CN114522169A

CN114522169A - Traditional Chinese medicine monomer composition for treating helicobacter pylori and preparation method and application thereof

Info

Publication number: CN114522169A
Application number: CN202210107625.2A
Authority: CN
Inventors: 黄衍强; 李如佳
Original assignee: Youjiang Medical University for Nationalities
Current assignee: Youjiang Medical University for Nationalities
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-24
Anticipated expiration: 2042-01-28
Also published as: CN114522169B

Abstract

A Chinese medicinal monomer composition for treating helicobacter pylori and a preparation method and application thereof are disclosed, and the method comprises the following steps: a. dissolving the medicine: respectively dissolving berberine, evodiamine, arctigenin and tanshinone I in anhydrous ethanol; b. the combination medicine comprises the following components: mixing the prepared ethanol solution according to the mass ratio of berberine to evodiamine to arctigenin to tanshinone I of 6:1 (0.75-6) to obtain the composition. The invention successfully prepares the traditional Chinese medicine monomer composition for treating the helicobacter pylori, the composition has very 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

Traditional Chinese medicine monomer composition for treating helicobacter pylori as well as preparation method and application thereof

Technical Field

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

Background

Helicobacter pylori (Hp) is a bacterium that persists in the human gastric mucosa and causes gastric infection, and is associated with various gastrointestinal diseases. At present, the Hp eradication at home and abroad is mainly treated by combining antibiotics with bismuth agents, and the antibacterial drugs mainly comprise metronidazole, clarithromycin, levofloxacin and the like. However, with the wide use and abuse of antibiotics, the Hp drug resistance is higher and lower, the eradication rate is lower and lower, and the health safety of the public is seriously threatened. The Hp treated by the traditional Chinese medicine has the advantages of definite curative effect, low possibility of drug resistance, low toxicity, high eradication rate and the like, but effective monomer components of a plurality of bacteriostatic traditional Chinese medicines are not clear, and even if the effective components of the monomer of the traditional Chinese medicine are found, the effective components are sometimes effective in vitro but not effective in vivo, and further experimental verification is needed.

The screening research of the traditional Chinese medicine antibacterial components shows that main components of coptis chinensis, fructus evodiae, fructus arctii and radix salviae miltiorrhizae for inhibiting Hp are berberine, evodiamine, arctigenin and tanshinone I respectively, MICs of the berberine, the evodiamine, the arctigenin and the tanshinone I are 16-32 mu g/mL, 1-2 mu g/mL and 0.5-1 mu g/mL respectively, and the action mechanisms of the monomeric compounds are different. Different monomer compounds are combined for antibiosis, so that the action target of the composition is increased, the antibacterial effect is improved, and the drug resistance of the compound is reduced. Therefore, according to the quality proportion of the Zuojin pill, the berberine, the evodiamine, the arctigenin and the tanshinone I are combined according to a certain quality proportion to inhibit Hp, the combination can synergistically enhance the inhibition effect on helicobacter pylori, the drug resistance is not easy to generate, and the Hp-inhibiting capsule has a very good effect on treating diseases related to helicobacter pylori infection.

The preparation method of the traditional Chinese medicine monomer composition related to the invention is not reported; the combination has specific inhibition effect on helicobacter pylori in vivo and in vitro, can better promote repair of gastric mucositis of mice with acute gastritis infected by helicobacter pylori, is not easy to generate drug resistance and high in safety, can be used as a candidate drug for treating diseases related to helicobacter pylori infection, and belongs to the first disclosure.

Disclosure of Invention

The technical problem to be solved is as follows: in order to improve the inhibition activity of traditional Chinese medicines on helicobacter pylori, the invention provides a traditional Chinese medicine monomer composition for treating the helicobacter pylori, a preparation method and application thereof.

The technical scheme is as follows: a preparation method of a traditional Chinese medicine monomer composition for treating helicobacter pylori comprises the following steps: a. dissolving the medicine: respectively dissolving berberine, evodiamine, arctigenin and tanshinone I in anhydrous ethanol; b. the combination medicine comprises the following components: mixing the prepared ethanol solution according to the mass ratio of berberine to evodiamine to arctigenin to tanshinone I of 6:1 (0.75-6) to obtain the composition.

The mass ratio of the berberine to the evodiamine to the arctigenin to the tanshinone I is 6:1:3: 3.

The Chinese medicinal monomer composition prepared by the method.

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

The medicine for treating helicobacter pylori infection related diseases comprises the effective component of the traditional Chinese medicine monomer composition.

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 helicobacter pylori, has high action specificity and small toxic and side effects, is not easy to generate drug resistance, can be used as a candidate drug for treating helicobacter pylori infection diseases, and effectively relieves the severe drug resistance problem of the helicobacter pylori.

Drawings

FIG. 1 toxicity evaluation of the formulated 6:1:3:3 composition on cells;

FIG. 2 formulation of 6:1:3:3 composition for the treatment of gastric, liver, spleen and kidney tissue injury in mice;

FIG. 3 evaluation of drug resistance of the formulation 6:1:3:3 composition;

FIG. 4 shows the amount of Hp159 colonized after the composition is formulated at a ratio of 6:1:3:3 to treat acute gastritis mice;

FIG. 5 shows that the composition with the ratio of 6:1:3:3 is used for treating the gastric mucosal tissue repair of mice with acute gastritis;

FIG. 6 shows that the composition of 6:1:3:3 is formulated to treat the decrease of inflammatory factors after the treatment of mice with acute gastritis.

