2-(2-phenethyl) chromone dimer in agilawood as well as preparation method and application of 2-(2-phenethyl) chromone dimer

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a 2-(2-phenethyl) chromone dimer in agilawood as well as a preparation method and application of the 2-(2-phenethyl) chromone dimer. The method comprises the following steps: by taking the agilawood corassonii as a raw material, carrying out reflux extraction with ethanol, extracting with petroleum ether to remove volatile components, separating and purifying with various chromatographic technologies such as silica gel column chromatography, ODS column chromatography and semi-preparative HPLC (High Performance Liquid Chromatography) to obtain a monomeric compound, and identifying the structure of the monomeric compound through spectroscopy methods such as UV (Ultraviolet), high-resolution mass spectrum, 1H and 13C nuclear magnetic resonance and the like in combination with physicochemical properties. The structure of a compound 1 is identified as a new 2-(2-phenethyl) chromone dimer, and the compound 1 is named as aquicrasone W. The invention also discloses a preparation method of the compound 1. A biological activity test verifies that the compound 1 shows anti-helicobacter pylori activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a 2-(2-phenylethyl) chromone dimer in eaglewood and a preparation method and application thereof. BACKGROUND

[0002] Eaglewood is a resin-containing wood produced by plants of Aquilaria or Gyrinopsis of Thymelaeaceae after being injured, has the effects of promoting qi and relieving pain, warming middle and relieving vomiting, and has the effects of promoting qi and relieving pain, warming middle and relieving vomiting, and is clinically used for chest and abdominal distending pain, stomach cold vomiting and hiccup, and kidney deficiency and asthma. At present, the research on the chemical components of eaglewood finds that the chemical components of eaglewood are very complex, and the main components are sesquiterpenes, 2-(2-phenylethyl) chromone and aromatic compounds. The 2-(2-phenylethyl) chromone is the main active ingredient, has antibacterial, anti-inflammatory, anti-depression, acetylcholinesterase inhibition and other biological activities. Due to the hot market of eaglewood and people's more attention to health, the pharmacological activity of eaglewood has been more and more widely concerned, developed and utilized. In order to further develop and utilize the pharmacological activity value of eaglewood, it is necessary to deeply study the chemical components and pharmacological activity of eaglewood, which has a very important promoting effect on the development of new prodrugs of eaglewood. SUMMARY

[0003] The application provides a 2-(2-phenylethyl) chromone dimer in eaglewood and a preparation method and application thereof.

[0004] The technical scheme of the application is as follows:

[0005] A 2-(2-phenylethyl) chromone dimer in eaglewood, the structure of which is shown as formula 1:

[0006]

[0007] Formula 1.

[0008] The preparation method of the 2-(2-phenylethyl) chromone dimer described above comprises the following steps:

[0009] (1) dry eaglewood is weighed, crushed, and then extracted 3 times by refluxing with 95% ethanol, the extract is combined and concentrated under reduced pressure to obtain an ethanol extract;

[0010] (2) the ethanol extract is dispersed in water, petroleum ether is added for extraction to remove low-polarity volatile components, and the lower aqueous phase is collected; the aqueous phase is concentrated under reduced pressure to obtain an extract;

[0011] (3) The extract was subjected to silica gel column chromatography, eluted with chloroform:methanol, and the same components were combined (the same below) by thin layer chromatography detection to obtain 20 fractions Fr.1 - Fr.20;

[0012] (4) Fr.14 (30.0 g) was selected as the target fraction and subjected to octadecylsilyl (ODS) column chromatography, gradient eluted with methanol-water, and 14 fractions Fr.14-1 - Fr.14-14 were collected;

[0013] (5) Fr.14-7 was further separated by silica gel chromatography, gradient eluted with chloroform-methanol, and 9 fractions Fr.14-7-1 - Fr.14-7-9 were collected;

[0014] (6) Fr.14-7-4 was further purified by twice semi-preparative high performance liquid chromatography (HPLC) to obtain compound 1 finally.

[0015] Further, in the step (1), the agarwood is Aquilaria crassna Pierre ex Lecomte, and the origin is Cambodia.

[0016] Further, in the step (3), the volume ratio of chloroform to methanol is 1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively.

[0017] Further, in the step (4), the column chromatography uses an octadecylsilyl silica gel column, and the concentration of methanol is 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively.

[0018] Further, in the step (5), the silica gel chromatography uses silica gel with a specification of 200-300 mesh; and the volume ratio of chloroform to methanol is 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, and 0:1, respectively.

[0019] Further, in the step (6), Fr.14-7-4 is purified by semi-preparative high performance liquid chromatography, using a C18 column, a mobile phase of methanol / water with a volume ratio of 57:43 to obtain component Fr.14-7-4-6 with a retention time of 51.0 min; and component Fr.14-7-4-6 is further purified by semi-preparative high performance liquid chromatography, using a 5PFP column, a mobile phase of methanol / water with a volume ratio of 56:44 to obtain compound 1 with a retention time of 44.2 min.

