Anti-inflammatory and anti-helicobacter pylori compound separated from agilawood and preparation method thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an anti-inflammatory and anti-helicobacter pylori compound separated from agilawood and a preparation method thereof. The method comprises the following steps: by taking the agilawood corasta as a raw material, carrying out reflux extraction with ethanol, extracting with petroleum ether to remove volatile components, separating and purifying by adopting various chromatographic technologies such as silica gel column chromatography, reversed-phase column chromatography and semi-preparative HPLC (High Performance Liquid Chromatography) to obtain a monomeric compound, and identifying the structure of the monomeric compound by adopting spectroscopy methods such as ultraviolet, high-resolution mass spectrometry and nuclear magnetic resonance. The structure of a compound 1 is identified as a new 2-(2-phenethyl) chromone dimer, and the compound 1 is named as aquicrasone V. The invention further discloses a preparation method of the compound 1. A biological activity test verifies that the compound has relatively strong anti-inflammatory activity and anti-helicobacter pylori activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to compounds isolated from agarwood that have anti-inflammatory and anti-Helicobacter pylori properties, and their preparation methods. Background Technology

[0002] Agarwood is the resinous wood produced by plants of the genus *Aquilaria* or *Gyrinopsis* (Thymelaeaceae). Clinically, it is used to treat chest and abdominal distension and pain, stomach cold, vomiting and hiccups, and kidney deficiency with shortness of breath. Studies have found that the main chemical components of agarwood are sesquiterpenes, 2-(2-phenylethyl)chromone, and aromatic compounds. With the continuous isolation and identification of chemical components in agarwood in recent years, and modern pharmacological activities, it has been discovered that in addition to its antibacterial, antiasthmatic, sedative, analgesic, and antispasmodic effects, agarwood also possesses anti-inflammatory, hypoglycemic, antidepressant, and acetylcholinesterase inhibitory biological activities. With the increasing popularity of agarwood and growing health awareness, its pharmacological activities have received widespread attention. Therefore, further in-depth research into the chemical components and pharmacological activities of agarwood is crucial for further exploring its medicinal value and developing new prodrugs. Summary of the Invention

[0003] This invention provides compounds isolated from agarwood that have anti-inflammatory and anti-Helicobacter pylori properties, as well as a method for their preparation.

[0004] The technical solution of this invention is implemented as follows:

[0005] A compound isolated from agarwood has the structure shown in Formula 1:

[0006] Formula 1.

[0007] The preparation method of the above-described compounds includes the following steps:

[0008] (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.

[0009] (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;

[0010] (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.

[0011] (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.

[0012] (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.

[0013] (6) Fr.14-7-4 was further purified by two semi-preparative high performance liquid chromatography (HPLC) processes to finally obtain compound 1.

[0014] Furthermore, the agarwood used in step (1) is Aquilaria crassna Pierre ex Lecomte, originating from Cambodia.

[0015] Furthermore, 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.

[0016] Furthermore, in step (4), the column chromatography uses an octadecylsilane-bonded silica column, and the methanol concentrations are 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0017] Furthermore, in step (5), the silica gel used for silica gel chromatography is of 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.

[0018] Further, 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 45.8 min.

[0019] The above-mentioned compounds are used in the preparation of anti-inflammatory agents.

[0020] The above-described compounds are used in the preparation of formulations that inhibit the formation of nitric oxide.

[0021] The above-described compounds are used in the preparation of anti-Helicobacter pylori preparations.

[0022] An anti-inflammatory preparation containing the compounds described above.

[0023] An anti-Helicobacter pylori preparation containing the compounds described above.

