CN113855737B

CN113855737B - Composition for treating helicobacter pylori and preparation method and application thereof

Info

Publication number: CN113855737B
Application number: CN202111202472.1A
Authority: CN
Inventors: 黄衍强; 李如佳; 周文婷; 李琛妍; 黄雯; 董蕙华; 黄凯黎; 何继凯; 吴童
Original assignee: Youjiang Medical University for Nationalities
Current assignee: Youjiang Medical University for Nationalities
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2022-06-03
Anticipated expiration: 2041-10-15
Also published as: CN113855737A

Abstract

A composition for treating helicobacter pylori and a preparation method and application thereof are disclosed, wherein an aqueous extract is prepared by the following steps: soaking coptis chinensis and fructus evodiae in water according to the weight ratio of 6:1, decocting, filtering to obtain filtrate, adding water, decocting again, combining the three filtrates, concentrating, drying in vacuum, and storing in a refrigerator at-20 ℃ for later use after drying; dissolving the medicine: weighing arctigenin and tanshinone I respectively, dissolving in anhydrous ethanol to make the final concentration be 4mg/mL, weighing the prepared water extract, and dissolving in sterile water to make the final concentration be 4 mg/mL; the combination medicine comprises the following components: mixing the water extract, the arctigenin and the tanshinone I together according to the mass ratio of 100:8:8 to obtain the composition. The invention successfully prepares the traditional Chinese medicine and traditional Chinese medicine component composition for treating the helicobacter pylori, the composition has obvious inhibition effect on the helicobacter pylori in vivo and in vitro, and has the advantages of single effect, low toxicity and difficult generation of drug resistance.

Description

Composition for treating helicobacter pylori and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a composition for treating helicobacter pylori, and a preparation method and application thereof.

Background

Helicobacter pylori (Hp) is an important cause of diseases such as gastritis, peptic ulcer and gastric cancer. Hp now infects more than about half of the global population. At present, the proposal for eliminating the Hp at home and abroad mainly comprises standard triple, non-bismuth agent tetrad (PPI +3 antibacterial agents), bismuth agent tetrad (PPI + bismuth agent +2 antibacterial agents) and the like, and the antibacterial agents mainly comprise metronidazole, clarithromycin, levofloxacin and the like. However, with the widespread use of antibiotics for treatment, the Hp drug resistance is higher and lower, the eradication rate is lower and lower, and the health and safety of the public are seriously threatened. The Hp treated by the traditional Chinese medicine has the advantages of definite curative effect, difficult generation of drug resistance, low toxicity, high eradication rate and the like. The ZUOJIN pill is derived from Danxi Heart method, and comprises Coptidis rhizoma and fructus evodiae at a dosage ratio of 6:1, the traditional Chinese medicine composition has the effects of clearing liver and purging fire, calming the adverse-rising energy and preventing vomiting, is mainly used for acute and chronic gastritis, helicobacter pylori infection and the like, but through medicine sensitivity detection, the Minimum Inhibitory Concentration (MIC) of a Zuojin pill water extract is 50-100 mu g/mL, the inhibitory effect on helicobacter pylori is not obvious, and the antibacterial effect is to be improved. The arctigenin and tanshinone I are respectively active ingredients of traditional Chinese medicines such as burdock and salvia miltiorrhiza, experiments prove that the arctigenin and tanshinone I have good inhibition effect on helicobacter pylori, MIC can reach 1 mu g/mL, and the action mechanisms are different, belong to auxiliary materials in Chinese pharmacopoeia, and have good safety. Therefore, the arctigenin and tanshinone I are added into the Zuojin pill, the inhibition effect on the helicobacter pylori can be synergistically enhanced through different action targets, the antibacterial effect of the Zuojin pill is enhanced, the action mechanism of the Zuojin pill is not influenced, and the synergistic effect on treating the helicobacter pylori infection related diseases is achieved.

The preparation method of the composition related to the invention is not reported; the composition has specific inhibition effect on helicobacter pylori in vivo and in vitro, the synergistic antibacterial effect is enhanced by 10 times compared with that of a Zuojin pill, the repair of gastric mucositis of mice can be better promoted, the drug resistance is not easy to generate, the safety is high, and the composition can be used as a candidate drug for treating helicobacter pylori infection-related diseases, and the application of the composition is disclosed for the first time.

