CN113425725B

CN113425725B - Application of schizophyllum commune and its extract in preparation of anti-helicobacter pylori medicines

Info

Publication number: CN113425725B
Application number: CN202110812516.6A
Authority: CN
Inventors: 赵明; 段金廒; 徐明明; 周俊飞; 毕洪凯
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2022-05-10
Anticipated expiration: 2041-07-19
Also published as: CN113425725A

Abstract

Screening experiments show that the azaanthraquinone benzo [ g ] isoquinoline-5,10-dione and/or oxaanthraquinone 1-hydroxybenzoisohroman quinone separated from the dehiscent fruits have higher in vitro antibacterial activity than positive drugs metronidazole and levofloxacin and can be used for preparing the anti-helicobacter pylori drugs.

Description

Application of schizophyllum commune and its extract in preparation of anti-helicobacter pylori medicines

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of schizophyllum commune and extracts thereof, namely azaanthraquinone and oxaanthraquinone, in preparation of a helicobacter pylori resistant medicine.

Background

Helicobacter pylori (Hp) is a spiral and microaerophilic gram-negative bacillus, is planted on gastric mucosa to play a pathogenic role, and is closely related to various digestive system diseases such as chronic active gastritis, gastroduodenal ulcer, gastric mucosa-related lymphoid tissue lymphoma, gastric cancer and the like. The Hp infection rate of natural population in the world is over 50 percent, the infection rates of different countries are different, and the infection rate of developing countries is obviously higher than that of developed countries. According to the latest meta-analysis, the infection rate of Hp in Chinese population is 55.82%, the number of infected people is close to 8 hundred million people, and the infection rate increases by 0.5% -1% every year. A carcinogen list published by International cancer research institution of world health organization in 2017 shows that Hp belongs to I-type carcinogens and can induce the generation of tumors such as gastric cancer and lymphoproliferative gastric lymphoma. Currently, the clinical treatment scheme for Hp infection is triple or quadruple therapy, i.e. taking a proton pump inhibitor (omeprazole, etc.) together with two antibiotics (two selected from clarithromycin, amoxicillin, tetracycline, metronidazole, etc.) or a bismuth agent (bismuth potassium citrate, etc.) together with a proton pump inhibitor (omeprazole, etc.) together with two antibiotics (two selected from clarithromycin, amoxicillin, tetracycline, metronidazole, etc.). However, with the long-term use of antibiotics, Hp has different degrees of drug resistance to antibiotics, so that the eradication rate of related eradication therapy is remarkably reduced and the clinical requirement is far from being met. According to the report, the drug resistance rates of Hp to metronidazole, clarithromycin, levofloxacin, rifampicin, amoxicillin and tetracycline are 63.4%, 52.6%, 54.8%, 18.2%, 4.4% and 7.3% respectively, and the cure effects of the related drugs are not significant, so that a new high-efficiency and safe Hp-resistant drug is urgently found.

Mitracarpus hirtus (L.) DC (MH) is a plant of the genus Geotrichum of the family Rubiaceae, and is widely distributed in tropical countries. The fruit of Ficus Carica is used in Western nontraditional medicine for treating nervous system diseases such as toothache, amenorrhea, dyspepsia, liver disease, venereal disease, sore throat, dermatosis, surgical wound, leprosy and headache, and also used as antidote for curare. Modern researches show that MH has good antibacterial and antifungal effects, but reports and researches on helicobacter pylori are not found. The research of the invention finds that the extract of the dehiscent fruit has better inhibiting effect on the helicobacter pylori, the main effective substances for resisting the helicobacter pylori in the dehiscent fruit are benzog isoquinoline-5,10-dione (1) and 1-hydroxybenzoisoquinonone (2) through activity tracing and separation, and the bacteriostatic activity in vitro is stronger than that of positive drugs metronidazole and levofloxacin.

