Chromone tripolymer for inhibiting activity of helicobacter pylori as well as preparation method and application of chromone tripolymer

Abstract

The invention relates to the technical field of natural medicines, in particular to a chromone tripolymer for inhibiting the activity of helicobacter pylori as well as a preparation method and application thereof, and is technically characterized in that the invention provides a compound for treating and / or inhibiting the activity of helicobacter pylori, and the compound has a structure as shown in a formula I and belongs to a chromone tripolymer with a novel structure. Evaluation of inhibition of generation of helicobacter pylori is carried out through MIC and MBC determination methods, and the compound shown in the formula I can inhibit generation of helicobacter pylori. Therefore, the compound provided by the invention can be used for preparing medicines or health care products for treating and / or inhibiting the activity of the helicobacter pylori, and has wide development and application prospects.

Description

Technical Field

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

[0002] Chronic gastritis is a common disease of the digestive system, and its pathological changes are mainly manifested as chronic inflammation of the gastric mucosa. According to the results published by the World Health Organization, the global incidence of gastric diseases is as high as 80%, among which chronic gastritis is the most common, accounting for 50% of the total number of gastric diseases. The incidence of gastric diseases in my country is higher than the global level, among which chronic gastritis accounts for a higher proportion of the overall gastric diseases than the global level, about 50% to 80%, which has a great impact on people's lives and work. The long-term persistence of this disease will also lead to the occurrence of gastric cancer. Relevant studies have shown that 80% to 95% of patients with chronic gastritis have Helicobacter pylori infection in the gastric mucosa. Helicobacter pylori (Hp) infection is the most important pathogenic factor of gastritis and the most important and controllable risk factor for preventing gastric cancer. Almost all Hp patients with current infection have varying degrees of gastric mucosal inflammation. Therefore, finding natural, safe and efficient Helicobacter pylori inhibitors has become one of the research hotspots.

[0003] Agarwood is a wood formed by the accumulation of resin secreted by plants of the genus Aquilaria or Gyrinops in the family Thymelaeaceae after being damaged by the outside world. Agarwood is known as the "king of incense" and is mainly distributed in Guangdong, Guangxi, Taiwan, Fujian and other places in my country. It has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting, and relieving asthma. It is used for chest and abdominal distension and pain, stomach cold vomiting and hiccups, and kidney deficiency and qi reversal and asthma. It is a precious traditional Chinese medicine for regulating qi commonly used in clinical Chinese medicine to treat stomach diseases. The main characteristic components of agarwood are sesquiterpenes and 2-(2-phenylethyl) chromone compounds, which have anti-inflammatory, antibacterial, anti-gastric ulcer, gastrointestinal motility, anti-tumor and cell pharmacological effects.

[0004] At present, there are no reports in the prior art on the isolation of compounds that inhibit the activity of Helicobacter pylori from Philippine silk agarwood. Therefore, the present application provides a chromone trimer that inhibits the activity of Helicobacter pylori and a preparation method and application thereof. Summary of the invention

[0005] The purpose of the present invention is to solve the prior art problems raised in the above background technology, and to provide a chromone trimer for inhibiting the activity of Helicobacter pylori, as well as a preparation method and application thereof.

[0006] The above-mentioned purpose of the present invention is achieved like this:

[0007] In one aspect, the present invention provides a chromone trimer for inhibiting the activity of Helicobacter pylori, wherein the chromone trimer is a compound comprising the following general formula I:

[0008]

[0009] Another aspect of the present invention provides a method for preparing a chromone trimer that inhibits the activity of Helicobacter pylori, comprising the following steps:

[0010] S1. After crushing the Philippine silk agarwood, heat and reflux and extract with 5L ethanol for 5 times to obtain an extract, which is combined and concentrated into an ethanol extract;

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

[0012] S3, taking the ethyl acetate extract and passing it through a vacuum normal phase silica gel column to elute with chloroform-methanol (1:0→0:1, V / V) to obtain 14 fractions, which are recorded as Fr.1 to Fr.14;

[0013] S4. Through liquid phase mass spectrometry analysis of components Fr.1 to Fr.14, it was found that Fr.13 contained chromone polymers with a molecular weight between 800-1000Da;

[0014] S5, fraction Fr.13 was passed through a silica gel column with chloroform-methanol (100:1→0:1, V / V) for gradient elution to obtain 28 fractions, which were recorded as Fr.13-1 to Fr.13-28;

[0015] S6, fraction Fr.13-16 was eluted through a Sephadex LH-20 gel column with chloroform:methanol (1:1) to obtain 6 fractions, which were recorded as Fr.13-16-1 to Fr.13-16-6;

