Chromone trimer for inhibiting activity of helicobacter pylori, preparation method and application thereof

Abstract

The present application relates to the technical field of natural medicine, in particular to a kind of chromone trimer for inhibiting the activity of helicobacter pylori and preparation method and application, and its technical points are: the present application provides the compound for treating and / or inhibiting the activity of helicobacter pylori, with the structure shown in formula I, belongs to the novel chromone trimer of structure.Inhibition helicobacter pylori production is evaluated by MIC and MBC determination method, and the compound shown in formula I can inhibit the production of helicobacter pylori.Therefore, it is shown that the compound provided by the present application can be used to prepare the medicine or health product for treating and / or inhibiting the activity of helicobacter pylori, and has broad development and application prospect.

Description

Technical Field

[0001] This invention patent relates to the field of natural medicine technology, specifically to a chromone trimer that inhibits the activity of Helicobacter pylori, its preparation method, and its application. Background Technology

[0002] Chronic gastritis is a common digestive system disease, primarily characterized by chronic inflammation of the gastric mucosa. According to the World Health Organization, the global incidence of stomach diseases is as high as 80%, with chronic gastritis being the most common, accounting for 50% of all stomach diseases. Studies have shown that 80%–95% of patients with chronic gastritis have Helicobacter pylori infection in their gastric mucosa. Helicobacter pylori Helicobacter pylori (Hp) infection is the most significant causative factor of gastritis and a crucial, controllable risk factor for preventing gastric cancer. Almost all individuals currently infected with Hp exhibit varying degrees of gastric mucosal inflammation. Therefore, the search for natural, safe, and highly effective Hp inhibitors has become a research hotspot.

[0003] Agarwood belongs to the genus Aquilaria in the family Thymelaeaceae. Aquilaria Or agarwood ( ) Gyrinops Agarwood is a type of wood formed from the resin accumulated and secreted by plants after they have been damaged by external factors. Known as the "King of Fragrances," agarwood is mainly distributed in Guangdong, Guangxi, Taiwan, and Fujian provinces of my country. It possesses properties that promote qi circulation and relieve pain, warm the stomach and stop vomiting, and regulate qi and relieve asthma. It is used for chest and abdominal distension and pain, stomach cold with vomiting and hiccups, and kidney deficiency with shortness of breath. It is a commonly used and precious traditional Chinese medicine for treating stomach ailments. The main characteristic components of agarwood are sesquiterpenes and 2-(2-phenylethyl)chromone compounds, which have pharmacological effects such as anti-inflammatory, antibacterial, anti-gastric ulcer, gastrointestinal motility promotion, anti-tumor, and cell-activating properties.

[0004] Currently, there are no reports in the prior art regarding the isolation of compounds that inhibit the activity of Helicobacter pylori from Philippine agarwood. Therefore, this application provides a chromone trimer that inhibits the activity of Helicobacter pylori, its preparation method, and its application. Summary of the Invention

[0005] The purpose of this invention is to solve the existing technical problems mentioned in the background section, and to provide a chromone trimer that inhibits the activity of Helicobacter pylori, its preparation method, and its application.

[0006] The above-mentioned objective of the present invention is achieved as follows:

[0007] One aspect of this invention provides a chromone trimer that inhibits the activity of Helicobacter pylori, wherein the chromone trimer is a compound comprising the following general formula I:

[0008]

[0009] The present application also provides a preparation method of the chromone trimer for inhibiting the activity of Helicobacter pylori, comprising the following steps:

[0010] S1, crushing the Philippines Aquilaria sinensis, and extracting 5 times with 5 L of ethanol under heating and reflux to obtain an extraction liquid, which is combined and concentrated into an ethanol extract;

[0011] S2, preparing a suspension of the ethanol extract and water at a volume ratio of 1:1, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and then concentrating each extract to obtain an extract;