Disclosure of Invention

The invention aims to provide the application value of the schizophyllum commune and the extracts thereof, namely the azaanthraquinone and the oxaanthraquinone in the preparation of anti-helicobacter pylori medicaments, and the invention also aims to provide the preparation method of the azaanthraquinone and the oxaanthraquinone.

The technical scheme is as follows: the invention separates aza anthraquinone (g) isoquinoline-5,10-dione) and oxa anthraquinone (1-hydroxynaphthochrome-anthraquinone) from plant dehiscent fruit, and provides the preparation method of the compound.

Experiments show that the azaanthraquinone and the oxaanthraquinone have stronger inhibition effects on drug resistance and sensitivity of helicobacter pylori, have stronger in-vitro antibacterial activity than positive drugs of metronidazole and levofloxacin, and can be used for preparing the anti-helicobacter pylori drugs.

The compounds of the present invention have the structural formula:

the azaanthraquinone and oxaanthraquinone are prepared by the following method:

(1) weighing a schizocapsa medicinal material, crushing, adding an ethanol or methanol aqueous solution to soak the medicinal material at room temperature, filtering, collecting filtrate, and concentrating under reduced pressure until no alcohol smell exists to obtain a schizocapsa concentrated solution;

(2) extracting the schizocapsa fruit concentrated solution obtained in the step (1) with ethyl acetate, combining ethyl acetate extraction parts, and concentrating under reduced pressure to obtain an ethyl acetate part;

(3) putting the ethyl acetate part obtained in the step (2) on a silica gel column, and eluting by using mixed solvents of petroleum ether and ethyl acetate with different volume ratios;

(4) and (3) mixing the petroleum ether obtained in the step (3) in a volume ratio of 85: 15: eluting fraction Fr.2 with ethyl acetate mixed solvent, performing MCI reversed phase medium pressure gradient elution, and collecting eluate of 6.3-6.9BV and 8.1-8.7 BV; preparing oxaanthraquinone 1-hydroxybenzoisochromanol from the effluent of 6.3-6.9BV by preparative high performance liquid chromatography; preparing aza anthraquinone benzole [ g ] isoquinoline-5,10-dione from the effluent of 8.1-8.7BV by preparative high performance liquid chromatography;

。

preferably, the preparation method of the azaanthraquinone and the oxaanthraquinone provided by the invention is carried out according to the following steps:

(1) weighing 2.83kg of Mitracarpus hirtus L. (MH) medicinal material, crushing, soaking the medicinal material in 10 times of the weight of the medicinal material at room temperature by using 70-80% ethanol or methanol aqueous solution for three times, wherein each time lasts for 2-3 hours, filtering, collecting filtrate, and concentrating under reduced pressure until no alcohol smell exists to obtain MH concentrated solution;

(2) extracting the liquid medicine obtained in the step (1) by using ethyl acetate with the same volume, extracting for four times, combining the extracted ethyl acetate parts, and concentrating under reduced pressure to obtain the ethyl acetate part.

(3) Taking an ethyl acetate part obtained in the step (2), and mixing the ethyl acetate part with the sample: silica gel 1:1.2 was stirred, and a 2-fold amount of silica gel was loaded on a negative pressure column (30.0 cm. times.9.5 cm), and the column volume was 500mL, and eluted with a mixed solvent of petroleum ether and ethyl acetate. Adopting petroleum ether: eluting 5 column volumes with a mixed solvent of ethyl acetate 95:5 to obtain fraction Fr.1; adopting petroleum ether: eluting 4 column volumes with a mixed solvent of ethyl acetate 85:15 to obtain fraction Fr.2; adopting petroleum ether: eluting 5 column volumes with a mixed solvent of 70:30 ethyl acetate to obtain fraction Fr.3; adopting petroleum ether: eluting 6 column volumes with a mixed solvent of ethyl acetate 60:40 to obtain fraction Fr.4; adopting petroleum ether: eluting 6 column volumes with a mixed solvent of ethyl acetate (30: 70) to obtain fraction Fr.5; elution with ethyl acetate gave fraction fr.6 for 4 column volumes.