Disclosure of Invention

The technical problem to be solved is as follows: in order to improve the inhibitory activity of the Zuojin pill on helicobacter pylori and not change the original action mechanism of the Zuojin pill, the invention provides the composition for treating the helicobacter pylori and the preparation method and the application thereof.

The technical scheme is as follows: a preparation method of a composition for treating helicobacter pylori comprises the following specific steps: step one, preparing an aqueous extract: mixing Coptidis rhizoma and fructus evodiae at a weight ratio of 6:1, soaking in water for 20-40min, decocting for 3 times, first decocting for 1 hr, second decocting for 45min, and third decocting for 30 min; adding water to supplement evaporated water, stirring, filtering after each decoction, obtaining filtrate, adding water, decocting again, mixing the three filtrates, concentrating, vacuum drying, and storing in a refrigerator at-20 deg.C for use; and step two, dissolving the medicine: weighing arctigenin and tanshinone I respectively, dissolving in absolute ethyl alcohol to make the final concentration be 4mg/mL, weighing prepared water extract, and dissolving in sterile water to make the final concentration be 4 mg/mL; step three, combining the medicaments: mixing the water extract, the arctigenin and the tanshinone I according to different mass ratios of 100 (1-16) to 1-16 to obtain the composition.

Preferably, the soaking time in water is 30 min.

Preferably, the aqueous extract, the arctigenin and the tanshinone I are mixed according to the proportion of 100:8:8 after preparing the same mother liquor concentration.

The composition prepared by the preparation method.

The application of the composition in preparing the medicine for treating the helicobacter pylori infection related diseases.

Has the advantages that: first, the present invention successfully prepares a composition; secondly, the composition prepared by the invention acts on inhibiting the growth of the helicobacter pylori, has better effect than that of Zuojin pills, has high action specificity and small toxic and side effect, is not easy to generate drug resistance, can be used as a candidate drug for treating the helicobacter pylori infection disease, and can effectively relieve the severe drug resistance problem of the helicobacter pylori.

Drawings

FIG. 1 formulation 100:8:8 composition toxicity assessment on cells;

FIG. 2 formulation of 100:8:8 composition for mouse stomach, liver, spleen and kidney tissue injury;

FIG. 3 evaluation of drug resistance of the formulated 100:8:8 composition;

FIG. 4 shows that the amount of Hp159 planted in mice with acute gastritis treated by the composition is 100:8: 8;

FIG. 5 shows that the composition with the ratio of 100:8:8 is used for treating the gastric mucosal tissue repair of mice with acute gastritis;

FIG. 6 shows that the composition of 100:8:8 is formulated to treat the decrease of inflammatory factors after the treatment of mice with acute gastritis.

Detailed Description

Example 1

Step one, preparing an aqueous extract: the Chinese medicines of coptis root, evodia fruit, great burdock achene and salvia miltiorrhiza are weighed accurately according to different proportions. Soaking in water for 30min, decocting for 3 times, the first time for 1 hr, the second time for 45min, and the third time for 30 min. Continuously adding water to supplement evaporated water, stirring with glass rod to avoid bottom burnt, filtering after each decoction, collecting filtrate, and adding appropriate amount of water to decoct again. Mixing the three filtrates, concentrating, vacuum drying, and storing in-20 deg.C refrigerator.

And step two, dissolving the medicine: the aqueous extract dried in the first step was dissolved in sterile water to a final concentration of 4mg/mL to give composition 1.

Example 2

Step one, preparing an aqueous extract: the Chinese medicines of coptis and evodia are accurately weighed according to the proportion of 6: 1. Soaking in water for 30min, decocting for 3 times, the first time for 1 hr, the second time for 45min, and the third time for 30 min. Continuously adding water to supplement evaporated water, stirring with glass rod to avoid bottom burnt, filtering after each decoction, collecting filtrate, and adding appropriate amount of water to decoct again. Mixing the three filtrates, concentrating, vacuum drying, and storing in-20 deg.C refrigerator.