[0012] S3, taking the ethyl acetate extract to pass through a normal pressure silica gel column under reduced pressure to elute with chloroform-methanol (1:0→0:1, V / V) to obtain 14 flow parts, denoted as Fr.1 ~ Fr.14;

[0013] S4, performing liquid chromatography-mass spectrometry analysis on the components of Fr.1 ~ Fr.14, and finding that Fr.13 contains chromone polymers with a molecular weight of 800-1000 Da;

[0014] S5, taking the flow part Fr.13 to pass through a silica gel column to elute with chloroform-methanol (100:1→0:1, V / V) by gradient elution to obtain 28 flow parts, denoted as Fr.13-1 ~ Fr.13-28;

[0015] S6, taking the flow part Fr.13-16 to pass through a Sephadex LH-20 gel column to elute with chloroform:methanol (1:1) to obtain 6 flow parts, denoted as Fr.13-16-1 ~ Fr.13-16-6;

[0016] S7, taking the flow part Fr.13-16-3 to pass through a Sephadex LH-20 gel column to elute with chloroform:methanol (1:1) to obtain 3 flow parts, denoted as Fr.13-16-3-1 ~ Fr.13-16-3-3;

[0017] S8, taking the flow part Fr.13-16-3-2 to pass through a silica gel column to elute with chloroform-methanol (25:1→0:1, V / V) to obtain 5 flow parts, denoted as Fr.13-16-3-2-1 ~ Fr.13-16-3-2-5;

[0018] S9, taking the flow part Fr.13-16-3-2-3 to perform semi-preparative high performance liquid chromatography to obtain 5 fractions Fr.13-16-3-2-3-1 ~ 13-16-3-2-3-5;

[0019] S10, eluting the flow fraction Fr.13-16-3-2-3-5 by semi-preparative high performance liquid chromatography to obtain a compound of formula I with a molecular weight between 800-900.

[0020] Further, the conditions of the high performance liquid chromatography in step S9 are as follows: C18 column; methanol / water, 58:42, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm.

[0021] Further, the conditions of the high performance liquid chromatography in step S10 are as follows: πNAP column; methanol / water, 68:32, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm.

[0022] Another aspect of the present application provides a preparation method of the chromone trimer for inhibiting the activity of Helicobacter pylori, and application of the chromone trimer prepared by the preparation method in the preparation of a product for inhibiting the activity of Helicobacter pylori.

[0023] Another aspect of the present application provides application of the chromone trimer or the chromone trimer prepared by the preparation method in the preparation of a medicine or health product for treating and / or relieving a disease related to Helicobacter pylori production.

[0024] Further, the disease related to Helicobacter pylori production includes one or more of chronic gastritis, atrophic gastritis and gastric ulcer.

[0025] Another aspect of the present application provides a product for inhibiting Helicobacter pylori production, which is a medicine or health product.

[0026] Further, the product includes the above-mentioned chromone trimer compound or the chromone trimer prepared by the above-mentioned preparation method and a pharmaceutically or health product acceptable adjuvant.

[0027] Further, the dosage form of the medicine is tablet, capsule, powder injection or suspension.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The group of compounds provided by the present application for treating and / or inhibiting the activity of Helicobacter pylori has the structure shown in formula I, which belongs to a novel chromone trimer. The compound shown in formula I provided by the present application can inhibit the production of Helicobacter pylori, and therefore, it is indicated that the above-mentioned compound provided by the present application can be used for preparing a medicine or health product for treating and / or inhibiting the activity of Helicobacter pylori. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1is a compound of the structure of Formula I prepared in the examples 1 H NMR spectrum;

[0031] Figure 2 is a compound of the structure of Formula I prepared in the examples 13 C NMR spectrum;

[0032] Figure 3 HSQC spectrum of a compound of the structure of Formula I prepared in the examples

[0033] Figure 4 is a compound of the structure of Formula I prepared in the examples 1 H- 1 H COSY spectrum;

[0034] Figure 5 HMBC spectrum of a compound of the structure of Formula I prepared in the examples

[0035] Figure 6 ROESY spectrum of a compound of the structure of Formula I prepared in the examples

[0036] Figure 7 HRESIMS spectrum of a compound of the structure of Formula I prepared in the examples. DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application.