[0020] Use of the 2-(2-phenylethyl)chromone dimer described above in the preparation of an anti-Helicobacter pylori preparation.

[0021] An anti-Helicobacter pylori preparation containing the 2-(2-phenylethyl)chromone dimer described above.

[0022] Advantages of the present application:

[0023] The present application takes the agarwood of the original plant Aquilaria crassna as raw material, extracts by ethanol reflux, and then uses petroleum ether extraction, silica gel column chromatography, ODS column chromatography, silica gel column chromatography, and semi-preparative HPLC and other chromatographic techniques for separation and purification to obtain one monomer compound. The structure of the compound 1 is identified as a new 2-(2-phenylethyl)chromone dimer, named aquicrassone W, through UV, high-resolution mass spectrometry, 1 H and 13 C nuclear magnetic resonance and other spectroscopic methods combined with physicochemical properties. The compound 1 shows anti-Helicobacter pylori activity, and the minimum inhibitory concentration (MIC) is 80 µM. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0025] Figure 1 . H NMR (500 MHz) spectrum of compound 1 in DMSO-d6. 1

[0026] Figure 2 . H NMR (500 MHz) spectrum of compound 1 in DMSO-d6. 13

[0027] Figure 3 . HSQC spectrum of compound 1 in DMSO-d6.

[0028] Figure 4 . H NMR (500 MHz) spectrum of compound 1 in DMSO-d6. 1 1 H COSY spectrum of compound 1 in DMSO-d6.

[0029] Figure 5 . HMBC spectrum of compound 1 in DMSO-d6.

[0030] ​​​Figure 6 . ROESY spectrum of compound 1 in DMSO-d6.

[0031] Figure 7 . HRESIMS spectrum of compound 1.

[0032] Figure 8 . Structural formula of compound 1. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative effort belong to the scope of protection of the present application.

[0034] Example 1

[0035] 1. Compound extraction and separation

[0036] Take 928.0 g of dried Aquilaria crassna, crush it, and then extract it with 95% ethanol by reflux extraction for 3 times. Combine the extract and concentrate it under reduced pressure to obtain 375.2 g of ethanol extract. Disperse the ethanol extract in water, add petroleum ether to extract to remove low-polarity volatile components, and collect the lower aqueous phase. Concentrate the aqueous phase under reduced pressure to obtain 374.8 g of extract. Perform silica gel column chromatography on the extract, elute it with chloroform:methanol, and the volume ratio of chloroform to methanol is 1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 0:1, respectively. Perform silica gel thin layer chromatography (silica gel type: GF254) on the eluate, and collect the fractions with Rf values of 0.5-0.6 and 0.3-0.4. Concentrate the collected fractions under reduced pressure to obtain 0.5 g of fraction 1 and 0.3 g of fraction 2. 254) and combined the same fractions (the same below) to obtain 20 fractions Fr.1 - Fr.20. Fr.14 (30.0 g) was selected as the target fraction for ODS (Octadecylsilyl) column chromatography, and gradient elution was performed with methanol-water (the concentration of methanol was 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively), and 14 fractions (Fr.14-1 - Fr.14-14) were collected. Fr.14-7 (752.1 mg) was selected for further separation, and silica gel chromatography (silica gel specification: 200-300 mesh) was performed with gradient elution of chloroform-methanol (the volume ratio of chloroform to methanol was 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, and 0:1, respectively), and 9 fractions (Fr.14-7-1 - Fr.14-7-9) were collected. Fr.14-7-4 (205.6 mg) was purified by semi-preparative high performance liquid chromatography (HPLC) using a C18 (250 mm x 4.6 mm, 5 µm) column with a mobile phase of methanol / water (volume ratio 57:43) to obtain component Fr.14-7-4-6 (43.8 mg, retention time 51.0 min). Component Fr.14-7-4-6 was purified again by semi-preparative high performance liquid chromatography using a 5PFP column with a mobile phase of methanol / water (volume ratio 56:44) to obtain compound 1 (9.1 mg, retention time 44.2 min).

[0037] 2. Structure identification

[0038] Compound 1 was a light yellow sticky substance, {[\alpha ]}^{25}_{D} +78 (c 0.10, methanol); UV (methanol) λ max (logε): 235 (4.65), 254 (4.39), 320 (4.32) nm; CD (methanol) λ max (Δε) 322 (−19.52), 295 (+8.68), 275(−5.99), 253 (+1.30) nm; The measured value by high resolution mass spectrometry (HR-ESI-MS) was m / z 649.2041 [M + Na] + , and its molecular formula was recommended as C 36 H 34 NaO 10 (theoretical value m / z 649.2044). The molecular formula of compound 1 was determined as C 1 H and 13C NMR (125 MHz) data (δ ppm) were used for structure identification, and the compound configuration was determined by NOE. Combined with the above spectral data (Table 1 and Figures 1-7 ), the structure of compound 1 was identified as a new 2-(2-phenylethyl) chromone dimer, and named as aquicrassone W (as shown in Figure 8 ).

