CN114984177A

CN114984177A - Application of dried ginger in preparation of helicobacter pylori urease inhibitor

Info

Publication number: CN114984177A
Application number: CN202210694711.8A
Authority: CN
Inventors: 李彩兰; 鲁强
Original assignee: Zhuhai Campus Of Zunyi Medical University
Current assignee: Zhuhai Campus Of Zunyi Medical University
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2022-09-02

Abstract

The invention relates to the technical field of medicines, and discloses application of dried ginger in preparation of a helicobacter pylori urease inhibitor. The invention provides a new application of dried ginger as a helicobacter pylori urease inhibitor in the field of medicines, and the dried ginger can be used for preparing medicines for treating and preventing gastritis, gastric ulcer and other stomach related diseases. Meanwhile, the dried ginger is used as the urease inhibitor of the helicobacter pylori, so that the selectable types of the existing urease inhibitor are enriched, and the drug resistance of the drug is reduced. In addition, the dried ginger is a natural Chinese medicinal material and has the characteristics of high efficiency and no toxicity, so the dried ginger has a good development prospect in the aspect of inhibiting the urease activity of helicobacter pylori.

Description

Application of dried ginger in preparation of helicobacter pylori urease inhibitor

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of dried ginger in preparation of a helicobacter pylori urease inhibitor.

Background

Helicobacter pylori (Helicobacter pylori) is one of the most common infectious pathogens worldwide, with over 50% of the population infecting worldwide. Helicobacter pylori infection is not only a main cause of peptic ulcer and chronic gastritis, but also closely related to the occurrence of gastric mucosa-associated lymphoma and gastric adenocarcinoma. Helicobacter pylori is the only species of microorganism known to be able to survive in the human stomach, although it is a bacterium that is very demanding on growth conditions. The stomach is a strong acid environment, the main reason that helicobacter pylori can survive in the stomach is urease activity, the pH value can be increased by hydrolyzing ammonia released by urea by the urease, and recent research shows that urea molecules in a receptor structure are key factors for sensing the helicobacter pylori and avoiding the gastric acid environment. The urease thus acts to create a suitable microenvironment for H.pylori. The pathogenic bacteria with urease activity either hydrolyze urea by urease to generate ammonia to provide a nitrogen source for own vital activities or utilize the alkalinity of ammonia to provide a proper microenvironment for the survival. However, the release of ammonia can cause cytotoxicity, inflammation or ulcer, and also can cause hepatic encephalopathy and the like due to hyperammonemia, and in addition, urease can stimulate the oxidative burst of human neutrophils through immune reaction to generate ammonia chloride to participate in cell damage and canceration induction, so that the urease is an important virulence factor of the pathogenic bacteria. Therefore, the urease activity is blocked, and the germs can be effectively inhibited and even killed, so that the aim of treating the diseases is fulfilled. Meanwhile, virulence factors are not essential for the survival of bacteria like DNA, protein and the like, so compared with traditional antibiotics, the virulence factors inhibiting bacteria are less prone to develop drug resistance, and the superiority shows that urease inhibitors can possibly become first-line drugs for treating the diseases.

At present, researchers at home and abroad research and use urease inhibitors for prevention and treatment aiming at the serious harm of urease to human production and life. Heretofore, urease inhibitors have been widely used in the fields of medicine, agriculture, animal husbandry and the like, and the types thereof mainly include urea analogues, hydroxamic acids, heavy metal ions, phosphoramides, quinones, polyphenols and the like. However, most of the existing urease inhibitors have the problems of poor stability, short action time, obvious toxic and side effects and the like, so that the practical application has certain limitation. Therefore, researches for screening safe, efficient and pollution-free urease inhibitors from natural medicines are receiving much attention.

Disclosure of Invention

The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides an application of dried ginger in preparing a helicobacter pylori urease inhibitor. The dried ginger has good effect of inhibiting helicobacter pylori urease, has the characteristics of low price, easy obtainment, safety, no toxicity and the like, has good application value and development prospect, can be used as the helicobacter pylori urease inhibitor, can be widely used for treating stomach diseases related to helicobacter pylori, such as peptic ulcer, gastritis, gastric mucosal lymphoma and the like, and further reduces the occurrence probability of drug resistance.

The invention provides an application of dried ginger in preparing a helicobacter pylori urease inhibitor in a first aspect.