And step two, dissolving the medicine: the commercially available compounds arctigenin and tanshinone I were weighed and dissolved in absolute ethanol to a final concentration of 4mg/mL, and the prepared aqueous extract was weighed and dissolved in sterile water to a final concentration of 4 mg/mL.

Thirdly, combining the medicines: mixing the water extract, arctigenin and tanshinone I according to the proportion of 100:1:1, 100:2:2, 100:4:4, 100:8:8 and 100:16:16 respectively to obtain the composition 2.

The inhibitory effect of the present invention on helicobacter pylori is further illustrated in detail by the following examples.

1. Material

1.1 sample

Compositions were prepared using examples 1 and 2, respectively.

1.2 strains

(1) Helicobacter pylori strains, standard strains 26695, NSH57, MSD132 and G27, were gifted by BihongKai teacher of Nanjing medical university; clinical metronidazole-resistant strains, clarithromycin-resistant strains, levofloxacin and metronidazole-resistant strains, clarithromycin and metronidazole-resistant strains, levofloxacin, clarithromycin and metronidazole multi-resistant strains (HPBS001, HPBS002, HPBS003 and the like) are provided by drug-resistant microbial infection prevention and treatment research centers of the national medical colleges in the Yangjiang province.

(2) Non-helicobacter pylori bacteria: staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Candida albicans, Enterobacter cloacae, Campylobacter jejuni, Bacillus subtilis, Proteus mirabilis, Lactobacillus curvatus, stenotrophomonas maltophilia, Morganella morganii, Candida tropicalis, Staphylococcus haemolyticus, Acetobacter, Saccharomyces cerevisiae, and Enterobacter hophattai were provided by the research center for the prevention and treatment of infection by drug-resistant microorganisms of the national institute of medicine, Youjiang.

1.3 Main Medium and reagents: columbia culture medium, brain heart infusion culture medium, nutrient agar culture medium, nutrient broth culture medium, MH culture medium, Sabouraud culture medium, and standard calf serum.

1.4 Main instruments: three-gas incubator, centrifuge, enzyme-labeling instrument, electronic balance, etc.

1.5 consumable: EP tubes, Tip heads, centrifuge tubes, etc.

2. Method and results

2.1 microdilution assay of the Minimal Inhibitory Concentration (MIC) of the composition prepared in example 1 against helicobacter pylori

2.1.1 microdilution assay of minimal inhibitory concentration (MIC, 100. mu.L system) of composition 1 against helicobacter pylori

(1) Preparing 4mg/mL of composition 1 (such as coptis chinensis (24g), evodia rutaecarpa (4g), great burdock achene (6g), salvia miltiorrhiza (6g), coptis chinensis (24g), evodia rutaecarpa (4g), great burdock achene (12g), salvia miltiorrhiza (12g) and the like) in different proportions for later use.

(2) Preparing an agar plate: antibacterial drugs with different concentrations are respectively added into a 50mL flask, so that the drug contents of the culture medium and the drugs after mixing are respectively 1, 0.8, 0.6, 0.4, 0.2 and 0.1mg/mL, and the total amount of the drugs and the culture medium is 20 mL.

(3) Preparing and inoculating bacterial liquid: taking bacteria growing in logarithmic phase on a solid plate, preparing into bacterial suspension by using corresponding culture medium, and adjusting the concentration of bacterial liquid to be 1 × 10 by Hp⁸CFU/mL (OD600 of 0.3), ready for use. mu.L of the suspension was inoculated onto the agar surface at about 10. mu.l/spot⁵And (4) CFU. And 1 mul of bacterial liquid is inoculated to the original culture medium of the strain as a positive control, and an agar plate containing the liquid is used as a negative control. Inoculating and placing the strain in a corresponding incubator for incubation.

(3) And (4) judging the result: the experiment only makes sense when the bacteria on the negative control plate containing the drug and without bacteria are not grown and the bacteria on the positive plate of the original culture medium of the strain are grown. The plate was placed on a dark, non-reflective object surface to determine the end of the test, with the lowest drug concentration inhibiting bacterial growth being the MIC. Slight bacterial growth was seen on the well agar surface, with the lowest drug concentration inhibiting bacterial growth by more than 80% compared to the growth control as the end-point concentration. If more than 2 colonies grow on the agar plate containing the drug at a concentration higher than the end level, or do not grow on the low-concentration drug agar plate but grow on the high-concentration drug agar plate, the culture purity should be checked or the test should be repeated.

