A phenanthrenone compound, a preparation method and use thereof

Abstract

The present application relates to a kind of phenanthrene ketone compound, its preparation method and purposes in the field of biological medicine technology, the compound is extracted from Hainan leave calyx wood, molecular formula is C 17 H 16 O3, it is four reduced diterpene type modified phenanthrene ketone component with 6 / 6 / 5 tricyclic structure.Extraction, first Hainan leave calyx wood dry medicinal material is extracted with 95% ethanol, filter, filter liquor is combined, again, concentrated under reduced pressure and dried, obtain 95% ethanol extract;Water is added to disperse, and sequentially extracted with petroleum ether, ethyl acetate, n-butanol, obtain corresponding part extract;Finally, ethyl acetate part is chromatographically separated and purified to obtain.The research finds that the phenanthrene ketone compound and the Hainan leave calyx wood extract containing the compound of the present application have a certain degree of inhibitory effect on the release of NO of macrophage RAW264.7 induced by LPS, and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to new compounds discovered from plants and their applications, specifically a compound extracted and isolated from *Euonymus alatus* hainanensis, its preparation method, and its uses. Background Technology

[0002] Blachia siamensis Gagnep., commonly known as Hainan Blachia, is a perennial shrub belonging to the genus Blachia in the family Euphorbiaceae. It is mainly distributed in Hainan and Guangdong provinces of China.

[0003] However, there are no reports on the chemical composition of *Euonymus alatus* from either domestic or international sources. Therefore, in-depth research on the chemical composition of *Euonymus alatus* is urgently needed to isolate its active ingredients. Summary of the Invention

[0004] Objective of the Invention: To address the aforementioned problems, this invention provides a phenanthrene compound, its preparation method, and its uses. It is extracted and isolated from *Euonymus alatus* hainanensis, and a simple, rapid, and highly pure extraction and separation method for this new compound is also provided. Studies have shown that this new compound significantly inhibits NO release from RAW264.7 cells.

[0005] Technical solution: This invention provides a phenanthrene compound with the molecular formula Cp. 17 H 16 O3, named siamensisone, has the following chemical structural formula:

[0006]

[0007] The above compound was isolated from *Euonymus alatus*, and its preparation method includes the following steps:

[0008] A) After extracting the dried medicinal material of Hainan Euphorbia hainanensis with 95% ethanol solution, the extract was filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain 95% ethanol extract.

[0009] B) Disperse the 95% ethanol extract in water and extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction;

[0010] C) The ethyl acetate fraction was purified by chromatography to obtain a pure new compound.

[0011] The steps in step C) above, namely the chromatographic separation and purification of the ethyl acetate fraction, are as follows:

[0012] 1) The ethyl acetate fraction was subjected to column chromatography on a silica gel column and eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of (1:0) to (1:1) to obtain the petroleum ether-ethyl acetate eluent; preferably, the petroleum ether-ethyl acetate gradient elution was performed at volume ratios of 50:1, 25:1, 12:1, 6:1, 3:1, and 1:1.

[0013] 2) The petroleum ether-ethyl acetate eluent was subjected to column chromatography on MCI and ODS, respectively, and eluted with a methanol-water gradient of volume ratio (3:7) to (1:0) to obtain a methanol-water eluent; preferably, the methanol-water gradient elution was 3:7, 5:5, 7:3, 9:1, or 1:0.

[0014] 3) The methanol-water eluent was purified by semi-preparative high performance liquid chromatography and eluted with acetonitrile-water at a volume ratio of 53:47 to obtain a pure new compound from the acetonitrile-water eluent.

[0015] Specifically, the chromatographic separation and purification of the ethyl acetate fraction in step C) is as follows: elution is performed using a petroleum ether-ethyl acetate gradient with a volume ratio of (1:0) to (1:1), and the fractions are combined to obtain 14 fractions; fractions 6-9 are combined and subjected to MCI and ODS column chromatography, eluted with a methanol-water gradient with a volume ratio of (3:7) to (1:0), and the fractions are combined to obtain 6 fractions; fraction 3 is purified by semi-preparative high-performance liquid chromatography, eluted with acetonitrile-water with a volume ratio of 53:47, and the main peak is collected to obtain the compound.

[0016] This invention also includes the application of the phenanthrene compound and the extract of *Symplocos hainanensis* in the preparation of anti-inflammatory drugs. Isocyclic analogs or pharmaceutically acceptable salts of the phenanthrene compound can also be used to prepare anti-inflammatory drugs.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] The isolation, purification, and bioactivity studies of the novel compounds from *Euonymus alatus* described in this invention have not been reported in existing academic papers. This invention provides novel compounds derived from *Euonymus alatus* and a method for the extraction, separation, and purification of these novel compounds. The method employs alcohol extraction, silica gel column chromatography, MCI, ODS column chromatography, and semi-preparative HPLC for separation and purification, successfully obtaining the novel compounds. The operation method is simple and rapid, and the compounds obtained by this method have high purity, greater than 95%. Furthermore, studies have shown that these compounds and *Euonymus alatus* extracts possess inhibitory activity against LPS-induced RAW264.7NO release from macrophages, and can be used to prepare anti-inflammatory drugs. Attached Figure Description

[0019] Figure 1 The compounds of this invention1 H-NMR spectrum.

