A hybrid of 2-(2-phenylethyl) chromone and lignan and its application

Abstract

The present invention belongs to the field of agarwood, and in particular to a kind of 2-(2-phenylethyl) chromone and lignan heterocompound and its application. The present invention uses the agarwood produced by thick-leaved agarwood tree as raw material, and after reflux extraction, successively adopts silica gel, Sephadex LH-20 gel, high performance liquid chromatography and other chromatographic techniques to separate and purify to obtain 1 monomer compound, and its structure is identified by spectral methods such as mass spectrometry and nuclear magnetic resonance and combined with physicochemical properties. It is retrieved as a new compound by SciFinder and named as thick-leaved agarwood alcohol (aquicrassnol), which is the first heterocompound formed by 2-(2-phenylethyl) chromone and lignan found in agarwood. It has been verified by experiment that the compound has an inhibitory effect on the production of nitric oxide in mouse mononuclear macrophage RAW264.7 induced by lipopolysaccharide, and its half-maximal inhibitory concentration value is 47.53±2.14μmol / L, which has anti-inflammatory activity.

Description

Technical Field

[0001] The present invention belongs to the field of agarwood, and particularly relates to a 2-(2-phenylethyl) chromone and lignan hybrid and application thereof. Background Art

[0002] Aquilaria sinensis belongs to the genus Aquilaria sinensis of the family Thymelaeaceae Aquilaria Lam. or Aquilaria Gyrinops The wood of the plant (Gaertn.) contains resin and is a natural spice and precious traditional medicinal material. At present, the main plant sources of agarwood traded on the international market are white agarwood [ A. sinensis (Lour.) Spreng.], Malay Agarwood ( A. malaccensis Lam.), silk agarwood[ A. filaria (Oken) Merr.] and Aquilaria sinensis (also known as Aquilaria kerasina, A. crassna Pierre ex Lecomte). In the past, the above four plant sources of agarwood and the Aquilaria vara ( A. walla Gaertn) and Aquilaria sinensis ( G. salicifolia Extensive research on agarwood produced by the Randall Aquilaria spp. (Ridl) has revealed that 2-(2-phenylethyl)chromone and sesquiterpenes are the two main characteristic components of agarwood. Early 2-(2-phenylethyl)chromone isolations from agarwood mostly occurred as monomers, with only a few occurring as dimers or trimers. These unusual compounds represent a relatively unique class of chemical components in agarwood. Therefore, the study of dimer and trimer hybrids in agarwood provides important clues for the discovery of more structurally novel compounds in agarwood. Summary of the Invention

[0003] The present invention provides a 2-(2-phenylethyl) chromone and lignan hybrid and its application. The compound is isolated from agarwood produced by the Aquilaria crassifolia tree and is the first heteropolymer formed by 2-(2-phenylethyl) chromone and lignan found in agarwood. It is a new compound retrieved by SciFinder and named aquicrassnol, and it exhibits anti-inflammatory activity.

[0004] The technical solution of the present invention is achieved as follows:

[0005] A 2-(2-phenylethyl) chromone and lignan hybrid, named linalool, has a structure as shown in Formula I:

[0006] Ⅰ.

[0007] The preparation method of the above-mentioned 2-(2-phenylethyl) chromone and lignan hybrid comprises the following steps:

[0008] (1) The dried and crushed agarwood wood is extracted by heating and refluxing with ethanol water having a volume concentration of 95%, and the ethanol is recovered under reduced pressure until there is no alcohol smell to obtain an ethanol extract. The ethanol extract is dispersed into a suspension with water, extracted with ethyl acetate, and concentrated to obtain an ethyl acetate extract;

[0009] (2) The ethyl acetate extract was subjected to vacuum column chromatography and eluted with chloroform-methanol to obtain 11 fractions Fr. 1 to 11;

[0010] (3) The eighth fraction Fr.8 was subjected to MCI column chromatography and gradient elution with methanol / water to obtain nine fractions Fr. 8-1 to 8-9; the eighth fraction Fr. 8-8 was subjected to reverse phase silica gel column chromatography and gradient elution with methanol-water to obtain five fractions Fr. 8-8-1 to 8-8-5;

[0011] The second fraction Fr. 8-8-2 was separated by gel column chromatography and eluted with chloroform-methanol to obtain three fractions Fr. 8-8-2-1 to 8-8-2-3. The first fraction Fr. 8-8-2-1 was purified and separated by semi-preparative high performance liquid chromatography (HPLC) to obtain a compound whose structure is shown in the above formula I.