The dried ginger is a traditional Chinese medicinal material used as both medicine and food, is a dried rhizome of Zingiber of ficnale Rosc of Zingiberaceae, and is mainly used for treating the symptoms of abdominal psychroalgia, vomiting and diarrhea, cold limbs and slight pulse, cold drink, cough and asthma and the like. Modern pharmacological research shows that the dried ginger has the functions of analgesia, anti-inflammation, anti-tumor and the like. The invention firstly discovers that the dried ginger has the function of inhibiting the urease activity of the helicobacter pylori, so the dried ginger can be applied to preparing the urease inhibitor of the helicobacter pylori.

In a second aspect, the present invention provides a helicobacter pylori urease inhibitor.

Specifically, the helicobacter pylori urease inhibitor comprises a dried ginger extract.

Preferably, the dried ginger extract is obtained by water extraction.

More specifically, the preparation method of the dried ginger extract comprises the following steps:

pulverizing Zingiberis rhizoma, soaking in water, reflux-extracting, and filtering to remove residue to obtain the Zingiberis rhizoma extract.

Preferably, the mass ratio of the dried ginger to the water is 1: (5-20).

Preferably, the soaking time is 0.2-1 h. The active ingredients in the dried ginger can be fully extracted by soaking and reflux extraction.

Preferably, the reflux extraction time is 1-3 hours.

Preferably, the helicobacter pylori urease inhibitor further comprises pharmaceutically acceptable salts or auxiliary materials of the dried ginger extract.

Preferably, the pharmaceutically acceptable adjuvant comprises at least one of a filler, a lubricant, a disintegrant, a buffer, a cosolvent, an antioxidant, an emulsifier, a binder, or a suspending agent.

Preferably, the helicobacter pylori urease inhibitor is prepared in the form of tablets, capsules, granules or coated pills.

The third aspect of the invention also provides the application of the helicobacter pylori urease inhibitor in preparing the medicine for treating stomach diseases.

Preferably, the gastric disease comprises gastritis, gastric ulcer or gastric cancer.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new application of dried ginger as a helicobacter pylori urease inhibitor in the field of medicines, and the dried ginger can be used for preparing medicines for treating and preventing gastritis, gastric ulcer and other stomach related diseases. Meanwhile, the dried ginger is used as the urease inhibitor of the helicobacter pylori, so that the selectable types of the existing urease inhibitor are enriched, and the drug resistance of the drug is reduced. In addition, the dried ginger is a natural Chinese medicinal material and has the characteristics of high efficiency and no toxicity, so the dried ginger has a good development prospect in the aspect of inhibiting the urease activity of helicobacter pylori.

Drawings

FIG. 1 shows urease inhibitory activity of the extract of dried ginger prepared in example 1;

FIG. 2 is a Lineweaver-Burk reciprocal plot of urease inhibiting activity of the dried ginger extract prepared in example 1;

FIG. 3 is a plot of intercept versus inhibitor concentration for the curves in the Lineweaver-Burk Bireciprocal plot of urease inhibiting activity of the dried ginger extract prepared in example 1;

FIG. 4 is a bar graph showing the effect of sulfhydryl containing compounds on dried ginger to inhibit urease activity;

FIG. 5 is a bar graph showing the effect of the order of addition of sulfhydryl compound, Zingiberis rhizoma and urease on the activity of urease.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1

Preparing a dried ginger extract: taking dried rhizoma Zingiberis, pulverizing into powder, weighing 50g, adding 10 times of distilled water, soaking for 0.5 hr, heating under reflux for 2 hr, filtering while hot, collecting filtrate, extracting the residue once again by the same operation, mixing the filtrates, concentrating the filtrate under reduced pressure in a rotary evaporator to obtain concentrated solution of rhizoma Zingiberis extract, and storing at 4 deg.C in dark place for use.

The extract can be used as urease inhibitor of helicobacter pylori.

Product effectiveness testing

1. Experimental materials

(1) Test drug

Urea, dithiothreitol and cysteine are all purchased from Solibao; glutathione was purchased from melphalan organisms; sodium fluoride and boric acid were purchased from mcelin. HEPES, sodium salicylate, sodium nitroprusside, sodium hydroxide, sodium hypochlorite and glycerol are purchased from Guangzhou chemical reagent factories and are all analytically pure.

(2) Culture of helicobacter pylori

At 37 ℃ with 5% O ₂ 、10％CO ₂ And 85% N ₂ The culture medium is Brookfield broth containing 10% inactivated horse serum, and the helicobacter pylori is cultured for 24h under the micro-aerobic condition. After 3 days of resuscitative culture, the identified and non-contaminated helicobacter pylori was subcultured: the bacterial colonies were scraped from the mother plate with an inoculating loop, inoculated on a fresh culture plate, gently and uniformly spread on the whole plate, and then subjected to canning culture for 3 days.

