groups showed comparable inhibitory effects on HpSS1 proliferation before 48 hours. However, over time, HpSS1 populations rebounded in the 128 µg / mL group (indicating that this concentration was unable to completely inhibit HpSS1 growth), while the 256 and 512 µg / mL groups maintained a stable inhibitory effect and sustained control of HpSS1 proliferation. In summary, Amomum villosum essential oil extract effectively inhibits HpSS1 population growth by delaying the logarithmic phase and reducing its division rate. High concentrations (≥256 µg / mL) of Amomum villosum essential oil extract exhibited superior bactericidal efficacy.

[0066] 3. Crystal violet staining experiment to examine the effect of Amomum villosum essential oil extract on HpSS1 biofilm

[0067] Test sample: the Amomum villosum essential oil extract of the present invention.

[0068] Test method: In a 48-well plate, 200 μL of 1×10 6 A suspension of HPSS1 with a CFU / mL was incubated at 37°C under microaerobic conditions and saturated humidity for 72 h to form a biofilm. The culture supernatant was then aspirated and fresh culture medium was added to wash away the suspended HPSS1. A blank group (culture medium only), a negative control group (containing a biofilm without drug), a solvent control group (containing 5% ethanol), and a positive drug control group (containing 2 µg / mL metronidazole) were set up. The experimental groups were supplemented with 512 µg / mL, 256 µg / mL, 128 µg / mL, and 64 µg / mL of Amomum villosum essential oil extract, respectively, and incubated under the same microaerobic conditions for 24 h. After the incubation period, the supernatant was aspirated and the wells were washed with 200 µL of PBS buffer to completely remove non-adherent HPSS1. After drying at room temperature, the wells were stained with 0.1% (w / v) crystal violet solution for 15 min. The staining solution was discarded and the wells were repeatedly rinsed with PBS buffer until no pigment remained. After drying at room temperature, the wells were added with 75% ethanol to dissolve the bound crystal violet, and finally the 595 was measured using a microplate reader. The absorbance at 40 nm wavelength was used to quantify HpSS1 biofilm.

[0069] like Figure 5 and Figure 6As shown in the results, compared with the negative control, 64 µg / mL of Amomum villosum essential oil extract significantly inhibited HpSS1 biofilm formation (P* < 0.05), and the degree of biofilm inhibition increased significantly with increasing concentrations of Amomum villosum essential oil extract (P** < 0.01). The optimal inhibitory effects were achieved at concentrations of 256 and 512 µg / mL. These results confirm that Amomum villosum essential oil extract can treat HpSS1 infection by inhibiting biofilm formation in a dose-dependent manner.

[0070] 4. The checkerboard method was used to determine the graded inhibitory concentration index (FIC) to evaluate the effect of Amomum villosum essential oil extract combined with four clinical antibiotics against HpSS1.

[0071] Test samples: the essential oil extract of Amomum villosum of the present invention, metronidazole, levofloxacin, amoxicillin, and clarithromycin.

[0072] Determination method: Amomum villosum essential oil extract was added to a 96-well plate in a gradient manner along the X-axis by a two-fold dilution method to a final concentration of 512 μg / mL, 256 μg / mL, 128 μg / mL, and 64 μg / mL, respectively. Antibiotics (metronidazole 0.125-2 μg / mL, levofloxacin 6.25×10 -2 ~0.5 µg / mL, amoxicillin 7.8×10 -3 ~6.25×10 -2 µg / mL and clarithromycin 4.88×10 -4 ~3.9×10 -3 µg / mL); all concentrations were obtained by adding 1×10 6 The final concentration of the system after the suspension of HpSS1 was 100 CFU / mL. After incubation of the culture plates at 37°C in a microaerobic environment for 3 days, the MIC values ​​of the drug combination were determined by visual observation and verified by measuring the absorbance at 600 nm. The above experiment was repeated three times.

[0073] The FIC was calculated using the following formula: (MIC of Amomum villosum essential oil extract combined with other drugs) / (MIC of Amomum villosum essential oil extract alone) + (MIC of antibiotic combination) / (MIC of antibiotic alone)

[0074] Judgment criteria: FIC ≤ 1 is synergistic; 1<FIC ≤ 2表示无关作用;FIC> 2 indicates antagonism.

[0075] As shown in Table 3, when the Amomum villosum essential oil extract prepared in the present invention was used in combination with four commonly used clinical antibiotics (metronidazole, levofloxacin, amoxicillin, and clarithromycin), all combinations exhibited a synergistic effect (FIC ≤ 1). Combined use reduced the dosage of Amomum villosum essential oil extract by 50% (256 → 128 µg / mL) and simultaneously reduced the dosage of antibiotics by 2-8 times, demonstrating a significant synergistic effect.

[0076] Table 3: Chessboard method for detecting the effects of Amomum villosum essential oil extract combined with antibiotics

[0077]

[0078] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. However, obvious variations or modifications derived from the essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A combined drug for treating Helicobacter pylori, characterized in that: It is composed of Amomum villosum essential oil extract and antibiotics; The Amomum villosum essential oil extract is prepared by the following steps: 1) Grinding the Amomum villosum fruit and mixing it with pure water at a material-liquid ratio of 1:8 to obtain an Amomum villosum liquid; 2) extracting the Amomum villosum solution of step 1) by steam distillation at 100-120° C. for 4 hours to obtain a crude Amomum villosum extract; 3) collecting the upper oil phase of the Amomum villosum crude extract obtained in step 2) through an oil-water separator, and removing residual water in the oil phase by a physical method to obtain a pure Amomum villosum essential oil extract; The antibiotic is one of metronidazole, levofloxacin, clarithromycin or amoxicillin.

2. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The mass ratio of the Amomum villosum essential oil extract to metronidazole is 1:2×10 -3 .

3. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The mass ratio of the Amomum villosum essential oil extract to levofloxacin is 1:9.8×10 -4 .

4. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The mass ratio of the Amomum villosum essential oil extract to amoxicillin is 1:6×10 -5 .

5. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The mass ratio of the Amomum villosum essential oil extract to clarithromycin is 1:3.8×10 -6 .

6. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The physical method of removing residual moisture in the oil phase specifically involves drying with anhydrous sodium sulfate to remove the residual moisture in the oil phase.

7. The anti-Helicobacter pylori combination drug according to claim 1, characterized in that: The active ingredients of the Amomum villosum essential oil extract include camphor, bornyl acetate, limonene, camphene and borneol.

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