[0012] Furthermore, in the step (1), the agarwood is agarwood produced by the Aquilaria thick-leaved plant.

[0013] Furthermore, in the step (1), reflux extraction is performed 3 times and extraction is performed 3 times.

[0014] Furthermore, in the step (2), the volume ratio of chloroform to methanol is 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1, respectively; in the step (3), the volume ratio of methanol to water used for eluting the fraction Fr.8 is 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, respectively; the volume ratio of methanol to water used for eluting the fraction Fr.8-8 is 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, respectively.

[0015] Furthermore, in the step (4), the fraction Fr. 8-8-2 was separated by Sephadex LH-20 gel column chromatography, and the volume ratio of chloroform to methanol used for elution was 1:1.

[0016] Furthermore, in step (4), the HPLC purification conditions are: C 18 The mobile phase was acetonitrile-water with a volume ratio of 2:3 at a flow rate of 4 mL / min; the retention time of the compound represented by formula I was 15.12 min.

[0017] The application of the 2-(2-phenylethyl) chromone and lignan hybrid in the preparation of anti-inflammatory preparations.

[0018] Furthermore, the 2-(2-phenylethyl) chromone and lignan hybrid can inhibit the production of nitric oxide in mouse mononuclear macrophage RAW264.7 cells induced by lipopolysaccharide.

[0019] An anti-inflammatory preparation containing the above-mentioned 2-(2-phenylethyl)chromone and lignan hybrid.

[0020] Beneficial effects of the present invention:

[0021] This invention uses aquilaria sinensis (Aquilaria crassifolia) as the raw material. After reflux extraction, the compound was separated and purified using various chromatographic techniques, including silica gel, Sephadex LH-20 gel, and high-performance liquid chromatography, to obtain a monomeric compound. Its structure was identified by mass spectrometry, nuclear magnetic resonance, and other spectroscopic methods, combined with physicochemical properties. A SciFinder search revealed that the compound was a new compound and named aquicrassnol. This compound is the first hybrid discovered in aquilaria sinensis, consisting of 2-(2-phenylethyl)chromone and lignans. Experimental studies have shown that the compound inhibits lipopolysaccharide-induced nitric oxide production in mouse mononuclear macrophage RAW264.7 cells, with a 50% inhibitory concentration of 47.53±2.14 μmol / L, demonstrating anti-inflammatory properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 For compound 1 1 H NMR spectrum (500 MHz, CDCl3).

[0024] Figure 2 Compound 1 13 C NMR spectrum (125 MHz, CDCl3).

[0025] Figure 3 Compound 1 1 H- 1 H COSY plot.

[0026] Figure 4 HSQC spectrum of compound 1.

[0027] Figure 5 HMBC pattern of compound 1.

[0028] Figure 6 ROESY pattern of compound 1.

[0029] Figure 7 are the key 2D NMR related signals of compound 1.

[0030] Figure 8 The CD spectrum and chromophore exciton chirality of compound 1.

[0031] Figure 9 is the chemical structure of compound 1. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1 Isolation and identification of compounds

[0034] 1. Materials

[0035] The agarwood samples were collected in Vientiane, Laos. The original plant was identified by Huang Shengzhuo, an associate researcher at the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, as Aquilaria sepia (Aquilaria sepia, Thymelaeaceae). A. crassna ), voucher specimen (No. 20140909), is preserved in the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0036] 2 Extraction and separation

[0037] Dried, crushed agarwood (2.2 kg) was extracted three times with 95% ethanol and water (volume concentration) by heating and refluxing, adding 10 L of ethanol each time. The ethanol was recovered under reduced pressure until the alcohol flavor was eliminated, yielding an ethanol extract (296.0 g). The ethanol extract was then dispersed in water to form a suspension and extracted three times with ethyl acetate and n-butanol, respectively. The ethyl acetate extract (191.6 g) was purified by vacuum column chromatography using chloroform-methanol (volume ratios of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1, respectively). Identical fractions were combined and analyzed by thin-layer chromatography (the same below), yielding 11 fractions, Fr. 1–11. The eighth fraction Fr.8 was subjected to MCI column chromatography and eluted with methanol / water (volume ratios of 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, 2.5 L each gradient) to obtain nine fractions Fr. 8-1 to 8-9.

[0038] The eighth fraction Fr. 8-8 (13.5 g) was then subjected to reverse-phase silica gel column chromatography and eluted with methanol-water (volume ratios of 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, 2.5 L each gradient) to obtain five fractions Fr. 8-8-1 to 8-8-5.