(3) Extraction of helicobacter pylori urease

Centrifuging 50mL of helicobacter pylori liquid at 5000g and 4 ℃, collecting helicobacter pylori, washing twice with phosphate buffer solution with pH of 7.4, depositing the helicobacter pylori at-80 ℃, preserving for 24h, taking out, standing to room temperature, adding 3mL of distilled water and protease inhibitor, performing ultrasonic treatment for 1min, centrifuging at 15000g and 4 ℃ for 10min, taking out supernatant, dialyzing and desalting to obtain a helicobacter pylori urease solution, adding glycerol with the same volume, and storing at 4 ℃.

(4) Preparation of dried ginger extract

The concentrated solution of the dried ginger extract prepared in example 1 was diluted with 20mM HEPES buffer solution (pH 7.5) to prepare solutions having different concentrations.

(5) Preparation of urea

An appropriate amount of urea was weighed, dissolved in 20mM HEPES buffer (pH 7.5) to prepare a 150mM urea solution, and stored in a refrigerator at 4 ℃ for further use.

(6) Preparation of Bertholot color developing solution

Solution A: respectively weighing appropriate amount of sodium nitroprusside and sodium salicylate powder, dissolving in 20mM HEPES buffer solution (pH 7.5) to prepare a developing solution A containing 9.73mM sodium nitroferricyanide and 700mM sodium salicylate, and storing at 4 deg.C in dark place for later use.

And B, liquid B: weighing 9g of sodium hydroxide, dissolving in 20mM HEPES buffer solution (pH 7.5), cooling, adding 12mL of sodium hypochlorite, mixing, diluting to 50mL, and storing at 4 ℃ in a dark place for later use.

2. Test

Test 1: dry ginger urease inhibition test of helicobacter pylori

Uniformly mixing helicobacter pylori urease solution with a certain concentration and rhizoma Zingiberis extract solution with a series of concentrations, incubating at 37 deg.C for 20min, adding urea solution at room temperature, reacting in dark for 20min, adding Bertholt color developing solution, developing in dark for 10min, sucking 200 μ L of incubation solution onto 96-well plate, and measuring OD at 595nm with microplate reader _{Absolute value} Value, find the corresponding OD _{Relative to each other} Value, blank pair with solvent added to materialIn parallel, 3 times were measured. Determining the corresponding OD according to equation 1 _{Relative to each other} The value is obtained. The residual enzyme activity is determined according to equation 2 and the corresponding half-inhibitory concentration IC is determined from the concentration-residual enzyme activity curve ₅₀ Expressed as mean ± standard error.

OD _{Relative to each other} ＝OD _Absolute -OD _{Blank space} (formula 1)

Residual enzyme activity (%) ═ OD _{Relative value} ^{Test article} /OD _{Relative to each other} ^{Blank space} X 100% (formula 2)

The results of the inhibition of the urease activity of helicobacter pylori by the dried ginger extract prepared in example 1 are shown in FIG. 1, and it can be seen from FIG. 1 that the half Inhibitory Concentration (IC) of the dried ginger extract against the urease activity of helicobacter pylori ₅₀ ) It was 7.17. + -. 0.59 mg/mL.

Test 2: study on type of inhibition of helicobacter pylori urease by dried ginger

Mixing helicobacter pylori urease at a certain concentration with a series of concentrations of Zingiberis rhizoma extract (0, 6, 12, 24mg/mL) prepared in example 1, and incubating at 37 deg.C for 20 min. Then adding a series of urea solutions (0.469-15mM) at room temperature for reaction in dark for 20min, and then developing to obtain OD according to the method of test 1 _Absolute Value and finding the corresponding OD _{Relative to each other} The value is obtained. The solvent to which the substance was added was used as a blank and 3 replicates were run. The experiment was conducted by counting the reciprocal of the reaction rate (1/v, i.e., 1/OD) _{Relative to each other} ) Reciprocal of substrate concentration (1/[ urea)]) Making a Lineweaver-Burk diagram, and finally obtaining a kinetic parameter K through an L-B curve combined with a formula 3 _M 、v _max And obtaining the inhibition constant K by secondary plotting through an L-B curve _is 。

The results are shown in FIGS. 2 and 3. As can be seen from the Lineweaver-Burk double reciprocal plot of FIG. 2, the kinetic parameter K of Zingiberis rhizoma for inhibiting urease of helicobacter pylori increases with the concentration of Zingiberis rhizoma _M And v _max All decreased, and it was therefore preliminarily concluded that the type of action of Zingiberis rhizoma against helicobacter pylori urease was of the antiparasitic inhibition type. In addition, Lineweaver-Burk double reciprocal plot was plotted twice for the type of urease inhibiting helicobacter pylori combined with Zingiberis rhizoma to obtain FIG. 3, which shows that the Zingiberis rhizoma extract inhibits the urease LineweIntercept of the curve in the aver-Burk Biobloid plot is plotted against inhibitor concentration. FIG. 3 shows that the inhibition constant K of Zingiberis rhizoma against urease of helicobacter pylori can be determined _is It was 1.60. + -. 0.32 mg/mL.