[0038] The implementation of the present application is described in detail below with reference to specific examples.

[0039] The compound provided by the present application has the activity of inhibiting H. pylori, and those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application are described by the following preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0040] The experimental materials used in the following examples of the present application are all ordinary commercially available products and can be purchased in the market.

[0041] Referring to Figures 1-7 The following are preferred examples provided by the present application.

[0042] The present application provides a chromone trimer for inhibiting the activity of Helicobacter pylori, and a preparation method and application thereof.

[0043]

[0044] The following is a specific implementation of the present application: Example 1:

[0045] 1.1, Instruments and reagents

[0046] Bruker AV-500 superconducting nuclear magnetic resonance spectrometer (Bruker, Switzerland); Autospec 300 mass spectrometer (VG, UK); analytical high performance liquid chromatograph (Agilent, USA); semi-preparative high performance liquid chromatograph (Agilent, USA); N-1000 (2 L) vertical rotary evaporator and CA-1111 cooling water circulating device (Shanghai Ailang Instrument Co., Ltd.); SHZ-D (III) circulating vacuum pump (Shanghai Longtuo Instrument and Equipment Co., Ltd.); AS 220.R2 one-millionth electronic scale (RADWAG Wagi Elektroniczne); Sephadex LH-20 gel (Merck Co. Ltd.); C 18 Reversed-phase silica gel (20-45 µm, Fuji Silysia Chemical Ltd, Japan); Cosmosil C 18 Chromatographic column (10.0 ID × 250.0 mm, Nacalai tesque); Cosmosil πNAP chromatographic column (10.0 ID × 250.0 mm, Nacalai tesque); silica gel for column chromatography and thin layer chromatography silica gel plate (Qingdao Marine Chemical Plant); deuterated reagents and chromatographic methanol (Merck, Germany); 95% ethanol, redistilled methanol, ethyl acetate, chloroform, petroleum ether, acetone and other commonly used organic reagents (Kemio, Tianjin, Fumian, Guangzhou Guanghua, etc.).

[0047] 1.2, Preparation and structural identification of compounds

[0048] The Philippines Agarwood sample (1.0 Kg) was purchased in Haikou City, Hainan Province in July 2020, and the original plant was identified by Professor Dai Haofu of the Institute of Tropical Biological Technology, Chinese Academy of Tropical Agricultural Sciences as Agarwood Aquilaria filaria (Oken) Merr. Voucher specimen (No. FLBS20200710) is kept in the Institute of Tropical Biological Technology, Chinese Academy of Tropical Agricultural Sciences.

[0049] The Philippines Agarwood sample (817.0 g) was crushed and extracted with 95% ethanol aqueous solution (5 L) by heating (micro-boiling state) for 5 times. The obtained extract was filtered and concentrated under reduced pressure at 45 ℃ to obtain ethanol extract (180.4 g). The extract was diluted with 5.0 L water to form a suspension, which was extracted with petroleum ether (5.0 L each time), ethyl acetate (5.0 L each time) and n-butanol (5.0 L each time) for 3 times, respectively. The extract was concentrated under reduced pressure at 45 ℃ to obtain petroleum ether phase extract, ethyl acetate phase extract and n-butanol phase extract, respectively.

[0050] The ethyl acetate extract (81.7 g) was subjected to gradient elution with chloroform-methanol (1:0→0:1, V / V) by reduced pressure normal phase silica gel column (silica gel H) to obtain 14 fractions, denoted as Fr.1 ~ Fr.14. The components of Fr.1 ~ Fr.14 were analyzed by liquid chromatography-mass spectrometry, and it was found that Fr.13 contained chromone trimers with molecular weight between 800-1000.