[0039] The second fraction Fr. 8-8-2 (1.2 g) was purified by Sephadex LH-20 gel column chromatography using chloroform-methanol (volume ratio 1:1) as eluent to obtain three fractions Fr. 8-8-2-1 to 8-8-2-3. Fr. 8-8-2-1 was purified by semi-preparative HPLC (C 18 Compound 1 (3.1 mg, t R =15.12 min).

[0040] 3 Structure identification

[0041] Compound 1 is a pale yellow amorphous solid. [α] 25 D +64.0 ( c 0.15, MeOH); (nm):212 (4.83), 260 (4.27), 283 (3.65); (cm −1 ): 3 414, 2 928, 1 657, 1 599,1 508, 1 453, 1 239, 1 121, 1 029, 476; ECD (MeOH) λ max (Δ ε ): 272 (+4.4), 235 (−3.8), 226 (−4.0) nm, 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0042] Table 1 Compound 1 1 H (500 MHz) and 13 C (125 MHz) NMR data (CDCl3, δ in ppm, J in Hz)

[0043]

[0044] High-resolution mass spectrometry HR-ESI-MS m / z 1 101.370 9 [M+Na] + (C 58 H 62 O 20 The quasi-molecular ion peak of Na, calculated value 1101.372 7) indicates that the molecular formula of compound 1 is C 58 H 62 O 20 , the unsaturation is 28. Combining the above data and Figure 1-6 The spectral data of compound 1 were finally determined to be composed of two 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromone units and a lignan unit connected by two ether bonds.

[0045] Furthermore, according to the key 2D NMR related signals of compound 1 ( Figure 7 ) and CD spectra and chromophore exciton chirality ( Figure 8 ), and finally the structure of compound 1 was determined as Figure 9 As shown in the figure, it was retrieved as a new compound by SciFinder and named aquicrassnol. Its system name was (5 S ,5' S ,6 R ,6' R ,7 S ,7' S ,8 R ,8' R )-8,8'-((((1 R ,3a S ,4 R ,6a S )-tetrahydro-1 H ,3H -furo[3,4-c]furan-1,4-diyl)bis(2,6-dimethoxy-4,1-phenylene))bis(oxo))bis(5,6,7-trihydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydro-4 H -chromen-4-one).

[0046] Example 2 Anti-inflammatory activity test

[0047] 1 Cytotoxic activity

[0048] The cytotoxic activity of compound 1 on mouse mononuclear macrophage RAW264.7 cells was tested by MTT assay. RAW264.7 cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. 5 Cells were inoculated into 96-well plates at a concentration of 50 μg / mL and cultured for 24 h. A blank group, a positive drug doxorubicin hydrochloride group, and an experimental group were set up respectively. After the cells were placed in a 37°C, 5% CO2 cell culture incubator for 72 h, 15 μL of 5 mg / mL MTT solution was added to each well of cells. After reacting at 37°C for 4 h, the supernatant was aspirated and 100 μL of DMSO was added to each well to fully dissolve the cells. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader ( A ) values, and each experiment was repeated three times. The cell growth inhibition rate was calculated according to the formula.

[0049] Inhibition rate (%) = (1- A / A 0) × 100%

[0050] A 0. A are the absorbance of the blank group and the experimental group, respectively.

[0051] The experimental results showed that the cell survival rate (1-inhibition rate) of the above compounds was 96% at a concentration of 100 µmol / L, indicating that they had no cytotoxic activity.

[0052] 2. Inhibit the activity of NO production induced by lipopolysaccharide (LPS) in mouse mononuclear macrophage RAW264.7 cells

[0053] The anti-inflammatory activity of compound 1 was tested in the LPS-induced mouse mononuclear macrophage RAW264.7 cell model. RAW264.7 cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. 5Cells were inoculated with 100 μL of Griess reagent (40 mg / mL) in a 96-well plate and cultured for 24 h. A control group (without LPS), an LPS-induced group, a positive drug indomethacin group, and groups with different doses of the compound (100, 50, 25, 12.5, and 6.25 μmol / L) were set up. After adhering to the wall in a 37°C, 5% CO2 incubator for 24 h, 100 μL was taken and placed in a new 96-well plate. 100 μL (40 mg / mL) of Griess reagent was then added to each well. After mixing, the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader ( A ), each experiment was repeated three times. The NO inhibition rate was calculated according to the formula. Finally, the data were processed using GraphPad Prism software to determine the median inhibition concentration (IC) of compound 1 on LPS-induced nitric oxide (NO) production in RAW264.7 cells. 50 ).