Test 3: influence of sulfhydryl-containing compound on inhibiting helicobacter pylori urease activity of rhizoma Zingiberis

Mixing helicobacter pylori urease solution, sulfhydryl compound-containing solution (dithiothreitol, cysteine or glutathione solution, concentration is 1.25mM) and 24mg/mL Zingiberis rhizoma solution, pre-incubating at 37 deg.C for 20min, adding urea at room temperature for reaction for 20min, and developing to obtain OD according to test 1 _Absolute Value and finding the corresponding OD _{Relative to each other} And the corresponding residual enzyme activity was determined. The solvent to which the substance was added was used as a blank and 3 replicates were run.

As a result, as shown in FIG. 4, when a mercapto compound (dithiothreitol, cysteine or glutathione) is contained in the reaction system, the helicobacter pylori urease can maintain a high enzyme activity even in the presence of dried ginger, and it can be presumed that one of the sites of action of dried ginger for inhibiting the helicobacter pylori urease activity is a mercapto group in the urease amino acid series.

Test 4: interaction test of Zingiberis rhizoma extract, sulfhydryl group, helicobacter pylori urease

Mixing urease solution of helicobacter pylori, sulfhydryl compound solution (dithiothreitol, cysteine or glutathione solution, concentration is 1.25mM) and 24mg/mL Zingiberis rhizoma solution, pre-incubating at 37 deg.C for 20min, adding the third solution, mixing, incubating at 37 deg.C for 20min, adding urea to the co-incubated solution at room temperature for 20min, and developing to obtain OD according to test 1 _Absolute Value and finding the corresponding OD _{Relative to each other} And the corresponding residual enzyme activity was determined. A blank of HEPES (high Performance particulate air) as a solvent to which the substance was added was used, and 3-fold measurements were carried out.

As shown in FIG. 5, the activity of helicobacter pylori urease was closely related to the order of addition of the thiol-group-containing compound, helicobacter pylori urease, and the extract of dried ginger. Firstly, reacting a sulfhydryl protective agent with urease, and adding a dried ginger extract ((urease + sulfhydryl) + dried ginger group), wherein the residual enzyme activity of the urease is highest; reacting a sulfhydryl protective agent with a dried ginger extract, and then adding urease ((dried ginger + sulfhydryl) + urease), wherein the residual enzyme activity of the urease is inferior; if the thiol-protecting agent ((urease + Zingiberis rhizoma) + thiol group) is added after the Zingiberis rhizoma extract has reacted with urease for a certain period of time, the residual enzyme activity of urease is the lowest.

The experiments show that the dried ginger can obviously inhibit the activity of helicobacter pylori urease. In addition, the research on the action mechanism of the dried ginger for inhibiting the helicobacter pylori urease proves that the dried ginger is an anti-competitive urease inhibitor which mainly inhibits the activity of the urease by acting on a sulfhydryl active group in the urease active site of the helicobacter pylori. The research on the mechanism can further prove that the dried ginger has the function of resisting helicobacter pylori urease and has obvious effect. The dried ginger has good application value and development prospect in the aspect of preparing the anti-helicobacter pylori medicament, is a potential urease inhibitor and has the effect of treating helicobacter pylori related gastrointestinal tract diseases.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Zingiberis rhizoma in preparing urease inhibitor of helicobacter pylori is provided.

2. A helicobacter pylori urease inhibitor is characterized by comprising an extract of dried ginger.

3. The helicobacter pylori urease inhibitor according to claim 2, wherein the dried ginger extract is obtained by water extraction.

4. The helicobacter pylori urease inhibitor according to claim 3, wherein the dried ginger extract is prepared by the following method:

5. The helicobacter pylori urease inhibitor according to claim 4, wherein the mass ratio of the dried ginger to the water is 1: (5-20).

6. The helicobacter pylori urease inhibitor according to claim 4, wherein the soaking time is 0.2-1 h; the reflux extraction time is 1-3 hours.

7. The helicobacter pylori urease inhibitor according to claim 2 or 3, wherein the helicobacter pylori urease inhibitor is formulated in the form of a tablet, capsule, granule or coated pill.