[0020] Figure 2 The compounds of this invention 13 C-NMR spectrum.

[0021] Figure 3 This is the HSQC spectrum of the compound of the present invention.

[0022] Figure 4 This is the HMBC spectrum of the compound of this invention.

[0023] Figure 5 This is the NOESY diagram of the compound of the present invention.

[0024] Figure 6 This is a high-resolution mass spectrum of the compound of the present invention.

[0025] Figure 7 This is a bar chart showing the inhibition rate of NO release by the compounds of this invention.

[0026] Figure 8 A bar chart showing the inhibition rate of NO release by extracts from different parts of *Euonymus alatus* hainanensis. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] Example 1

[0029] Preparation of new compounds

[0030] The material was collected in Ledong County, Hainan Province in April 2021, and the specimen is preserved in the Herbarium of Chinese Medicinal Herbs, Department of Pharmacy, Medical School, Yangzhou University.

[0031] Extraction and separation: 17.934 kg of Hainan Euonymus alatus medicinal material was chopped and extracted three times by reflux with 95% ethanol at 85℃. The extracts were combined, concentrated and dried under reduced pressure to obtain a total extract (766.3 g). The total extract was dispersed in 10 times its volume of distilled water and extracted successively with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction (56.17 g), ethyl acetate fraction (75.33 g), and n-butanol fraction (136.18 g). The ethyl acetate fraction was subjected to silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of (1:0) to (1:1), and the fractions were combined to obtain 14 fractions. Fractions 6-9 were combined and subjected to MCI and ODS column chromatography, eluted with a methanol-water gradient at a volume ratio of (3:7) to (1:0), and the fractions were combined to obtain 6 fractions. Fraction 3 was purified by semi-preparative high performance liquid chromatography, eluted with acetonitrile-water at a volume ratio of 53:47, and the main peak was collected to obtain the compound.

[0032] Example 2

[0033] Structural identification of new compounds

[0034] The novel compound prepared in Example 1 of this invention is a yellow solid. High-resolution mass spectrometry yielded a quasi-molecular ion peak at m / z 291.0980 [M+Na]+, combined with... 1 H-NMR and 13 C-NMR determined the molecular formula of the compound to be C 17 H 16 O3, with an unsaturation degree of 10. 1 H, 13 C-NMR and HSQC show four methyl groups (including one methoxy group), four methine groups, and nine quaternary carbons (including two carbonyl groups). In the HMBC spectrum, HMBC correlations exist first from H-1 to C-2, C-3, and Me-2 (C-16), from H-4 to C-3, C-5, and C-6, and from C-16 to C-1, C-2, and C-3, thus establishing a methyl group (δ-) at C-2. H 2.31, δ C 16.7) substitution and methoxy group (δ) at C-3 H 4.04, δ C 56.6) substituted benzene ring A. Secondly, the HMBC correlation between H-8 and C-6, C-10; H-1 to C-7 (δ C 184.2), H-4 to C-10 (δ) C 155.6) The carbonyl-substituted six-membered aromatic ring B at C-7, sharing C-5 and C-6 with ring A, was determined. Furthermore, HMBC correlations of Me-13 (C-14 or C-15) to C-9 and C-13, and NOESY correlations between Me-13 (C-14 or C-15) and H-8, as well as characteristic NMR data (δ¹⁵) of typical gem-dimethyl groups, were obtained. H 1.31,6H,s; δ C 23.5) is a geminal dimethyl group attached to C-9. The remaining carbonyl group (δ) C 208.4) and geminal dimethyl can form a five-membered ring C with a ketone structure, sharing C-9 and C-10 with the B ring.

[0035] Therefore, the structure of Example 1 can be determined and named siamensisone, which is a tetra-norditerpenoid phenanthrene ketone with a 6 / 6 / 5 tricyclic system. Table 1 shows the structure of the new compound. 1 H-NMR and 13 C-NMR data.