Detailed Description

Example 1

First, dissolving the drug: weighing berberine, evodiamine, arctigenin and tanshinone I respectively, and dissolving in anhydrous ethanol to obtain final concentration of 4 mg/mL.

Step two, combining the medicines: mixing berberine, evodiamine, arctigenin and tanshinone I according to different mass ratios of 6:1:0.75:0.75 to obtain a composition 1.

Example 2

Step two, combining the medicines: mixing berberine, evodiamine, arctigenin and tanshinone I according to different mass ratios of 6:1:1.5:1.5 to obtain a composition 2.

Example 3

First, dissolving the drug: weighing berberine, evodiamine, arctigenin and tanshinone I, respectively, and dissolving in anhydrous ethanol to obtain final concentration of 4 mg/mL.

Step two, combining the medicines: mixing berberine, evodiamine, arctigenin and tanshinone I according to different mass ratios of 6:1:3:3 to obtain a composition 3.

Example 4

First step, dissolving the drug: weighing berberine, evodiamine, arctigenin and tanshinone I respectively, and dissolving in anhydrous ethanol to obtain final concentration of 4 mg/mL.

Step two, combining the medicines: mixing berberine, evodiamine, arctigenin and tanshinone I according to different mass ratios of 6:1:6:6 to obtain a composition 4.

Example 5

Step two, combining the medicines: mixing berberine, evodiamine, arctigenin and tanshinone I according to different mass ratios of 6:1:1.5:5.5 to obtain a composition 5.

The inhibitory action of the present invention against helicobacter pylori is further illustrated in detail by the following examples

1. Material

1.1 sample

Compositions

1, 2, 3, 4, 5 were prepared using examples 1, 2, 3, 4, 5, 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 hopcaligenes are provided by the research center for preventing and treating drug-resistant microbial infection of the pharmaceutical institute of the Dejiangyuan.

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 chessboard method for detecting synergistic inhibition of helicobacter pylori by pairwise mixing of Chinese medicinal monomers (FIC, 100 μ L system)

(1) Preparing the ethanol solution of berberine, evodiamine, arctigenin and tanshinone I, wherein the concentration is 4mg/mL for later use.

(2) MIC plate preparation first step: on a 96-well plate, 167.2 mu L of culture medium is firstly added into B1 and A2 wells, then 12.8 mu L of antibacterial agent is respectively added, and 90 mu L of culture medium is added into the other wells, and the medicines are diluted by a multiple ratio from A2 to A12 and from B1 to H1 in sequence; the second step is that: adding 90 mu L of culture medium into A2-A12 and B1-H1 respectively, and carrying out the third step: first, longitudinal dilution from A to H, and then transverse dilution from 1 to 12.

(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) The inoculated bacterial suspension was added to each well (the concentration of bacterial suspension per well was about 1.0X 10) in an amount of 10. mu.L, except for the A1 well⁶CFU/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. Calculating FIC by using a formula, wherein the formula is FIC-MICA/A + MICB/B (FIC: partial antibacterial combination index), wherein A and B are respectively MIC values when two medicines are singly used, MICA and MICB are MIC values when the two medicines are combined, and FIC is less than or equal to 0.5, and the synergistic effect is judged; 0.5-FIC is less than or equal to 1; the FIC is more than 1 and less than or equal to 2, and the effect is judged to be irrelevant; FIC > 2 was judged as antagonistic, and the test was repeated 3 times per drug.

(6) As a result: the combination of evodiamine and berberine is synergistic, the combination of arctigenin and tanshinone I is irrelevant, and the combination of the rest is additive, and the results are shown in Table 1.

TABLE 1 checkerboard FIC (. mu.g/mL) assay

2.2 microdilution assay of the minimum inhibitory concentration (MIC, 100. mu.L system) of the composition on helicobacter pylori

(1) Preparing 4mg/mL ethanol solutions of berberine, evodiamine, arctigenin and tanshinone I, and mixing the berberine, the evodiamine, the arctigenin and the tanshinone I according to the mass ratio of 6:1:0.75:0.75, 6:1:1.5:1.5, 6:1:3:3, 6:1:6:6 and 6:1:1.5:5.5 respectively 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.

(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 result was judged to have the lowest drug concentration that completely inhibited bacterial growth in the wells as the MIC. 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 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: the inhibition effect on helicobacter pylori is most obvious when the ratio of berberine to evodiamine to arctigenin to tanshinone I is 6:1:3:3 (namely the composition 2), and the results are shown in Table 2.