[0054] Inhibition rate = ( A 2- A 1) / ( A 2- A 0)

[0055] A 0. A 1. A 2 were control group, drug group, and LPS-induced group A value.

[0056] The experimental results showed that compound 1 has the activity of inhibiting LPS-induced NO production in mouse mononuclear macrophage RAW264.7 cells, IC 50 The value was (47.53±2.14) μmol / L. The IC of the positive control indomethacin 50 The value was (25.28±1.14) μmol / L.

[0057] The present invention is the first to extract the A. crassna Aquicrassnol, a new hybrid formed by 2-(2-phenylethyl) chromone and lignans, was found in the agarwood produced by the Chinese Academy of Sciences. This compound has a certain inhibitory activity on the production of nitric oxide in RAW264.7 cells induced by lipopolysaccharide. IC 50 The value was (47.53±2.14) μmol / L, which showed anti-inflammatory activity.

[0058] Previously isolated heteropolymers from agarwood were composed of a 2-(2-phenylethyl)chromone unit linked to a sesquiterpene, flavonoid, or benzyl acetone unit. Compound 1, described in this invention, is a heteropolymer composed of two 2-(2-phenylethyl)chromone units linked to a lignan unit via a COC linkage, representing the first isolation from agarwood. The lignan unit is (+)-syringol, a component previously unisolated from agarwood.

[0059] 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 in the scope of protection of the present invention.

Claims

1. A 2-(2-phenylethyl) chromone and lignan hybrid, characterized in that: It is named as linalool, and its structure is shown in Formula I: Ⅰ。 2. The method for preparing a hybrid of 2-(2-phenylethyl)chromone and lignans according to claim 1, characterized in that: The following steps are involved: (1) The dried and crushed agarwood wood is extracted by heating and refluxing with ethanol water having a volume concentration of 95%, and the ethanol is recovered under reduced pressure until there is no alcohol smell to obtain an ethanol extract. The ethanol extract is dispersed into a suspension with water, extracted with ethyl acetate, and concentrated to obtain an ethyl acetate extract; (2) The ethyl acetate extract was subjected to vacuum column chromatography and eluted with chloroform-methanol to obtain 11 fractions Fr. 1 to 11; (3) The eighth fraction Fr.8 was subjected to MCI column chromatography and gradient elution with methanol / water to obtain nine fractions Fr. 8-1 to 8-9; the eighth fraction Fr. 8-8 was subjected to reverse phase silica gel column chromatography and gradient elution with methanol-water to obtain five fractions Fr. 8-8-1 to 8-8-5; (4) The second fraction Fr. 8-8-2 was separated by gel column chromatography and eluted with chloroform-methanol solution to obtain three fractions Fr. 8-8-2-1 to 8-8-2-3. The first fraction Fr. 8-8-2-1 was purified and separated by semi-preparative high performance liquid chromatography to obtain a compound whose structure is shown in Formula I. In the step (1), the agarwood is agarwood produced by the Aquilaria thick-leaved plant.

3. The preparation method according to claim 2, wherein In the step (1), reflux extraction is performed 3 times and extraction is performed 3 times.

4. The preparation method according to claim 2, wherein In the step (2), the volume ratio of chloroform to methanol is 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1, respectively; in the step (3), the volume ratio of methanol to water used for eluting the fraction Fr.8 is 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, respectively; the volume ratio of methanol to water used for eluting the fraction Fr.8-8 is 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0, respectively.

5. The preparation method according to claim 2, wherein In the step (4), the fraction Fr. 8-8-2 was separated by Sephadex LH-20 gel column chromatography, and the volume ratio of chloroform to methanol used for elution was 1:

1.

6. The preparation method according to claim 2, wherein In the step (4), the HPLC purification conditions are: C 18 The mobile phase was acetonitrile-water with a volume ratio of 2:3 at a flow rate of 4 mL / min; the retention time of the compound represented by formula I was 15.12 min.

7. Use of the 2-(2-phenylethyl)chromone and lignan hybrid according to claim 1 in the preparation of an anti-inflammatory preparation.

8. The use according to claim 7, characterized in that The 2-(2-phenylethyl) chromone and lignan hybrid can inhibit the production of nitric oxide in mouse mononuclear macrophage RAW264.7 cells induced by lipopolysaccharide.

9. An anti-inflammatory preparation, characterized in that The invention contains the hybrid of 2-(2-phenylethyl)chromone and lignan according to claim 1.

Images