[0024] The beneficial effects of this invention are:

[0025] This invention uses agarwood from the native plant *Aquilaria crassna* as raw material. After ethanol reflux extraction, a single monomeric compound is obtained through a series of chromatographic techniques, including petroleum ether extraction, silica gel column chromatography, ODS column chromatography, silica gel column chromatography, and semi-preparative HPLC. The compound is then purified using UV chromatography, high-resolution mass spectrometry, and other methods. 1 H and 13 The structure of compound 1 was identified by spectroscopy methods such as nuclear magnetic resonance (NMR) combined with physicochemical properties, and was determined to be a novel 2-(2-phenylethyl)chromone dimer, named aquicrassone V. Experimental verification showed that this compound inhibited nitric oxide production in lipopolysaccharide-induced mouse RAW264.7 mononuclear macrophages, with a half-maximal inhibitory concentration (IC50) of [value missing]. 50 The concentration of 14.37 ± 2.36 µM indicates that compound 1 has strong anti-inflammatory activity; compound 1 also exhibits anti-Helicobacter pylori activity, with a minimum inhibitory concentration (MIC) of 80 µM. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Compound 1 in DMSO-d6 1 1H NMR (500 MHz) spectrum.

[0028] Figure 2 Compound 1 in DMSO-d6 13 C NMR (125 MHz) spectrum.

[0029] Figure 3 HSQC spectrum of compound 1 in DMSO-d6.

[0030] Figure 4 Compound 1 in DMSO-d6 1H- 1 H COSY spectrum.

[0031] Figure 5 HMBC spectrum of compound 1 in DMSO-d6.

[0032] Figure 6 ROESY spectrum of compound 1 in DMSO-d6.

[0033] Figure 7 HRESIMS diagram of compound 1.

[0034] Figure 8 Structural formula of compound 1. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Compound extraction and separation

[0038] 928.0 g of dried Aquilaria crassna was weighed, pulverized, and extracted three times by reflux with 95% ethanol. The extracts were combined and concentrated under reduced pressure to obtain 375.2 g of ethanol extract. The ethanol extract was dispersed in water, and petroleum ether was added for extraction to remove low-polarity volatile components. The lower aqueous phase was collected; the aqueous phase was concentrated under reduced pressure to obtain 374.8 g of extract. The extract was subjected to silica gel column chromatography, eluted with chloroform:methanol at volume ratios of 1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively. Thin-layer chromatography was performed on silica gel (silica gel type: GF). 254Chromatographic analysis was performed to combine identical components (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 bonded silica) column chromatography with gradient elution of methanol-water (methanol concentrations of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), and 14 fractions (Fr.14-1 - Fr.14-14) were collected. Fr.14-7 (752.1 mg) was further separated using silica gel chromatography (silica gel size: 200-300 mesh) with gradient elution of chloroform-methanol (chloroform and methanol volume ratios of 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, and 0:1, respectively), yielding nine fractions (Fr.14-7-1 - Fr.14-7-9). Fr.14-7-4 (205.6 mg) was purified by semi-preparative high-performance liquid chromatography (HPLC) using a C18 column (250 mm × 4.6 mm, 5 µm) with methanol / water (volume ratio 57:43) as the mobile phase, yielding fraction Fr.14-7-4-6 (43.8 mg, retention time 51.0 min). The 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 (v / v 56:44) to give compound 1 (8.5 mg, retention time 45.8 min).

[0039] 2. Structural identification

[0040] Compound 1 is a pale yellow viscous substance, {[\alpha ]}^{25}_{D} +14 (c 0.10, methanol), UV (methanol) λ max (log ε):253 (4.75), 255 (4.41), 320 (4.17) nm; CD (methanol) λ max (Δε) 319 (−9.58), 289 (+9.64), 269 (−1.12), 255 (+1.13) nm; measured by high-resolution mass spectrometry (HR-ESI-MS) at m / z 649.2044 [M + Na] + The recommended molecular formula is C. 36 H 34 NaO 10 (Theoretical value is m / z 649.2044). (Through...) 1 H and 13Structural identification was performed using 3C NMR (DMSO-d6) data (Table 1 and...). Figures 1-7 Based on the above spectral data, the structure of compound 1 was identified as a novel 2-(2-phenylethyl)chromone dimer, and named aquicrassone V (e.g., Figure 8 ).