[0039] Table 1.1H NMR (500 MHz) and 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data (δ ppm)

[0040]

[0041] 3. Anti-Helicobacter pylori activity

[0042] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound 1 against Helicobacter pylori (Hp) were determined using the ATCC 43504 strain as the experimental object. First, the strain was inoculated in a Columbia agar medium containing 5% sterile sheep blood and cultured at 37°C in a 5% oxygen three-gas incubator (containing 10% carbon dioxide and the rest nitrogen) for 4 days. Then, the culture was transferred to a brain heart infusion agar (BHA) containing 10% fetal bovine serum and cultured at 37°C under the condition of 150 rpm rotary shaking for 3 days. Compound 1 was dissolved in dimethyl sulfoxide (DMSO) and then diluted with the culture medium to obtain gradient concentrations of 160 µM, 80 µM, 40 µM, 20 µM, 10 µM and 5 µM. Then, 100 µL of each sample at a certain concentration was added to a 96-well plate containing 100 µL of a Helicobacter pylori suspension (concentration 1×10 8 CFU / mL) and incubated at 37°C in a microaerobic environment. After 48 hours, the minimum concentration that inhibited the visible growth of bacteria was recorded, which was the MIC. 10 µL of the culture solution in each well was spotted on a rapid urease test (RUT) reagent, and the minimum concentration that reduced the bacterial activity by 99.9% was defined as the MBC. Clarithromycin (CLA) was used as a positive control, and all experiments were repeated three times.

[0043] The results of the activity test showed that compound 1 exhibited anti-Helicobacter pylori activity, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 80 µM and 100 µM, respectively.

[0044] Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound 1 against Helicobacter pylori (H. pylori)

[0045]

[0046] The above descriptions are only the preferred embodiment of the application, not intended to limit the application and any modification, equivalent replacement and improvement made within the principle and technical scope of the application should be included in the protection scope of the application.

Claims

1. A 2-(2-phenylethyl)chromone dimer in agarwood, characterized in that, Its structure is shown in Equation 1: Formula 1.

2. The method for preparing the 2-(2-phenylethyl)chromone dimer as described in claim 1, characterized in that, Includes the following steps: (1) Weigh the dried agarwood, pulverize it, and extract it three times by reflux with 95% ethanol. Combine the extracts and concentrate them under reduced pressure to obtain the ethanol extract. (2) Disperse the ethanol extract in water, add petroleum ether for extraction to remove low-polarity volatile components, and collect the lower aqueous phase; concentrate the aqueous phase under reduced pressure to obtain the extract; (3) The extract was subjected to silica gel column chromatography and eluted with chloroform:methanol. The same components were combined by thin-layer chromatography to obtain 20 fractions Fr.1 - Fr.

20. (4) Fr.14 (30.0 g) was selected as the target fraction and subjected to octadecylsilyl bonded silica gel (ODS) column chromatography with methanol-water gradient elution. Fourteen fractions, Fr.14-1 - Fr.14-14, were collected. (5) Fr.14-7 was selected for further separation. Silica gel chromatography was used with gradient elution of chloroform-methanol to collect 9 fractions: Fr.14-7-1 - Fr.14-7-9. (6) Fr.14-7-4 was further purified by two semi-preparative high performance liquid chromatography (HPLC) processes to finally obtain compound 1.

3. The preparation method according to claim 2, characterized in that, The agarwood used in step (1) is Aquilaria crassna Pierre ex Lecomte.

4. The preparation method according to claim 2, characterized in that, In step (3), the volume ratio of chloroform to methanol is 1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively.

5. The preparation method according to claim 2, characterized in that, In step (4), the column chromatography used is an octadecylsilane-bonded silica column, and the methanol concentration is 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

6. The preparation method according to claim 2, characterized in that, In step (5), the silica gel used for silica gel chromatography is 200-300 mesh; the volume ratio of chloroform and methanol is 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, and 0:1, respectively.

7. The preparation method according to claim 2, characterized in that, In step (6), Fr.14-7-4 was purified by semi-preparative high-performance liquid chromatography using a C18 column and a mobile phase of methanol / water at a volume ratio of 57:43 to obtain component Fr.14-7-4-6 with a retention time of 51.0 min. Component Fr.14-7-4-6 was purified again by semi-preparative high-performance liquid chromatography using a 5PFP column and a mobile phase of methanol / water at a volume ratio of 56:44 to obtain compound 1 with a retention time of 44.2 min.

8. The use of the 2-(2-phenylethyl)chromone dimer as described in claim 1 in the preparation of anti-Helicobacter pylori formulations.

9. An anti-Helicobacter pylori preparation containing the above-described 2-(2-phenylethyl)chromone dimer.

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