[0051] The fraction Fr.13 (30.9 g) was separated by ODS column chromatography (4.0 cm×30.0 cm) eluted with methanol / water (40%→100 %) to obtain 28 fractions, denoted as Fr.13-1~Fr.13-28.

[0052] The fraction Fr.13-16 (1.76 g) was subjected to elution with [chloroform:methanol (1:1)] by Sephadex LH-20 gel column to obtain 6 fractions, denoted as Fr. 13-16-1~Fr. 13-16-6; Fr.13-16-3 (1.36 g) was subjected to elution with [chloroform:methanol (1:1)] by Sephadex LH-20 to obtain 3 sub-fractions Fr.13-16-3-1~13-16-3-3.

[0053] The fraction Fr. 13-16-3-2 (919.8 mg) was subjected to elution with chloroform-methanol (25:1→0:1, V / V) by normal phase silica gel column (silica gel H) to obtain 5 fractions, denoted as Fr.13-16-3-2-1~Fr.13-16-3-2-5; Fr.13-16-3-2-3 (213.7 mg) was subjected to semi-preparative high performance liquid chromatography (C 18; methanol / water, 58:42, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm) to give five fractions Fr. 13-16-3-2-3-1 ~13-16-3-2-3-5; Fr. 13-16-3-2-3-5 (16.7 mg) was subjected to semi-preparative HPLC (NAP; methanol / water, 68:32, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm) to give a compound of formula I with a molecular weight between 800-900 ( t R 54.0 min; 3.3 mg).

[0054] The compound of formula I is shown below:

[0055]

[0056] The compound of formula I prepared in Example 1 was structurally identified, and the results are shown in Figures 1-7 The high resolution mass spectrum of the compound m / z 905.2782 [M+Na] + , with a molecular formula of C 51 H 46 O 14 ; the 1 H (500 MHz) and 13 C NMR (125 MHz) data of the compound are shown in Table 1:

[0057] Table 1. The 1 H and 13 C NMR data of the compound delta in ppm, J in Hz, in CD3OD)

[0058]

[0059]

[0060]

[0061]

[0062] Example 2: Determination of the activity of the compound in inhibiting H. pylori

[0063] The frozen Helicobacter pylori strain is thawed at room temperature, and the bacterial liquid is inoculated on solid plate culture medium for recovery, and is cultured in an incubator at 5% O2, 10% CO2, 85% N2 and 37℃ for 5-7 days. The cultured colonies are scraped into liquid culture medium and cultured for 4-5 days. The cultured Hp bacterial liquid is aspirated, diluted with liquid culture medium (BHI) and mixed, and the concentration of the bacterial liquid is adjusted to 1×10 8 CFU / mL. The compounds are dissolved with DMSO, and the initial screening drug concentration is set to 100 μmol·L −1 . The anti-Hp activity of all the compounds is detected at this concentration. The inhibition rate test method adopts the micro-dilution method, and the re-screening removes the compounds with an inhibition concentration greater than 100 μmol·L −1 . The compound concentration is adjusted to 3.125-100 μmol·L −1 by using the double dilution method, and the final volume is 200 μL / well. Clarithromycin (CLA) is used as a positive drug, BHI and DMSO are used as blank controls, and 3 repetitions are set for each experimental group. The 96-well plate is placed in a 37℃, micro-oxygen, 150 rpm shaking culture for 48 h. The absorbance is measured by an enzyme marker at 600 nm, and the turbidity in the well is combined. The MIC is the minimum concentration at which no bacterial growth is observed, and the MBC is the minimum concentration at which the bacteria in the culture medium can be killed.

[0064] The OD 600 value reflects the concentration of the bacterial liquid, and is calculated according to the following formula:

[0065] The inhibition rate (%) = (OD 600 of the control bacterial liquid - OD 600 of the treated bacterial liquid) / OD 600 of the control bacterial liquid x 100.