(6) As a result: the inhibition effect of the composition added with the burdock and the salvia miltiorrhiza in different proportions on the helicobacter pylori is not better than that of the Zuojin pill (coptis chinensis + evodia rutaecarpa), even the inhibition effect is reduced, so that the inhibition effect of the Zuojin pill on the helicobacter pylori can be hardly improved by adding other traditional Chinese medicines, and the results are shown in tables 1, 2 and 3.

TABLE 1 MIC detection (mg/mL) of 159 strain in Zuojin pill after adding fructus Arctii and radix Salviae Miltiorrhizae

TABLE 2 MIC detection (mg/mL) of 26695 strain for Zuojin pill after adding fructus Arctii and radix Salviae Miltiorrhizae

TABLE 3 MIC detection (mg/mL) of G27 strain in Zuojin pill after adding fructus Arctii and radix Salviae Miltiorrhizae

2.1.2 microdilution assay of the Minimal Inhibitory Concentration (MIC) of the composition prepared in example 2 against helicobacter pylori

(1) Preparing 4mg/mL of water extract of Zuojin pill, arctigenin and tanshinone I, and respectively mixing according to a ratio of 100:1:1, 100:2:2, 100:4:4, 100:8:8 and 100:16:16 for later use.

(2) Preparing a MIC plate, namely adding 175 mu L of culture medium into a first well, adding 5 mu L of antibacterial agent into the first well, and diluting the mixture to a 7 th well in a multiple ratio; no drug was added to well 8, and 90. mu.L of the medium was retained as a control with and without drug added.

(3) Bacterial liquid preparation helicobacter pylori growing in logarithmic phase on solid plate is prepared into bacterial suspension by BHI culture medium, and the OD concentration is adjusted₆₀₀Is 0.3 (1X 10)⁸CFU/mL), 10-fold dilution at 1X 10⁷CFU/mL, spare.

(4) 10. mu.L of inoculum solution was added to wells 1-8 (the concentration of inoculum solution per well was about 1.0X 10)⁶CFU/mL). And culturing for 72h to judge the result.

(5) The results judged the MIC as the lowest drug concentration that completely inhibited bacterial growth in the wells. It is only significant when bacteria clearly grow in the 8 th well of the positive control well (i.e., no antibiotic) and the negative control well (i.e., sterile, medium only) does not grow. When a single jump hole occurs in the microdilution method, the highest concentration of drug that inhibits bacterial growth should be recorded. If a plurality of jump holes appear, the result should not be reported, and the test needs to be repeated. Each drug was tested in 3 replicates.

(6) As a result: in the composition prepared in example 2, the traditional Chinese medicine active ingredients of arctigenin and tanshinone I are added to the aqueous extract of the Zuojin pill, the composition has the optimal antibacterial effect at a ratio of 100:8:8, and the result is shown in Table 4; moreover, the minimum inhibitory concentration of the 100:8:8 composition is reduced by about 10-30 times compared with that of the original Zuojin pill, the inhibitory effect is obviously improved, and the result is shown in Table 5.

TABLE 4MIC (μ g/mL) assay for H.pylori for compositions of different ratios

TABLE 5 MIC (. mu.g/mL) assay for different strains of H.pylori for 100:8:8 compositions

2.2 drug toxicity testing

2.2.1 cytotoxicity assays

(1) Ges-1 and MGC823 cell suspensions were prepared, adjusted to a concentration of 1X 10⁵。

(2) Plating into 96-well plates: 100 μ L per well, and 3 replicates of the same sample were made.

(3) Incubated at 37 ℃ for 24 hours in an incubator.

(4) Adding the composition 2 with different proportions, and adding the medicine without adding the cell group.

(5) Incubated at 37 ℃ for 24 hours in an incubator.

(6) Add 10. mu.L of CCK8, mix well by tapping and incubate for 4 hours.