(4) Subjecting fraction Fr.2 obtained in step (3) to MCI (MCI particle size of 75-150 μm, 3.0cm × 60.0cm, column volume of 400mL) reverse phase medium pressure gradient elution, and collecting effluent of 6.3-6.9BV and 8.1-8.7 BV; preparing 1-hydroxybenzoisochromanoquinone from the effluent of 6.3-6.9BV by preparative high performance liquid chromatography; 8.1-8.7BV of effluent is treated by preparative high performance liquid chromatography to prepare benzog isoquinoline-5, 10-dione.

Preferably, said benzol [ g ] in step (4)]The preparation conditions of the isoquinoline-5,10-dione and 1-hydroxybenzoisohromanone medium pressure preparative chromatography are as follows: gradient elution with pure water (a) -methanol (B) as mobile phase, elution gradient: 0.01-10.00 min, 30% -30% B; 10.00-25.00 min, 40% -40% B; 25.00-40.00 min, 50% -50% B; 40.00-60.00 min, 60% -60% B; 60.00-80.00 min, 70% -70% B; 80.00-100.00 min, 80% -80% B; 100.00-120.00 min, 90% -90% B; 120.00-130.00 min, 0% -100% B; 130.00-140.00 min; collecting 1-hydroxybenzoisohromanquinone stock solution in 62-72min, and collecting benzol [ g ] in 95-104 min]Stock solutions of isoquinoline-5, 10-dione. benzol [ g ]]The isoquinoline-5,10-dione stock solution is subjected to preparative high performance liquid chromatography to obtain a monomeric compound (H)₂MeOH 32:68), 1-hydroxybenzoisochromanoquinone stock solution is prepared to form highObtaining the monomeric compound (H) by liquid chromatography₂O: MeOH 55: 45); the detection wavelengths are both 250nm and 310 nm.

A pharmaceutical composition comprises Ficus Carica extract or azaanthraquinone (g) isoquinone-5, 10-dione) and oxaanthraquinone (1-hydroxybenzoisohrom-anthraquinone) and pharmaceutically acceptable adjuvants. The pharmaceutically acceptable auxiliary materials are selected from one or more of starch, dextrin, sucrose, lactose, vegetable oil, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, crospovidone, sodium hydroxymethyl starch, polyethylene glycol, polyvinylpyrrolidone, magnesium stearate, vitamin powder silica gel, glucose, mannitol and xylitol.

A pharmaceutical preparation is prepared from the above medicinal composition and pharmaceutically acceptable adjuvants by making into granule, tablet, hard capsule, soft capsule, drop, oral liquid, aerosol, nasal drop or injection.

Has the advantages that:

(1) the present invention provides a method for preparing aza anthraquinone (g) isoquinoline-5,10-dione and oxaanthraquinone (1-hydroxybenzoisochrom-anthraquinone) with anti-helicobacter pylori action.

(2) The invention discovers that the schizocapsa extract (aza- (g) isoquinoline-5,10-dione) and oxa-anthraquinone (1-hydroxynaphthochrome-anthraquinone) have better inhibition effect on helicobacter pylori through a large number of experimental screens.

(3) The invention discovers for the first time that the compounds benzoquinoline-5, 10-dione and 1-hydroxybenzoisohromanone have stronger inhibition effect on drug resistance and sensitive helicobacter pylori, and the in vitro antibacterial activity is stronger than that of positive drugs metronidazole and levofloxacin, so that the invention is a novel anti-helicobacter pylori drug.

Detailed Description

Example 1

1. Apparatus and materials

1.1 instruments

1.2. Experimental Material

Hedera ODS preparative chromatography column (10nm, 5 μm, 10 x 250 mm); waters ACQUITY UPLC BEH C₁₈(2.1 mm. times.100 mm, 1.7 μm) chromatography column; MCI GEL (CHP 20/P120), MCI (CHP20,75-150 mu m), GEL Sephadex LH-20, column chromatography silica GEL (200-300 meshes); chromatographic grade acetonitrile, methanol and formic acid were purchased from merck, usa, analytical grade petroleum ether, ethyl acetate, etc. were purchased from south beijing chemicals, inc.

