CN116813681B - A kind of 21,24-cyclized lanolin alkane triterpenoid compound and its preparation method and use - Google Patents

Abstract

The invention belongs to the technical field of natural medicines, and particularly relates to a 21, 24-cyclized lanolin alkane type triterpene compound in inonotus obliquus, and a preparation method and application thereof. In vitro anti-inflammatory activity experiments show that the compound has remarkable inhibition effect on inflammatory factor NO, and can effectively inhibit the gene expression level of the inflammatory mediators increased in RAW264.7 cells. The invention provides an alternative compound for developing new anti-inflammatory medicaments, which has very important significance for comprehensive development and utilization of inonotus obliquus.

Description

A 21,24-cyclized lanolin alkane triterpenoid compound and its preparation method and use

Technical field

The invention relates to the technical fields of natural products and pharmaceuticals, and in particular to a 21,24-cyclized lanolin alkyl triterpenoid compound extracted from Inonotus obliquus and its preparation method and use.

Background technique

Inonotus obliquus is a medicinal and edible fungus of the genus Fuscoporia, family Hymenochaetaceae, order Hymenochaetales. Among the people, Inonotus obliquus can be used to prevent and treat heart disease, diabetes and various cancers (gastric cancer, liver cancer, intestinal cancer, etc.). Research on the phytochemistry of Inonotus obliquus shows that its chemical components are mainly lanolin alkanes among tetracyclic triterpenoids, and there are also small amounts of pentacyclic triterpenes, steroids, sugars, phenols and other compounds. . Modern pharmacological research has found that Inonotus obliquus has good blood sugar regulation, anti-tumor, anti-inflammatory, and antioxidant effects, as well as anti-platelet aggregation and blood pressure lowering effects.

Therefore, it is of great significance for the comprehensive development and utilization of Inonotus obliquus to extract, isolate and develop new compounds with medicinal value from natural Inonotus obliquus.

Contents of the invention

In view of this, the present invention provides a 21,24-cyclized lanolin alkane triterpenoid compound isolated and purified from Inonotus obliquus and its preparation method and application.

The invention provides a new 21,24-cyclized lanolin alkane-type triterpenoid compound, which is isolated and purified from Inonotus obliquus, and its structural formula is as shown in formula (I):

The invention also provides a method for preparing the above-mentioned 21,24-cyclized lanolin alkane triterpenoid compound, which includes the following steps:

S1. Weigh the dried Inonotus obliquus, crush it, extract it with ethanol, and then concentrate the extract under reduced pressure to obtain ethanol extract;

S2. Dissolve the ethanol extract with methanol, then add petroleum ether for extraction, and concentrate the methanol part under reduced pressure to obtain the methanol extract;

S3. Dissolve the methanol extract again with a 9:1 (v/v) mixture of water and methanol, then add ethyl acetate for extraction, and concentrate the ethyl acetate part under reduced pressure to obtain the ethyl acetate extract;

S4. Perform normal-phase silica gel column chromatography on the ethyl acetate extract, gradient elution, and use TLC to detect and combine similar components to obtain 14 components (A ~ N);

S5. Perform medium-pressure reversed-phase column chromatography on the obtained K component, perform gradient elution, and use TLC to detect and combine similar components to finally obtain 14 components (K-1 ~ K-14);

S6. After component K-10 is separated by semi-preparative high performance liquid chromatography, the compound represented by formula (I) is obtained.

Further, in step S4, the conditions for gradient elution of the ethyl acetate extract are: using petroleum ether and ethyl acetate in a volume ratio of 20:1, 10:1, 9:1, 8:2, 7: 3, 6:4, 1:1, 0:1 for gradient elution.

Further, in step S5, the conditions for gradient elution of component K are: use water and methanol to perform gradient elution according to the volumes of 90:10, 70:30, 50:50, 30:70, and 0:100.

Further, in step S6, the high performance liquid chromatography conditions of component K-10 are: using acetonitrile and water to perform isocratic elution at a volume ratio of 45:55 and a flow rate of 3 mL/min.

The above-mentioned 21,24-cyclized lanolin alkane triterpenoids can be used to prepare anti-inflammatory drugs.