(7) Measuring the absorbance at 450nm, and calculating the survival rate according to the formula: the cell survival rate is [ (As-Ab) ]/[ (Ac-Ab) ] × 100%, As is a well containing cell culture medium, drug and CCK-8, Ac is a well containing cell culture medium, CCK-8 and no drug, Ab is a well containing no cell and drug, only culture medium and CCK-8. And establishing a survival curve according to the survival rate.

(8) Results example 2 the 8-fold MIC of the formulated 100:8:8 composition did not significantly damage Ges-1 and BGC823 cells, was not significantly different from that of zuojin pill, and was less toxic, as shown in fig. 1.

2.2.2 animal toxicity test

(1) Preparation of mice: c57BL/6 mice, 6-8 weeks old, were purchased and randomly assigned to 10 of the drug and negative control groups per group.

(2) Intragastric administration: 10 times of the therapeutic dose is administered, the therapeutic dose of the composition (100:8:8) is 7mg/kg, namely 70mg/kg for mouse toxicity detection, and the administration is performed for 3 days and 1 time per day; the negative control group is given PBS, and the frequency and amount are the same as those of the administration group.

(3) Weighing: mice were weighed starting 1 day before dosing and were weighed 7 consecutive days.

(4) And (3) detection of drug effect: mice in the infection group on day 3 after drug withdrawal were weighed and the average body weight was calculated, blood was collected from the eyeballs, dislocation and neck amputation were performed to kill, and stomach, kidney, liver and spleen tissues were taken, and pathological sections were prepared and HE staining was performed.

(5) As a result: a to D represent the damage of the stomach, liver, spleen and kidney of the normal mice in the PBS group, and E to H represent the damage of the stomach, liver, spleen and kidney of the composition (100:8:8) prepared in the gastric perfusion example 2 to the stomach, liver, spleen and kidney, compared with the two groups, the administration of 10 times of the treatment dose of the composition (100:8:8) prepared in the example 2 does not obviously damage the stomach, liver, spleen and kidney of the mice, and the toxicity is very low, as shown in figure 2.

2.3 microdilution assay for Minimal Inhibitory Concentration (MIC) of the 100:8:8 composition formulated in example 2 against non-H.pylori

(1) Prepare 4mg/mL of the composition (100:8:8) and mix for use.

(2) Preparing a MIC plate, namely adding 175 mu L of culture medium into a first hole, adding 5 mu L of antibacterial agent into the first hole, and diluting the mixture to a 7 th hole in a multiple ratio; no drug was added to well 8, and 90. mu.L of the medium was retained as a control with and without drug added.

(3) Preparation of bacterial solution bacteria growing in logarithmic phase on solid plate are prepared into bacterial suspension by using corresponding culture medium, and the adjustment concentration OD600 of bacteria is 0.3 (1X 10)⁸CFU/mL), 100-fold dilution at 1X 10⁶CFU/mL, fungal adjustment concentration OD600 of 0.5 (5X 10)⁶CFU/mL), diluted 1000-fold at 5X 10³CFU/mL, spare.

(4) 10. mu.L of inoculum solution was added to wells 1-8 (the concentration of inoculum solution per well was about 1.0X 10)⁶CFU/mL). And culturing for 72h to judge the result. The concentrations of the drugs (Zuojin pill + arctigenin + tanshinone I) from the 1 st to the 6 th holes are respectively 86.21+6.9+6.9, 43.1+3.45+3.45, 21.55+1.72+1.72, 10.78+0.86+0.86, 5.39+0.43+0.43 and 2.69+0.22+0.22 mu g/mL.

(5) The results judged the MIC as the lowest drug concentration that completely inhibited bacterial growth in the wells. The test is meaningful when bacteria in the 7 th well (i.e., no antibiotic) of the positive control well grow significantly and the 8 th (sterile) well does not grow. When a single jump hole occurs in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If a plurality of jump holes appear, the result should not be reported, and the test needs to be repeated. Each drug was tested in 3 replicates.

(6) As a result: composition 2(100:8:8) had a poor inhibitory effect on most non-H.pylori bacteria, indicating that it has a narrow antibacterial spectrum, a strong specificity, and a specific effect on H.pylori bacteria, and the results are shown in Table 6.

