Composition capable of resisting helicobacter pylori synergistically with antibiotics and preparation method of composition

Abstract

The invention discloses a composition with a synergistic anti-helicobacter pylori effect with antibiotics and a preparation method of the composition, and belongs to the technical field of traditional Chinese medicines. The composition is prepared from clove, kaki calyx, ginseng and ginger according to a specific proportion through water or ethanol extraction. Experiments show that the composition has remarkable bacteriostasis and sterilization effects on helicobacter pylori standard strains and clinical drug-resistant strains, and can generate a synergistic effect (FICI < = 0.5) with antibiotics such as clarithromycin and the like. The invention provides a traditional Chinese medicine anti-HP scheme which is safe, effective and not prone to drug resistance, and has a good development prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine, and particularly relates to a composition with synergistic effect of antibiotics against Helicobacter pylori and a preparation method thereof. BACKGROUND

[0002] Helicobacter pylori (HP) is a gram-negative bacterium with a very high infection rate worldwide, and is closely related to chronic gastritis, peptic ulcer and gastric cancer. The current mainstream treatment is triple or quadruple therapy containing antibiotics, but the clinical efficacy is limited due to the increasing antibiotic resistance, poor patient compliance and frequent side effects.

[0003] Dingxiang Qizixian is a classic prescription first recorded in Tiaoyin Mai Zhi, and is traditionally used for warming middle-jiao, descending adverse qi, stopping vomiting and tonifying deficiency. However, its modern pharmacological research is still insufficient, especially there is no systematic report on its anti-Helicobacter pylori and synergistic effect with antibiotics. SUMMARY

[0004] To solve the above technical problems, the present application provides a composition with synergistic effect of antibiotics against Helicobacter pylori and a preparation method thereof, which solves the problems of strong drug resistance and large side effects of existing antibiotic therapy.

[0005] In the first aspect, the present application provides a composition with synergistic effect of antibiotics against Helicobacter pylori, which is composed of the following raw materials by weight: clove 2 parts, persimmon peel 2 parts, ginseng 1 part, and fresh ginger 3 parts.

[0006] The composition is a water extract or an alcohol extract, wherein the alcohol extract uses ethanol with a concentration of 55%-95% as the extraction solvent.

[0007] In the second aspect, the present application further provides a preparation method of the above-mentioned composition, which comprises the following steps:

[0008] The clove, persimmon peel, ginseng and fresh ginger are mixed in a proportion of 2:2:1:3, and then subjected to twice of heating reflux extraction, and the extraction solutions are combined to obtain the composition;

[0009] The solvent used in the first extraction and the second extraction is water or ethanol.

[0010] In the first extraction, the mass ratio of the raw material to the solvent is 1:10, the extraction temperature is 80-90℃, and the extraction time is 0.9-1.1 hours; in the second extraction, the mass ratio of the filter residue to the solvent is 1:10, the extraction temperature is 80-90℃, and the extraction time is 0.9-1.1 hours.

[0011] The method further comprises filtering, concentrating and freeze-drying the extraction solution to obtain the composition freeze-dried powder.

[0012] In a third aspect, the present application provides a pharmaceutical composition comprising the composition and a pharmaceutically acceptable carrier.

[0013] In a fourth aspect, the present application provides use of the composition in the preparation of a medicament for anti-Helicobacter pylori.

[0014] In a fifth aspect, the present application provides use of the composition in the preparation of a medicament for anti-Helicobacter pylori in combination with an antibiotic.

[0015] The antibiotic is clarithromycin.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) Significant direct antibacterial activity: In vitro experiments have confirmed that the water extract and the alcohol extract of the composition of the present application have clear bacteriostatic (MIC value is between 0.16-0.64 mg / mL) and bactericidal effects (MBC value can reach 1.28 mg / mL) on Helicobacter pylori including standard strains (such as 700392, SS1) and various clinically isolated antibiotic-resistant strains (such as CS01, FY-13, etc.), indicating that it is an effective anti-HP agent itself.

[0018] (2) Strong antibiotic synergistic effect: When combined with the clinically commonly used antibiotic clarithromycin (CLR), it shows a significant synergistic effect (FICI=0.25) and can greatly improve the antibacterial efficacy of clarithromycin, providing a new combined drug regimen for solving the problem of antibiotic resistance.

[0019] (3) Effective against drug-resistant strains: The composition still maintains excellent antibacterial activity against various clinically isolated strains that have been resistant to clarithromycin, providing an effective alternative or auxiliary treatment strategy for the treatment of drug-resistant HP infection.

[0020] (4) Flexible dosage form and diverse effects: It can be prepared into traditional water extract and alcohol extract, and the activities of the two extracts are equivalent, providing flexibility for different formulation needs; at the same time, the composition is derived from a classic famous prescription, and has the dual effects of "antibacterial" and "warming middle and replenishing qi", which is in line with the overall treatment concept of traditional Chinese medicine.