2. Experimental methods

2.1 preparation of dehiscent fruit samples

(4) Subjecting the fraction Fr.2 obtained in step (3) to MCI reversed-phase medium-pressure preparation (MCI particle size is 75-150 μm, 3.0cm × 60.0cm, column volume is 400mL), gradient elution, and eluting with methanol-pure water as mobile phase, wherein A is pure water, and B is methanol; elution gradient: 0.01-10.00 min, 30% -30% B; 10.00-25.00 min, 40% -40% B; 25.00-40.00 min, 50% -50% B; 40.00-60.00 min, 60% -60% B; 60.00-80.00 min, 70% -70% B; 80.00-100.00 min, 80% -80% B; 100.00-120.00 min, 90% -90% of B; 120.00-130.00 min, 0% -100% B; 130.00-140.00 min. The elution flow rate is 20mL/min, and the detection wavelengths are 250nm and 310 nm; after high performance liquid chromatography, 8 fractions Fr.2-1 to Fr.2-8 are obtained by concentration and combination by a rotary evaporator.

Fr.2-4 (effluent of 6.3-6.9 BV) passes through C₁₈Preparative high performance liquid chromatography (Hedera ODS,5 μm, 20X 250mm) with a gradient elution using pure water (A) -methanol (B) as the mobile phase, gradient elution: 0.01-54.00 min, 60-75%, collecting 1-hydroxybenzoisochromanol in 35-39 min; subjecting the stock solution to preparative high performance liquid chromatography to obtain monomer compound 1-hydroxybenzoisochromanoquinone (H)₂O, MeOH is 64:36), the pressure of the high performance liquid chromatography column is 9.8MPa, the column temperature is 22-26 ℃, the sample injection amount is 120 mu L, the flow rate is 5mL/min, and the detection wavelength is 250nm and 310 nm.

Fr.2-6 (effluent of 8.1-8.7 BV) pass through C₁₈Preparative high performance liquid chromatography (Hedera ODS,5 μm, 20X 250mm) with a gradient elution using pure water (A) -methanol (B) as the mobile phase, gradient elution: 0.01-60.00 min, 65-80%, collecting benzol [ g ] in 43-47min]isoquinoline-5,10-dione stock solution; the stock solution is processed by preparative high performance liquid chromatography to obtain a monomeric compound benzog]isoquinoline-5,10-dione (H₂O, MeOH 70:30), the high performance liquid chromatography column pressure is 9.8MPa, the column temperature is 22-26 ℃, the sample injection amount is 120 mu L, the flow rate is 5mL/min, and the detection wavelength is 250nm and 310 nm.

Identification of the compounds benzoquinoline-5, 10-dione and 1-hydroxybenzoisohromone: the compound 1 is benzo [ g ] isoquinoline-5, 10-diketone and the compound 2 is 1-hydroxybenzoishrome anthraquinone which are identified by nuclear magnetic resonance and high resolution mass spectrum analysis. The compound structure is analyzed as follows:

compound 1 is a pale yellow powder, HR-ESI-MS M/z 210.0541[ M + H [ ]]⁺。¹H-NMR(500MHz, Chloroform-d)δ:9.58(1H,s,H-1),9.11(1H,d,J＝5.0Hz,H-3),8.10(1H,d,J＝5.0 Hz,H-4),8.33(2H,ddd,J＝9.0,7.6,4.1Hz,H-6,9),7.87(2H,m,H-7,8)。¹³C-NMR (125MHz, Chloroform-d) delta: 155.3(C-1),149.7(C-3),119.3(C-4),126.5(C-4a), 182.5(C-5),133.2(C-5a),135.3(C-6),127.5(C-7),127.6(C-8),134.8(C-9),133.2 (C-9a),182.6(C-10),138.7(C-10 a). The above data are essentially consistent with literature reports, and compound 1 was identified as benzog]isoquinoline-5,10-dione。