TABLE 6 formulation 100:8:8 composition MIC (μ g/mL) assay for non-H.pylori

Note: 100:8: group 8 ">" means greater than 86.21+6.9+ 6.9; the ZUOJIN pill group ">" means greater than 100

2.4 detection of drug resistance in the 100:8:8 composition formulated in example 2

(1) The H.pylori G27 strain is used for detecting the drug resistance of the forsythiaside derivatives. First, the MICs of metronidazole and composition 2(100:8:8) were measured as 2. mu.g/mL and 5.39+0.43+ 0.43. mu.g/mL, respectively, and induction was performed with 1/4MIC concentration, once every 3 days for 24 days. The induction concentration is adjusted along with the change of MIC, for example, when the MIC of metronidazole is changed to 16 mug/mL, the induction concentration is adjusted to 4 mug/mL.

(2) After induction for 24 days, metronidazole is obviously resistant, and MIC is increased by 64 times; the 100:8:8 composition formulated in example 2 had no change in MIC and did not readily develop resistance as shown in figure 3.

2.5 construction of animal model in vivo assay of inhibitory effect of composition on H

The Zuojin pill, the arctigenin, the tanshinone I, the omeprazole, the amoxicillin and the clarithromycin are dissolved and diluted to 10 mg/mL. Experimental animals: c57 BL/6.

(1) Animal grouping: the experimental group averagely divides the infection group (BHK159) successfully molded into 5 groups, namely an omeprazole group, amoxicillin and clarithromycin group (triple therapy group), an omeprazole group and Zuojin pill group (7mg/kg), an omeprazole group and 100:8:8 composition (7mg/kg) and a PBS group, wherein each group comprises 10 patients; 10 mice not infected with h.

(2) Animal administration: the experimental group is administered by intragastric administration, the group with omeprazole is administered with omeprazole firstly, other medicines are administered after 30min, and after the medicines are administered, the patient is fasted and is forbidden to drink for 4 hours; the weight of the mouse is calculated according to the average 20 g/mouse, the dosage is 138.2mg/kg of omeprazole, 28.5mg/kg of amoxicillin and 14.3mg/kg of clarithromycin, and the administration is carried out for 1 time every day and 3 times continuously; the negative control group was given PBS solution, and the volume and frequency were the same as above.

(3) And (3) detection of drug effect: mice in the infected group on day 2 after drug withdrawal were weighed and the average body weight was calculated, blood was sampled from the eyeballs, neck was sacrificed by amputation, gastric tissue was taken, disrupted, isolated culture and identification of h.pyleri and the amount of permanent planting was calculated, and the treatment effect of the composition group containing 100:8:8 was significantly superior to that of the triple therapy and zuojin pill group, as shown in fig. 4. Paraffin section is carried out on partial stomach tissue, H & E staining, TUNEL immunohistochemistry and fluorescence immunoassay are carried out, the stomach tissue repair effect of the composition group containing 100:8:8 is good, and inflammatory factors are obviously reduced, as shown in fig. 5 and fig. 6.

Claims

1. A preparation method of a composition for inhibiting helicobacter pylori is characterized by comprising the following specific steps: step one, preparing an aqueous extract: mixing Coptidis rhizoma and fructus evodiae at a weight ratio of 6:1, soaking in water for 20-40min, decocting for 3 times, first decocting for 1 hr, second decocting for 45min, and third decocting for 30 min; adding water to supplement evaporated water, stirring, filtering after each decoction, obtaining filtrate, adding water, decocting again, mixing the three filtrates, concentrating, vacuum drying, and storing in a refrigerator at-20 deg.C for use; and step two, dissolving the medicine: weighing arctigenin and tanshinone I respectively, dissolving in anhydrous ethanol to make the final concentration be 4mg/mL, weighing the prepared water extract, and dissolving in sterile water to make the final concentration be 4 mg/mL; step three, combining the medicaments: mixing the water extract, the arctigenin and the tanshinone I together according to the mass ratio of 100:8:8 to obtain the composition.

2. The method of claim 1, wherein the soaking time in water is 30 min.

3. The composition obtained by the production process according to claim 1 or 2.

4. Use of a composition according to claim 3 for the manufacture of a medicament for the treatment of acute gastritis caused by helicobacter pylori.