[0066] Table 2: Compound inhibition of Helicobacter pylori activity results

[0067]

[0068] a Positive control, P 0.05 vs. positive control group, P 0.01 vs. positive control.

[0069] From the above examples 1 and 2, it can be seen that the present application finds a new class of chromone trimers with the activity of inhibiting Helicobacter pylori; the compound is derived from Lignum Aquilariae Resinatum from the Philippines, and has a good application prospect in developing drugs or health products for treating gastritis caused by Helicobacter pylori infection.

[0070] The above merely describes preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A chromone trimer that inhibits the activity of Helicobacter pylori, characterized in that, The chromone trimer is a compound of the following general formula I:

2. The method for preparing a chromone trimer that inhibits Helicobacter pylori activity according to claim 1, characterized in that, Includes the following steps: S1. After crushing the Philippine silk agarwood, heat and reflux it with 5 L of ethanol 5 times to obtain the extract, and combine and concentrate it into an ethanol extract. S2. Prepare a suspension by mixing the ethanol extract with water at a volume ratio of 1:1, and extract it sequentially with petroleum ether, ethyl acetate and n-butanol. Then concentrate each extract to prepare an extract. S3. The ethyl acetate extract was passed through a normal-phase silica gel column under reduced pressure and eluted with chloroform-methanol 1:0→0:1, V / V to obtain 14 fractions, denoted as Fr.1 ~ Fr.

14. S4. Liquid chromatography-mass spectrometry analysis of the Fr.1 ~ Fr.14 components revealed that Fr.13 contained a chromone polymer with a molecular weight between 800-1000 Da. S5. The fraction Fr.13 was subjected to gradient elution with chloroform-methanol 100:1→0:1, V / V through a silica gel column to obtain 28 fractions, which were denoted as Fr.13-1~Fr.13-28. S6. The fraction Fr.13-16 was eluted with chloroform:methanol 1:1 through a Sephadex LH-20 gel column to obtain 6 fractions, which were designated as Fr.13-16-1 to Fr.13-16-6. S7. The fraction Fr.13-16-3 was eluted with chloroform:methanol 1:1 through a Sephadex LH-20 gel column to obtain 3 fractions, denoted as Fr.13-16-3-1 to Fr.13-16-3-3; S8. Elute fraction Fr.13-16-3-2 through a silica gel column with chloroform-methanol 25:1→0:1, V / V to obtain 5 fractions, denoted as Fr.13-16-3-2-1~Fr.13-16-3-2-5; S9. The fraction Fr.13-16-3-2-3 was prepared by semi-preparative high performance liquid chromatography to obtain 5 fractions Fr.13-16-3-2-3-1~13-16-3-2-3-5; S10. The fraction Fr.13-16-3-2-3-5 was eluted by semi-preparative high performance liquid chromatography to obtain a compound with a molecular weight between 800 and 900 of Formula I. The high-performance liquid chromatography (HPLC) conditions in step S9 are as follows: C18 column; methanol / water, 58:42, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm. The conditions for high performance liquid chromatography in step S10 are as follows: πNAP column; methanol / water, 68:32, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm.

3. The use of the chromone trimer for inhibiting Helicobacter pylori activity as described in claim 1 in the preparation of a drug for inhibiting Helicobacter pylori activity.

4. The use of the chromone trimer according to claim 1 in the preparation of medicaments for treating and / or alleviating symptoms associated with Helicobacter pylori.

5. The application according to claim 4, characterized in that, Helicobacter pylori-related diseases are selected from one or more of the following: chronic gastritis, atrophic gastritis, and gastric ulcer.

6. A drug for inhibiting the activity of Helicobacter pylori, characterized in that, The pharmaceutical product includes the chromone trimer as described in claim 1 and its pharmaceutically acceptable excipients.

7. The drug for inhibiting Helicobacter pylori activity according to claim 6, characterized in that, The dosage form of the drug is tablets, capsules, powder for injection, or suspension.

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