Compound 2 is a pale yellow powder, HR-ESI-MS M/z 213.0666[ M + H ]]⁺。¹H-NMR(500 MHz,Chloroform-d)δ:5.39(1H,t,J＝3.6Hz,H-1),4.77(1H,dt,J＝18.5,3.1Hz, H-3a),4.63(1H,dt,J＝18.6,2.3Hz,H-3b),2.78(1H,dq,J＝18.7,3.3Hz,H-4a), 2.66(1H,m,H-4b),7.69(2H,m,H-6,9),8.03(2H,m,H-7,8)。¹³C-NMR (125MHz, Chloroform-d) delta 90.5(C-1),57.8(C-3),28.2(C-4),139.7(C-4a),183.7(C-5),131.8 (C-5a),141.5(C-6),126.2(C-7),126.5(C-8),133.9(C-9),132.0(C-9a),183.3(C-10), 133.9(C-10 a). The above data are essentially consistent with literature reports, and compound 2 was identified as 1-hydroxybenzoisochromanone.

EXAMPLE 2 anti-helicobacter pylori test

1. Material

1.1 sample

Compound 1: benzoquinone [ g ] 5,10-dione (purity > 98%) and compound 2: 1-hydroxybenzoisochrom-anthraquinone (purity greater than 98%) by experimental preparation.

1.2 strains

Helicobacter pylori G27, Hp159 and JRES00015 were provided by professor BihongKai, university of Nanjing medical science.

1.3 reagents

Media and main reagents: columbia culture medium, brain heart infusion culture medium, selective antibiotics (metronidazole, levofloxacin, polymyxin B and trimethoprim), serum, omeprazole, amoxicillin, clarithromycin, gram staining solution, bacterial genome DNA extraction kit, CagA gene primer specific to helicobacter pylori and the like.

2. Method and results

2.1 microdilution assay of minimal inhibitory concentration (MIC, 100. mu.L system) of Compounds 1 and 2 against helicobacter pylori

(1) The concentrations of the samples of the compound 1 and the compound 2 were 2 mg/mL.

(2) Preparing a MIC plate, namely adding 173.6 mu L of culture medium into a first well, adding 6.4 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. The drug concentrations in the 1 st to 7 th wells are 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 μ g/mL, respectively.

(5) The results judged the MIC as the lowest drug concentration that completely inhibited bacterial growth in the wells. The test is meaningful when the bacteria grow significantly in the 8 th well (i.e., no antibiotic) of the positive control well. 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 results are shown in Table 1.

TABLE 1 minimal inhibitory concentration (MIC: μ g/mL) of Compounds 1 and 2 against helicobacter pylori

Bacterial strains	G27	Hp159	JRES00015
				Compound 1	0.125	0.0625	0.0625
Compound 2	0.125	0.125	0.0625
				MTZ	2	16	16
CLR	0.008	2	2
				LVX	0.25	8	16

MTZ: a nail polish file; CLR; clarithromycin; LVX: levofloxacin.

The experimental results show that the compounds of benzog isoquinoline-5,10-dione and 1-hydroxybenzoisohromanquinone have stronger inhibition effect on drug resistance and sensitive helicobacter pylori, the in vitro antibacterial activity is stronger than that of positive drugs of metronidazole and levofloxacin, and a novel anti-helicobacter pylori drug can be developed.

The invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent substitutions are within the scope of the invention as claimed.

Claims

1. Use of an azaanthraquinone (g) isoquinoline-5,10-dione and/or oxaanthraquinone 1-hydroxyquinophthalone in the preparation of a medicament against helicobacter pylori.

2. The use according to claim 1, wherein the azaanthraquinone benzo [ g ] isoquinone-5, 10-dione and/or oxaanthraquinone 1-hydroxynaphthoquinonoid is formulated with pharmaceutically acceptable excipients into granules, tablets, capsules, drops, oral liquids, aerosols or injections.