[0016] S7, eluting the fraction Fr.13-16-3 through a Sephadex LH-20 gel column with chloroform:methanol (1:1) to obtain three fractions, recorded as Fr.13-16-3-1 to Fr.13-16-3-3;

[0017] S8. The fraction Fr.13-16-3-2 was eluted through a silica gel column with chloroform-methanol (25:1→0:1, V / V) to obtain five fractions, which were recorded as Fr.13-16-3-2-1 to Fr.13-16-3-2-5;

[0018] S9, fraction Fr.13-16-3-2-3 was prepared by semi-preparative high performance liquid chromatography to obtain five fractions Fr.13-16-3-2-3-1 to 13-16-3-2-3-5;

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

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

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

[0022] Another aspect of the present invention provides a method for preparing a chromone trimer capable of inhibiting the activity of Helicobacter pylori, and use of the prepared chromone trimer in preparing a product capable of inhibiting the activity of Helicobacter pylori.

[0023] Another aspect of the present invention provides the use of a chromone trimer or a chromone trimer prepared by the preparation method in the preparation of a medicine or health product for treating and / or alleviating reactions related to Helicobacter pylori.

[0024] Furthermore, Helicobacter pylori-related reactions include one or more of chronic gastritis, atrophic gastritis, and gastric ulcer.

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

[0026] Furthermore, the product includes the chromone trimer compound described above or the chromone trimer prepared by the preparation method described above and its pharmaceutically or health-care acceptable excipients.

[0027] Furthermore, the dosage form of the drug is tablets, capsules, powder injections or suspensions.

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

[0029] The present invention provides a group of compounds for treating and / or inhibiting the activity of Helicobacter pylori, which have a structure shown in Formula I and are chromone trimers with novel structures. The compounds shown in Formula I provided by the present invention are evaluated for inhibiting the production of Helicobacter pylori by MIC and MBC assays. The compounds shown in Formula I provided by the present invention can inhibit the production of Helicobacter pylori, thus indicating that the above-mentioned compounds provided by the scheme of the present invention can be used to prepare medicines or health products for treating and / or inhibiting the activity of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the compound of formula I prepared in the embodiment 1 H NMR spectrum;

[0031] Figure 2 is the compound of formula I prepared in the embodiment 13 C NMR spectrum;

[0032] Figure 3 is the HSQC spectrum of the compound of formula I prepared in the example;

[0033] Figure 4 is the compound of formula I prepared in the embodiment 1 H- 1 H COSY spectrum;

[0034] Figure 5 is the HMBC spectrum of the compound of formula I prepared in the example;

[0035] Figure 6 is the ROESY spectrum of the compound of formula I prepared in the example;

[0036] Figure 7 It is the HRESIMS spectrum of the compound of formula I prepared in the example. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0039] The embodiments of the present invention provide a compound with the activity of inhibiting Helicobacter pylori. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention are described by the following preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] The experimental materials used in the following examples of the present invention are all common commercial products and can be purchased in the market.

[0041] Reference Figure 1-Figure 7 As shown, the following are preferred embodiments provided by the present invention.

[0042] The embodiment of the present invention provides a chromone trimer for inhibiting the activity of Helicobacter pylori and a preparation method and application thereof. The general structural formula of the chromone trimer is:

[0043]

[0044] The following is a specific implementation of the implementation scheme of the present invention:

[0045] Embodiment 1:

[0046] 1.1 Instruments and reagents

[0047] 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 (2L) vertical rotary evaporator and CA-1111 cooling water circulation device (Shanghai Ailang Instrument Co., Ltd.); SHZ-D (III) circulating vacuum pump (Shanghai Longtuo Instrument Equipment Co., Ltd.); AS220.R2 1 / 10,000 electronic scale (RADWAGWagiElektroniczne); Sephadex LH-20 gel (Merck Co., Ltd.); C 18 Reverse phase silica gel (20-45 μm, Fuji Silysia Chemical Ltd., Japan); Cosmosil C 18Chromatographic column (10.0ID×250.0mm, Nacalaitesque Company) CosmosilπNAP chromatographic column (10.0ID×250.0mm, Nacalaitesque Company); silica gel for column chromatography and thin layer chromatography silica gel plate (Qingdao Ocean Chemical Plant); deuterated reagents and chromatographic methanol (Merck Company, Germany); 95% ethanol, redistilled methanol, ethyl acetate, chloroform, petroleum ether, acetone and other commonly used organic reagents (Tianjin Komio, Tianjin Fuchen, Guangzhou Guanghua and other companies).