[0021] (5) High safety and low cost: The prescription is composed of food-medicinal or traditional Chinese medicines, and is expected to have low adverse reactions and high patient acceptance; and the raw materials are easy to obtain, and the preparation process is simple, which has great potential to develop into a safe, effective and inexpensive anti-HP drug or health care product. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application but not all the embodiments.

[0023] Example 1: Preparation of the composition

[0024] Dingxiang 7.46 g, Shitidi 7.46 g, Renshen 3.73 g (ground through a 40-mesh sieve), Shengjiang 11.73 g (cut into pieces) were weighed, and then 10 times the mass of solvent (water or 75% ethanol) was added. After soaking for 0.5-1 hour, twice of heating reflux extraction was performed:

[0025] Water extraction condition: temperature 85-90℃, 1 hour each time;

[0026] Alcohol extraction condition: temperature 80-85℃, 1 hour each time.

[0027] The twice of extraction solutions were combined, filtered, concentrated using a rotary evaporator at 70-75℃ and 0.08 MPa, pre-frozen, and then freeze-dried to obtain a composition freeze-dried powder (water extract AE or alcohol extract HE).

[0028] Example 2: In vitro anti-Helicobacter pylori activity determination

[0029] 1. Strain selection: HP reference strain: 700392, SS1, 43504

[0030] HP clinical strain: ICDC111001, CS01, QYZ-001, QYZ-003, QYZ-004, FY-13, FY-14

[0031] 2. Experimental grouping: water extract group, 75% alcohol extract group, negative control group, clarithromycin (CLR) quality control group, and growth control group. Water extract group / 75% alcohol extract group: 50ul of drug solution + 50ul of bacterial solution; negative control group: 50ul of drug solution + 50ul of broth; CLR quality control group: 50ul of CLR + 50ul of bacterial solution; growth control group: 50ul of bacterial solution + 50ul of broth. The drug concentration of water extract / alcohol extract (1280ug / mL, 640ug / mL, 320ug / mL, 160ug / mL, 80ug / mL, 40ug / mL); the positive control drug CLR concentration (0.032ug / mL, 0.016ug / mL, 0.008ug / mL, 0.004ug / mL, 0.002ug / mL).

[0032] 3. Experimental method

[0033] 3.1 Determination of MIC

[0034] Broth microdilution method: The drug solution of the composition was diluted twice with BHI medium to obtain a series of concentrations, and 50 μL of the medium containing the drug was added to a 96-well plate. Each drug concentration was repeated 3 times, and negative control groups (containing only drug solution) and growth control groups (containing only bacterial solution) were set up, as well as a quality control group (clarithromycin treatment group). HP was scraped from a blood agar plate cultured for 48 h with a sterile moistened cotton swab and prepared into a bacterial suspension in BHI. The turbidity was adjusted to about 1 McFarland unit (McF), and after adjusting the turbidity of the bacterial suspension, it was diluted 10 times, and 50 μL was added to a 96-well plate. The final concentration of the bacterial solution was 10 6 CFU / mL, and placed in a three-gas incubator in a microaerobic environment (5% O2, 10% CO2, 85% N2) for constant temperature oscillation culture for 3 d. The results were available when the growth control was significantly turbid, and the drug concentration when the bacterial solution was significantly clear was the MIC. (Each group of experiments was repeated more than 3 times)

[0035] 3.2 Determination of MBC: 100 μL of medium from the MIC, 2MIC, and 4MIC wells was inoculated on a Columbia blood plate, and the plate was incubated in a microaerobic environment at 37°C for 4-5 d. The drug concentration without bacterial colony growth was the minimum bactericidal concentration (MBC). Clarithromycin treatment was used as a quality control for each determination, and the MIC value was determined within 10 generations, and the same number of generations was completed.

[0036] 4. Experimental results:

[0037] In the experiment, the MICs of the water extract and 75% alcohol extract of the composition against 10 different HP strains (3 standard antibiotic-sensitive strains and 7 clinically isolated antibiotic-resistant strains) were determined by broth microdilution method, and the results are shown in Tables 1 and 2. The MIC values of the water extract and alcohol extract of the composition against the HP strains were between 0.16 mg / mL and 0.64 mg / mL, showing certain bacteriostatic effects, and the bacteriostatic concentrations of the water extract and alcohol extract were close. The water extract and alcohol extract also had bacteriostatic effects on clinically resistant strains.

[0038] Table 1 MIC determination results of water extract of the composition on each HP strain (n≥2)

[0039] Note: The HP breakpoint value in EUCAST 2023 is CLR: S ≤ 0.25 (sensitive), R > 0.25 (resistant).

[0040] Table 2 MIC determination results of alcohol extract of the composition on each HP strain (n≥2)

[0041] Note: EUCAST 2023 HP breakpoint value CLR: S ≤ 0.25 (sensitive), R > 0.25 (resistant).