TABLE 2 MIC test of compositions in different proportions

2.3 microdilution assay for minimal inhibitory concentration (MIC, 100. mu.L system) of 6:1:3:3 composition against helicobacter pylori of different origins

(1) Prepared by mixing berberine, evodiamine, arctigenin and tanshinone I4 mg/mL according to the ratio of 6:1:3:3 for standby

(6) As a result: the berberine, evodiamine, arctigenin and tanshinone I have obvious inhibition effect on sensitive and drug-resistant helicobacter pylori when the ratio of the berberine to the evodiamine to the arctigenin to the tanshinone I is 6:1:3:3, and the results are shown in a table 3.

TABLE 36 1:3:3 composition MIC assays

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, the same samples were replicated 3 times.

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

(4) Adding composition 3 in different proportions, and adding medicine without adding 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 3 an 8-fold MIC of the formulated 6:1:3:3 composition did not cause significant cell damage to Ges-1 and BGC823 cells, was not significantly different from that of the zuojin pill, and was less toxic, as shown in fig. 1.

2.2.2 animal toxicity test

(1) Preparation of mice: c57BL/6 mice were purchased at 6-8 weeks of age and randomly assigned to groups of 10 mice and negative control groups.

(2) Intragastric administration: the composition (6:1:3:3) is administered in a therapeutic dose of 7mg/kg, namely 70mg/kg for 3 consecutive days, 1 time per day; the negative control group was given PBS, and the frequency and amount were the same as those of the group administered.

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

(4) And (3) detecting the 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 are the damage conditions of the stomach, the liver, the spleen and the kidney of a normal mouse in a PBS perfusion group, E to H are the damage conditions of the stomach, the liver, the spleen and the kidney of a perfusion composition (6:1:3:3), and compared with the two groups, the prepared composition (6:1:3:3) is administrated in an amount which is 10 times of the treatment amount, has no obvious damage to the stomach, the liver, the spleen and the kidney of the mouse, and has very low toxicity, and the toxicity is shown in figure 2.

2.3 microdilution assay Minimum Inhibitory Concentration (MIC) of the formulated 6:1:3:3 composition for non-H.pylori

(1) Prepare 4mg/mL of the composition (6:1:3:3) and mix until needed.

(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 for adding bacteria and no drug.

(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 adjusted concentration OD600 of 0.5 (5X 10)⁶CFU/mL), diluted 1000-fold to 5X 10³CFU/mL, spare.

(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 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: the composition (6:1:3:3) has a poor inhibitory effect on most of non-helicobacter pylori bacteria, which indicates that the composition has a narrow antibacterial spectrum and strong specificity, and can specifically act on helicobacter pylori, and the results are shown in Table 3.

TABLE 3 formulation 6:1:3:3 composition for MIC (μ g/mL) detection of non-H.pylori

Note: 6:1:3: group 3 ">" means greater than 46.1+7.7+23.1

2.4 detection of drug resistance in the 6:1:3:3 formulated composition

(1) The H.pylori G27 strain was used to test the resistance of the forsythiaside derivatives. Firstly, the MICs of metronidazole and the composition (6:1:3:3) are respectively detected to be 2 mug/mL and 0.72+0.12+0.36+0.36 mug/mL, and induction is carried out by 1/4MIC concentration, and the detection is carried out once every 3 days for 24 days. The induction concentration is adjusted along with the change of MIC, for example, when the Metronidazole MIC 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 formulated 6:1:3:3 composition 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: experimental groups the successfully molded infectious group (BHK159) was equally divided into 5 groups of omeprazole + amoxicillin + clarithromycin (triple therapy group), omeprazole + composition 6:1:3:3(7mg/kg) and PBS, 10 per group; 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 the 2 nd day after drug withdrawal are weighed and the average body weight is calculated, blood is collected from eyeballs, neck is cut off and killed, stomach tissues are taken and crushed, and the separated culture and identification of H.pyrori and the planting amount are calculated, wherein the treatment effect of the composition group containing 6:1:3:3 is obviously superior to that of the triple therapy treatment and the Zuojin pill group, as shown in figure 4. And paraffin sections are carried out on partial stomach tissues, H & E staining, TUNEL immunohistochemistry and fluorescence immunoassay are carried out, the stomach tissue repair effect of the composition group with the ratio of 6:1:3:3 is good, and inflammatory factors are obviously reduced, as shown in figures 5 and 6.

Claims

1. A preparation method of a traditional Chinese medicine monomer composition for treating helicobacter pylori is characterized by comprising the following steps: a. dissolving the medicine: respectively dissolving berberine, evodiamine, arctigenin and tanshinone I in anhydrous ethanol; b. the combination medicine comprises the following components: mixing the prepared ethanol solution according to the mass ratio of berberine to evodiamine to arctigenin to tanshinone I of 6:1 (0.75-6) to obtain the composition.

2. The preparation method according to claim 1, wherein the mass ratio of the berberine, the evodiamine, the arctigenin and the tanshinone I is 6:1:3: 3.

3. A traditional Chinese medicine monomer composition prepared by the method of any one of claims 1-2.

4. The use of the monomeric composition of a Chinese medicinal material as claimed in claim 3 in the preparation of a medicament for the treatment of a disease associated with helicobacter pylori infection.

5. The medicament for treating the diseases related to helicobacter pylori infection is characterized in that the effective component is the traditional Chinese medicine monomer composition of claim 3.