[0048] 1.2 Preparation and structural identification of compounds

[0049] The Philippine silk agarwood sample (1.0 kg) was purchased in Haikou City, Hainan Province in July 2020. It was identified as the silk agarwood Aquilaria filaria (Oken) Merr. by Researcher Dai Haofu from the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences. The voucher specimen (No. FLBS20200710) is preserved in the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0050] After the Philippine silk agarwood sample (817.0g) was crushed, it was heated (slightly boiling) and refluxed for extraction 5 times with a volume concentration of 95% ethanol aqueous solution (5L), and the obtained extracts were filtered and combined, and concentrated under reduced pressure at 45°C to obtain an ethanol extract extract (180.4g). The extract was diluted with 5.0L of water to a suspension, and extracted 3 times with petroleum ether (5.0L each time), ethyl acetate (5.0L each time) and n-butanol (5.0L each time), and the extraction layers were evaporated and concentrated at 45°C to obtain petroleum ether phase extract, ethyl acetate phase extract and n-butanol phase extract.

[0051] The ethyl acetate extract (81.7 g) was passed through a normal phase silica gel column (silica gel H) under reduced pressure and gradient eluted with chloroform:methanol (1:0→0:1, V / V) to obtain 14 fractions, which were recorded as Fr.1 to Fr.14. Liquid chromatography mass spectrometry analysis of the Fr.1 to Fr.14 components revealed that Fr.13 contained a chromone trimer with a molecular weight of 800-1000.

[0052] Fraction Fr.13 (30.9 g) was separated by ODS column chromatography (4.0 cm×30.0 cm) and eluted with methanol / water (40%→100%) to obtain 28 fractions, which were designated as Fr.13-1 to Fr.13-28;

[0053] Fraction Fr.13-16 (1.76 g) was eluted through a Sephadex LH-20 gel column with [chloroform: methanol (1:1)] to obtain 6 fractions, recorded as Fr.13-16-1 to Fr.13-16-6; Fr.13-16-3 (1.36 g) was eluted through a Sephadex LH-20 [chloroform: methanol (1:1)] to obtain 3 subfractions Fr.13-16-3-1 to 13-16-3-3.

[0054] The fraction Fr.13-16-3-2 (919.8 mg) was eluted with chloroform-methanol (25:1→0:1, V / V) through a normal phase silica gel column (silica gel H) to obtain 5 fractions, which were recorded as Fr.13-16-3-2-1 to Fr.13-16-3-2-5; Fr.13-16-3-2-3 (213.7 mg) was purified by 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) was used for elution to obtain 5 fractions Fr.13-16-3-2-3-1 to 13-16-3-2-3-5; Fr.13-16-3-2-3-5 (16.7 mg) was eluted by semi-preparative high performance liquid chromatography (πNAP; methanol / water, 68:32, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm) to obtain a compound of formula I with a molecular weight between 800 and 900 (t R 54.0min; 3.3mg).

[0055] The compound of formula I is shown below:

[0056]

[0057] The structure of the compound of formula I prepared in Example 1 was identified, and the test results are as follows: Figure 1 to Figure 7 As shown, the high-resolution mass spectrum of the compound is m / z905.2782[M+Na] + , molecular formula is C 51 H 46 O 11 Compound 1 H(500MHz) and 13 CNMR (125MHz) data are shown in Table 1:

[0058] Table 1 Compounds 1 H and 13 CNMR data (δinppm, JinHz, inCD3OD)

[0059]

[0060]

[0061]

[0062] Example 2: Determination of the activity of compounds in inhibiting Helicobacter pylori

[0063] Thaw the frozen Helicobacter pylori strain at room temperature, inoculate the bacterial solution on a solid plate medium to revive, and incubate it upside down in an incubator at 5% O2, 10% CO2, 85% N2, and 37°C for 5 to 7 days. Scrape the cultured colonies and culture them in liquid culture medium for 4 to 5 days. Take the cultured Hp bacterial solution, dilute and mix it with liquid culture medium (BHI), and adjust the bacterial solution concentration to 1×10 8 CFU / mL. The compounds were dissolved in DMSO and the initial screening drug concentration was set at 100 μmol·L -1 The anti-Hp activity of all compounds was tested at this concentration. The microdilution method was used to test the antibacterial rate, and the inhibitory concentration was greater than 100 μmol·L -1 The compound concentration was adjusted to 3.125-100 μmol·L by serial dilution method. -1 , the final volume is 200μL / well. Clarithromycin (CLA) was used as the positive drug, BHI and DMSO were used as blank controls, and each experimental group was repeated 3 times. The 96-well plate was placed at 37°C, microaerobic, and shaken at 150 rpm for 48 hours. The absorbance was measured at 600nm using an ELISA reader. Combined with the turbidity in the wells, the MIC was the lowest concentration at which no bacterial growth was observed; the MBC was the lowest concentration that could kill bacteria in the culture medium.