[0041] Table 1. Compound 1 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data (δ ppm)

[0042]

[0043] 3. Bioactivity

[0044] 3.1. Anti-inflammatory activity

[0045] The anti-inflammatory activity of compound 1 was detected using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage model. Quercetin and indomethacin were used as positive controls, and DMEM medium containing DMSO (Thermo Fisher Scientific) was used as a negative control. Cells were cultured in a humidified incubator at 37°C with 5% CO2 / 95% air. Initial screening revealed that compound 1 had anti-inflammatory activity. Further testing was conducted at six concentration gradients: 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, and 1.562 µM. The cell count was 5 × 10⁻⁶ cells / ... 4 Cells were seeded at 100 cells / mL in 96-well plates and stimulated with 500 ng / mL LPS (Thermo Fisher Scientific) for 24 h. 100 µL of the supernatant was then reacted with 100 µL of 40 mg / mL Griss reagent (Thermo Fisher Scientific). The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) system (Thermo Fisher Scientific), and the IC50 was calculated. 50 .

[0046] Activity assays showed that compound 1 significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, with an inhibitory concentration (IC50) of 1 / 3.5%. 50 The concentration was 14.37 ± 2.36 µM, indicating that compound 1 has strong anti-inflammatory activity.

[0047] Furthermore, cytotoxicity was detected by the MTT assay (Yan, XY, Li, W., Wang, H., Dong, WH, Zeng, J., Wu, F., Mei, WL, Liang, LF, Dai, HF, 2025. Three new dimeric 2-(2-phenethyl)chromones from the agarwood of Aquilaria malaccensis. Phytochem. Lett. 66, 81–85.). The results showed that compound 1 at concentrations up to 100 µM showed no cytotoxicity after 24 h of LPS treatment, indicating that the anti-inflammatory activity of compound 1 was not due to cytotoxicity.

[0048] Table 2. Inhibitory activity of compound 1 on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells (IC50). 50 )

[0049]

[0050] 3.2. Anti-Helicobacter pylori activity

[0051] Using ATCC 43504 strain as the experimental subject, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound 1 against Helicobacter pylori (Hp) were determined. First, the strain was inoculated onto Columbia agar medium supplemented with 5% sterile sheep blood and cultured for 4 days at 37°C in a 5% oxygen tri-gas incubator (containing 10% carbon dioxide and the remainder nitrogen). Then, the culture was transferred to brain-heart extract agar (BHA) supplemented with 10% fetal bovine serum and cultured for 3 days at 37°C and 150 rpm. Compound 1 was dissolved in dimethyl sulfoxide (DMSO) and then diluted with culture medium to a gradient concentration of 160 µM, 80 µM, 40 µM, 20 µM, 10 µM, and 5 µM. 100 µL of each concentration sample was taken and added to a 100 µL suspension of Helicobacter pylori (concentration 1×10⁻⁶). 8 96-well plates (CFU / mL) were incubated in a microaerophilic environment at 37°C. After 48 hours, the lowest concentration that inhibited visible bacterial growth was recorded as the MIC. 10 µL of culture from each well was spotted onto a rapid urease test (RUT) reagent; the lowest concentration that reduced bacterial viability by 99.9% was defined as the MBC. Clarithromycin (CLA) was used as a positive control, and all experiments were repeated three times.

[0052] Activity test results showed that compound 1 exhibited anti-Helicobacter pylori activity, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 80 µM and 100 µM, respectively.

[0053] Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound 1 against Helicobacter pylori.

[0054]

[0055] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound isolated from agarwood, characterized in that, Its structure is shown in Equation 1: Formula 1.

2. The method for preparing the compound according to 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 was selected as the target fraction and subjected to octadecylsilane-bonded silica gel 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, In step (1), the agarwood 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 45.8 min.

8. The use of the compound of claim 1 in the preparation of anti-inflammatory, nitric oxide-inhibiting and / or Helicobacter pylori-resistant preparations.

9. An anti-inflammatory preparation, characterized in that, It contains the compound as described in claim 1.

10. An anti-Helicobacter pylori preparation, characterized in that, It contains the compound as described in claim 1.

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