The beneficial effects brought by the technical solution provided by the present invention are: the present invention separates and purifies the ethanol extract of the medicinal fungus Inonotus obliquus to obtain a new compound, which is determined to be 21 by comprehensive application of multiple spectral analysis methods. , 24-cyclized lanolin alkane-type triterpenoids; through in vitro anti-inflammatory activity experiments on the obtained compounds, it was found that it has a significant inhibitory effect on the inflammatory factor NO, and can downregulate the inflammatory mediators TNF-α and IL -1β, IL-6, iNOS and COX-2 mRNA expression levels; the present invention provides candidate compounds for the development of new anti-inflammatory drugs, and is of great significance to the comprehensive development and utilization of Inonotus obliquus.

Description of the drawings

Figure 1 is a flow chart for the extraction and separation of 21,24-cyclized lanolin alkane triterpenoids prepared in Example 1 of the present invention;

Figure 2 is the ¹ H-NMR (500MHz, CD ₃ OD) spectrum of the compound prepared in Example 1 of the present invention;

Figure 3 is the ¹³ C-NMR (125MHz, CD ₃ OD) spectrum of the compound prepared in Example 1 of the present invention;

Figure 4 is the DEPT (Distortionless Enhancement by Polarization Transfer, distortion-free polarization transfer technology) (θ=90°) spectrum of the compound prepared in Example 1 of the present invention;

Figure 5 is the DEPT (θ=135°) spectrum of the compound prepared in Example 1 of the present invention;

Figure 6 is the HSQC (Heteronuclear Singular Qantum Correlation) spectrum of the compound prepared in Example 1 of the present invention;

Figure 7 is the HMBC (1H detected heteronuclear multiple bond correlation, 1H heteronuclear multiple carbon correlation spectrum) spectrum of the compound prepared in Example 1 of the present invention;

Figure 8 is the ¹ H- ¹ H COSY (Correlation spectroscopy, correlation spectrum) spectrum of the compound prepared in Example 1 of the present invention;

Figure 9 is the ROESY (Rotating Frame OverhauserEffect Spectroscopy, rotating coordinate system NOE spectrum) spectrum of the compound prepared in Example 1 of the present invention;

Figure 10 is the UV spectrum of the compound prepared in Example 1 of the present invention;

Figure 11 is the HR-ESI-MS spectrum of the compound prepared in Example 1 of the present invention;

Figure 12 shows the effects of different concentrations of the compounds of Example 1 on the viability of RAW264.7 cells;

Figure 13 shows the effect of the compounds of Example 1 on NO release, where CON: control group; LPS: model group; statistical data are expressed as mean ± SEM, ^### p<0.001, # indicates control group vs model group ; ***p<0.001, **p<0.01, *p<0.05, * indicates model group vs. drug administration group;

Figure 14 shows the effect of the compound of Example 1 on the expression of inflammatory mediator mRNA, where CON: control group; LPS: model group; DEX: dexamethasone; statistical data are expressed as mean ± SEM, ^### p<0.001 , the # sign indicates the control group vs the model group; ***p<0.001, **p<0.01, *p<0.05, the * sign indicates the model group vs the medication group.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the implementation of the present invention will be further described below with reference to specific examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1

This embodiment provides a preparation process for extracting 21,24-cyclized lanolin alkane triterpenoids from Inonotus obliquus. The specific steps are:

Step S1: Weigh 10.25kg of dried Inonotus obliquus, crush it and extract it with 95% (v/v) ethanol three times, soak it at room temperature for 24 hours each time, filter it after each soaking, and extract it three times. The filtered extracts were combined and concentrated under reduced pressure to obtain a total extract (170g); the Inonotus obliquus used in this example was purchased from Yichang City, Hubei Province and was identified as Dermatospora rusti by Associate Professor Liu Xinqiao of the School of Pharmacy, South Central University for Nationalities. Order Inonotus obliquus in the family Dermatomycetes.

Step S2: Dissolve the above total extract with methanol, then add petroleum ether for extraction, and concentrate the methanol part under reduced pressure to obtain a methanol extract (160g).

Step S3: Dissolve the methanol extract again with a 9:1 (v/v) mixture of water and methanol, then add ethyl acetate for extraction, and concentrate the ethyl acetate part under reduced pressure to obtain ethyl acetate extract (80g) .

Step S4: Perform normal phase silica gel column chromatography on the ethyl acetate extract, using petroleum ether-ethyl acetate gradient elution (petroleum ether:ethyl acetate=20:1, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1, v/v), using TLC (the developing solvent is chloroform: toluene: ethyl acetate: methanol = 10:4:1: (0.5～1.5) , v/v; the amount of methanol increases with the increase in the polarity of the compound) similar components were combined and 14 components were obtained, labeled A-N in order from small to large polarity, and the volume of petroleum ether-ethyl acetate was collected. The eluent with a ratio of 6:4, numbered K, was concentrated to dryness under reduced pressure and set aside.