[0036] Analysis of structure-related signals:

[0037]

[0038] Table 1: The novel compound siamensisone of this invention 1 H-NMR (600MHz, CD3COCD3) and 13 C-NMR (150MHz, CD3COCD3) data

[0039] 1 129.5 7.93,s 2 131.1 3 162.6 4 108.0 7.73,s 5 132.4 6 125.7 7 184.2 8 121.3 6.45,s 9 162.4 10 155.6 11 129.1 7.27,s 12 208.4 13 46.3 14 23.5 1.31,s 15 23.5 1.31,s 16 16.7 2.31,s OMe-3 56.6 4.04,s

[0040] Example 4

[0041] Anti-inflammatory activity testing of new compounds

[0042] RAW264.7 cells (mouse mononuclear macrophage leukemia cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. RAW264.7 cells were seeded into 96-well plates and cultured overnight. The prepared drug was added and pre-incubated for 1 hour, followed by the addition of 10 μL of LPS (1 μg / mL). After 16 hours, 50 μL of cell culture supernatant was transferred to a new 96-well plate, and 50 μL each of Griess reagent A and B were added to each well. The absorbance was measured. A NO standard solution was prepared, and a linear equation was derived from the absorbance values ​​to calculate the NO concentration. The inhibition percentage of NO production was calculated using the formula (AL-AC) / (AL-A0) multiplied by 100%, where AL is the absorbance of the LPS-treated group, A0 is the absorbance of the normal control, and AC is the absorbance of the test compound and the LPS-treated group. The IC50 of each compound was calculated using a linear regression equation. 50 value.

[0043] The inhibition rate of NO production = (AL-AC) / (AL-A0)×100%. Where AL is the absorbance of the LPS-treated group, A0 is the absorbance of the normal control, and AC is the absorbance of the test compound and extract compared with the LPS-treated group.

[0044] Experimental results show that the novel compound of this invention has an effect on the IC50 of RAW264.7 cells. 50 The value was 4.57±1.49μM, indicating that siamensisone has in vitro anti-inflammatory activity against the above-mentioned inflammatory cells and can be used to prepare anti-inflammatory drugs.

[0045] The new compound siamensisone exhibits a concentration-dependent inhibition rate of NO release, such as... Figure 7 As shown.

[0046] To explore the medicinal value of *H. hainanensis*, based on modern herbal medicine development models and traditional application methods, we designed ethanol extracts (95% ethanol reflux, J1 and Y1) and water extracts (boiling water extraction, J2 and Y2) from the stems (HNLEM-J) and leaves (HNLEM-Y). The water and ethanol extracts from the stems and leaves were used for preliminary screening of NO inhibitory activity in LPS-induced RAW 264.7 cells at a concentration of 40 mg / mL. The results are as follows... Figure 8 As shown, both the stems and leaves, extracted with water or alcohol, exhibit certain anti-inflammatory effects; the water extract is relatively better than the alcohol extract. In particular, the petroleum ether extract (HNLEM-A) and ethyl acetate extract (HNLEM-B) from the alcohol extract were found to have strong anti-inflammatory effects, and the n-butanol extract (HNLEM-C) was also more effective than the total extracts from the water or alcohol extracts of the stems or leaves. Therefore, extracts from *Symplocos hainanensis* can be used to prepare anti-inflammatory drugs.

[0047] This invention is not limited to the above embodiments. Based on the disclosure of this invention, those skilled in the art can foresee without creative effort that similar ring systems or pharmaceutically acceptable salts of the phenanthrene compounds disclosed in this invention have anti-inflammatory effects and can be used to prepare anti-inflammatory drugs.

Claims

1. A phenanthrene compound, characterized in that, The molecular formula of the compound is C 17 H 16 O3, chemical structural formula as follows: 。 2. The method for preparing a phenanthrene compound according to claim 1, characterized in that, Includes the following steps: A) After extracting the dried medicinal material of Hainan Ephedra with 95% ethanol, the extract was filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain a 95% ethanol extract. B) Disperse the 95% ethanol extract in water and extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; C) The ethyl acetate fraction was purified by chromatography to obtain the compound; the chromatographic purification steps were as follows: 1) The ethyl acetate fraction was subjected to column chromatography on silica gel and eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of (1:0) to (1:1) to obtain the petroleum ether-ethyl acetate eluent. 2) The petroleum ether-ethyl acetate eluent was separated by MCI column and ODS column chromatography, and eluted with a methanol-water gradient at a volume ratio of (3:7) to (1:0) to obtain the methanol-water eluent. 3) The methanol-water eluent was purified by semi-preparative high performance liquid chromatography and eluted with acetonitrile-water at a volume ratio of 53:47 to obtain the pure compound from the acetonitrile-water eluent.

3. The method for preparing the phenanthrene compound according to claim 2, characterized in that: The specific steps for chromatographic separation and purification of the ethyl acetate fraction in step C) are as follows: elution is performed using a petroleum ether-ethyl acetate gradient with a volume ratio of (1:0) to (1:1), and the fractions are combined to obtain 14 fractions; fractions 6-9 are combined and subjected to MCI and ODS column chromatography, eluted with a methanol-water gradient with a volume ratio of (3:7) to (1:0), and the fractions are combined to obtain 6 fractions; fraction 3 is purified by semi-preparative high-performance liquid chromatography, eluted with acetonitrile-water with a volume ratio of 53:47, and the main peak is collected to obtain the compound.

4. The use of the phenanthrene compound of claim 1 in the preparation of an anti-inflammatory drug.

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