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

[0065] Inhibition rate (%) = (OD 600 Control bacterial solution-OD 600 Treated bacterial solution) / OD 600 Control bacterial solution ×100.

[0066] Table 2 Results of the inhibitory activity of compounds against Helicobacter pylori

[0067]

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

[0069] It can be seen from the above Examples 1 and 2 that the present invention has discovered a new class of chromone trimers with the activity of inhibiting Helicobacter pylori; the compound is derived from Philippine silk agarwood and has good application prospects in the development of drugs or health products for treating gastritis caused by Helicobacter pylori infection.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

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

2. The method for preparing a chromone trimer for inhibiting the activity of Helicobacter pylori according to claim 1, characterized in that: The following steps are involved: S1. After crushing the Philippine silk agarwood, heat and reflux and extract with 5L ethanol for 5 times to obtain an extract, which is combined and concentrated into an ethanol extract; S2, preparing a suspension by mixing the ethanol extract and water in a volume ratio of 1:1, extracting with petroleum ether, ethyl acetate and n-butanol in sequence, and then concentrating each extract to prepare an extract; S3, taking the ethyl acetate extract and passing it through a vacuum normal phase silica gel column to elute with chloroform-methanol (1:0→0:1, V / V) to obtain 14 fractions, which are recorded as Fr.1 to Fr.14; S4. Through liquid phase mass spectrometry analysis of components Fr.1 to Fr.14, it was found that Fr.13 contained chromone polymers with a molecular weight between 800-1000Da; S5, fraction Fr.13 was passed through a silica gel column with chloroform-methanol (100:1→0:1, V / V) for gradient elution to obtain 28 fractions, which were recorded as Fr.13-1 to Fr.13-28; S6, fraction Fr.13-16 was eluted through a Sephadex LH-20 gel column with chloroform:methanol (1:1) to obtain 6 fractions, which were recorded as Fr.13-16-1 to Fr.13-16-6; S7, eluting the fraction Fr.13-16-3 through a Sephadex LH-20 gel column with chloroform:methanol (1:1) to obtain three fractions, recorded as Fr.13-16-3-1 to Fr.13-16-3-3; S8. The fraction Fr.13-16-3-2 was eluted through a silica gel column with chloroform-methanol (25:1→0:1, V / V) to obtain five fractions, which were recorded as Fr.13-16-3-2-1 to Fr.13-16-3-2-5; S9, fraction Fr.13-16-3-2-3 was prepared by semi-preparative high performance liquid chromatography to obtain five fractions Fr.13-16-3-2-3-1 to 13-16-3-2-3-5; S10. Elute the fraction Fr.13-16-3-2-3-5 by semi-preparative high performance liquid chromatography to obtain a compound having a structure of formula I with a molecular weight between 800-900.

3. The method for preparing a chromone trimer for inhibiting the activity of Helicobacter pylori according to claim 2, characterized in that: The conditions of the high performance liquid chromatography in step S9 are: C18 column; methanol / water, 58:42, V / V; flow rate 4.0 mL / min; UV detection wavelength 210 / 254 nm.

4. The method for preparing a chromone trimer for inhibiting the activity of Helicobacter pylori according to claim 2, characterized in that: The conditions of the high performance liquid chromatography in step S10 are: πNAP column; methanol / water, 68:32, V / V; flow rate 4.0 mL / min; ultraviolet detection wavelength 210 / 254 nm.

5. Use of the chromone trimer prepared according to the method for preparing a chromone trimer for inhibiting the activity of Helicobacter pylori according to any one of claims 2 to 4 in preparing a product for inhibiting the activity of Helicobacter pylori.

6. Use of the chromone trimer according to claim 1 or the chromone trimer prepared according to the preparation method according to any one of claims 2 to 4 in the preparation of drugs or health products for treating and / or alleviating reactions related to Helicobacter pylori.

7. The use according to claim 6, characterized in that: Reactions associated with Helicobacter pylori include one or more of chronic gastritis, atrophic gastritis, and gastric ulcer.

8. A product for inhibiting the production of Helicobacter pylori, characterized in that: The product is a medicine or a health product.

9. A product for inhibiting the production of Helicobacter pylori according to claim 8, characterized in that: The product comprises the chromone trimer compound according to claim 1 or the chromone trimer prepared by the preparation method according to any one of claims 2 to 4 and its pharmaceutically or health-care acceptable excipients.

10. A product for inhibiting the production of Helicobacter pylori according to claim 9, characterized in that: The dosage form of the medicine is tablet, capsule, powder injection or suspension.

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