Step S5: Dry load the K component (3.50g), use medium-pressure reversed-phase column chromatography, and use methanol and water (water:methanol = 90:10, 70:30, 50:50, 30:70 , 0:100, v/v) for gradient elution, through TLC (thin-layer chromatography, thin layer chromatography, the developing solvent is chloroform: toluene: ethyl acetate: methanol = 10:4:1: (0.5~ 1.5), v/v; the amount of methanol increases as the polarity of the compound increases) similar components were detected and merged, and finally 14 components were obtained, labeled K-1 to K- in ascending order of polarity. 14. Collect the eluent with a water-methanol volume ratio of 30:70, numbered K-10, concentrate to dryness under reduced pressure, and set aside.

Step S6: Use the semi-preparative column YMC-Pack ODS-A (250 × 10 mm, 5 μm) to purify the obtained K-10 component (27.48 mg) by HPLC (High Performance Liquid Chromatography, high performance liquid chromatography), and use acetonitrile and Water is used for isocratic elution at a flow rate of 3 mL/min at a volume ratio of 45:55, and the substance with a retention time t _R = 31.53 min is the 21,24-cyclized lanolin alkane triterpene of the present invention. compound, its weight is 0.80 mg.

The flow chart of the above steps is shown in Figure 1.

The compounds prepared in this example were subjected to high-resolution mass spectrometry, ultraviolet spectrum, optical rotation, and nuclear magnetic resonance analysis to determine the structure of the compound.

The physical and chemical data and spectral data of the compounds are as follows:

White amorphous powder; HR-ESI-MS m/z 473.36261[M+H] ⁺ (calcd for C ₃₀ H ₄₉ O ₄ ,473.36254); (c 0.05, MeOH); UV(MeOH) λ _max (logε): 240 (3.50); NMR data of the compound are shown in Table (2). ^{The 1} H NMR (500MHz, CD ₃ OD) spectrum is shown in Figure 2, ^{the 13} C-NMR (125MHz, CD ₃ OD) spectrum is shown in Figure 3, the DEPT (θ=90°) spectrum is shown in Figure 4, and the DEPT (θ= 135°) spectrum is shown in Figure 5, HSQC spectrum is shown in Figure 6, HMBC spectrum is shown in Figure 7, ¹ H- ¹ H COSY spectrum is shown in Figure 8, ROESY spectrum is shown in Figure 9, UV spectrum is shown in Figure 10, HR -ESI-MS spectrum is shown in Figure 11.

Table 1: Chinese and English names of compounds

Table 2: NMR data of compounds (Record in CD ₃ OD)

Measured at _a) CD ₃ OD

Example 2: In vitro anti-inflammatory activity test

In vitro anti-inflammatory activity experiments were performed on the compounds prepared in Example 1:

Materials and reagents: lipopolysaccharide (LPS), dexamethasone and dimethyl sulfoxide were purchased from Sigma Company in the United States; high-glucose DMEM medium was purchased from Gibco Company in the United States; penicillin-streptomycin solution was purchased from GE Company in the United States; fetal bovine serum It was purchased from GE Company of the United States; the CCK-8 kit was purchased from Guangzhou Biosharp Company; the nitric oxide detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

The tumor cell line tested: macrophage RAW264.7, purchased from Wuhan Pronosai Life Technology Co., Ltd. (CatNO: CL-0190).

(1) CCK-8 detection of cell viability

experimental method:

RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (containing penicillin and streptomycin double antibodies) until the growth was about 80% and passaged, and then RAW264.7 cells (1×10 ⁴ /well ) was inoculated into a 96-well plate with a final volume of 100 μL, and finally cultured in a 37°C cell culture incubator for 12 h. The compound of Example 1 was prepared with DMSO, the concentration of the stock solution was 10mM, and then diluted to different concentrations (50, 100μM) with DMEM culture medium, and added to the above-mentioned 96-well plate, with a final volume of 200μL. The experiment also set up a blank group (without cells, only adding DMEM medium) and a control group (adding DMEM medium, with cells). After incubating in the incubator for 24 hours, add 10 μL of CCK-8 solution, continue to incubate in the incubator in the dark for 1 hour, and then measure the OD value of each well at a wavelength of 450 nm. Calculate the cell survival rate (see Figure 12). The calculation formula of the cell survival rate is (OD _{experimental group} -OD _{blank group} )/(OD _{control group} -OD _{blank group} )×100%.