[0042] From the results of Table 3 and Table 4, the water extract of the composition has a certain bactericidal effect on 700392, SS1, CS01, QYZ-004, FY-13, FY-14 strains; the alcohol extract of the composition has a certain bactericidal effect on 700392, SS1, CS01, QYZ-003, QYZ-004, FY-13, FY-14 strains, and the water extract and alcohol extract of the composition have a certain bactericidal effect on some strains.

[0043] Table 3 MBC determination results of water extract of the composition on each HP strain (n≥2)

[0044] Table 4 MBC determination results of alcohol extract of the composition on each HP strain (n≥2)

[0045] Example 3: Analysis of the synergistic effect of the composition and clarithromycin

[0046] 1. Strain selection: 700392

[0047] 2. Drug concentration setting: drug concentration of water extract / alcohol extract of the composition (5120 ug / mL, 2560 ug / mL, 1280 ug / mL, 640 ug / mL, 320 ug / mL, 160 ug / mL); CLR concentration (0.128 ug / mL, 0.064 ug / mL, 0.032 ug / mL, 0.016 ug / mL, 0.008 ug / mL, 0.004 ug / mL)

[0048] 3. Experimental method

[0049] 1 / 4 MIC-4 MIC alcohol extract and water extract of the composition were prepared by BHI double dilution method, and antibiotic liquid was prepared by the same method. After the preparation of the drug solution was completed, 30 μL of each was added to the 96-well plate. After measuring the turbidity of the 700392 bacterial suspension in the growth period, 1 Mac's turbidity unit was diluted 10 times, and 60 μL was added to the drug-containing well. The final concentration of each well was 30 μL of drug solution + 30 μL of antibiotic solution + 60 μL of bacterial suspension. The 96-well plate was immediately placed in a microaerobic incubator at 37°C, 150 rpm, and cultured for 72 h. The results were observed after taking out. The results can only be used when the growth control is obviously turbid. The turbidity of the clear well was calculated, and the corresponding clove extract drug concentration, antibiotic drug concentration, and MIC of the combination of the two drugs were calculated. The FICI was calculated according to the following formula:

[0050] FICI= + ;

[0051] When FICI≤0.5, it is considered that there is a synergistic effect between the two drugs; when 0.5 < FICI≤4, it is considered that there is no effect between the two drugs; when FICI>4, it is considered that there is an antagonistic effect between the two drugs.

[0052] 4. Experimental results

[0053] The results are shown in the following table 5, and the FICI value of the composition water extract or alcohol extract combined with clarithromycin is 0.25 (≤0.5), indicating that the two have a synergistic effect on anti-Helicobacter pylori.

[0054] Table 5

[0055]

[0056] In summary, the composition of the present application not only has good in vitro anti-Helicobacter pylori activity, but also can synergistically enhance the curative effect with antibiotics, reduce the dosage of antibiotics and the risk of drug resistance, and has important clinical application value.

[0057] The preferred embodiments of the present application disclosed above are only used to help explain the present application. The preferred embodiments do not describe all the details and limit the present application to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of the present application.

Claims

1. A composition having a synergistic anti-Helicobacter pylori activity with antibiotics, characterized in that, It consists of the following ingredients in parts by weight: 2 parts cloves, 2 parts persimmon calyx, 1 part ginseng, and 3 parts ginger.

2. The composition according to claim 1, characterized in that, The composition is an aqueous extract or an alcoholic extract, wherein the alcoholic extract uses ethanol with a concentration of 55%-95% as the extraction solvent.

3. A method for preparing the composition according to claim 1 or 2, characterized in that, Includes the following steps: Cloves, persimmon calyx, ginseng, and ginger were mixed in a certain proportion and subjected to two heating and reflux extractions. The extracts were combined to obtain the composition. The solvents used for the first and second extractions are water or ethanol.

4. The method according to claim 3, characterized in that, In the first extraction, the mass ratio of raw material to solvent is 1:10, the extraction temperature is 80℃–90℃, and the extraction time is 0.9–1.1 hours; in the second extraction, the mass ratio of filter residue to solvent is 1:10, the extraction temperature is 80℃–90℃, and the extraction time is 0.9–1.1 hours.

5. The method according to claim 3 or 4, characterized in that, The method further includes filtering, concentrating, and freeze-drying the extract to obtain a freeze-dried powder of the composition.

6. A pharmaceutical composition, characterized in that, It comprises the composition of claim 1 or 2 and a pharmaceutically acceptable carrier.

7. Use of the composition of claim 1 or 2 in the preparation of a medicament for treating Helicobacter pylori.

8. Use of the composition of claim 1 or 2 in the preparation of a medicament for use in combination with an antibiotic to synergistically combat Helicobacter pylori.

9. The application according to claim 8, characterized in that, The antibiotic in question is clarithromycin.

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