Experimental results:

As can be seen from Figure 12, the cytotoxicity screening results show that the compound has no obvious toxic effect on cells at concentrations of 50 and 100 μM.

(2) NO activity measurement

experimental method:

RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal calf serum (containing penicillin and streptomycin double antibodies) until the growth was about 80% and passaged, and then 1×10 ⁴ cells/well of RAW264.7 cells were cultured. Cells were seeded in 96-well plates and cultured in a 5% CO ₂ , 37°C cell culture incubator for 6 h. DMEM medium was used to prepare different concentrations (5, 10, 20, 40 μM) of the compound of Example 1 and 1 μg/mL lipopolysaccharide (LPS). Discard the old culture medium in the well, add only DMEM culture medium (without cells) to the blank group, add DMEM culture medium (with cells) to the control group, add 1 μg/mL LPS to the model group, and add 1 μg/mL LPS and different concentrations to the drug group. Add 100 μL of the compound of Example 1 to each well and incubate in an incubator for 12 hours. Then take 50 μL of the supernatant, detect the nitric oxide content in the supernatant according to the Griess method, and calculate its IC ₅₀ value. The experimental results are shown in Figure 13.

Experimental results:

Through the CCK-8 experiment, the concentrations of anti-inflammatory activity were set to 5, 10, 20, and 40 μM. Compared with the control group, the release of NO in the model group significantly increased after stimulating inflammatory cells with LPS. At the same time, compared with the model group, the release of NO decreases as the concentration of the compound increases, indicating that the compound of the present invention has an inhibitory effect on NO, and the calculated IC ₅₀ value is 20.3 μM.

(3) Determination of anti-inflammatory activity

experimental method:

RAW264.7 cells were cultured in DMEM high-glucose medium (containing penicillin and streptomycin double antibodies) containing 10% fetal bovine serum until the growth was about 80% and passaged, and then RAW 264.7 macrophages (3 × 10 ⁴ /mL) was inoculated into a 12-well plate, and after incubation for 24 hours in a cell culture incubator with 5% CO ₂ and 37°C, different concentrations (10, 20, 40 μM) of the compound of Example 1 were added, and after continuing to incubate for 1 hour, 1 μg/mL was added. Lipopolysaccharide, and then incubated for 3 hours, the compound of Example 1 and lipopolysaccharide were used to prepare solutions in DMEM culture medium. Remove the culture medium, collect the cells, use RNAiso Plus to extract total RNA from RAW264.7, then use a reverse transcription kit to reverse transcribe the RNA into cDNA, and use real-time fluorescence quantitative PCR method to detect the expression level of inflammatory factor mRNA of the test compound. Impact. The relative RNA expression levels were calculated by 2 ^-ΔΔCt and normalized to β-Actin. The gene-specific primers used for qPCR are shown in Table (3). The experiment also set up a blank group (only adding DMEM medium, no cells), a control group (adding DMEM medium, with cells), a model group (adding 1 μg/mL LPS) and a positive group (adding 20 μM dexamethasone).

Table 3 Gene-specific primer sequences for qPCR

Experimental conclusion: Compared with the control group, the mRNA levels of inflammatory cytokines TNF-α, IL-1β, IL-6, COX-2 and iNOS were significantly increased in the model group ( ^### p<0.001). Compared with the model group, the low, medium and high dose groups of the compound can down-regulate the elevated mRNA levels of TNF-α, IL-1β, IL-6 and iNOS in the model group. Therefore, the compound of the present invention can effectively inhibit RAW264.7 Elevated gene expression levels of inflammatory mediators in cells (Figure 14).

The above-described embodiments and features in the embodiments herein may be combined with each other if there is no conflict.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (6)

1. A 21, 24-cyclized lanolin alkane type triterpene compound, which has the structural formula:

2. the 21, 24-cyclized lanolin alkane type triterpene compound according to claim 1, wherein said compound has a structural formula:

3. a process for the preparation of a compound as claimed in claim 1 or 2, comprising the steps of:

s1, weighing and drying inonotus obliquus, crushing, leaching with ethanol, and concentrating the extracting solution under reduced pressure to obtain ethanol extract;

s2, dissolving the ethanol extract with methanol, then adding petroleum ether for extraction, and concentrating the methanol part under reduced pressure to obtain a methanol extract;

s3, dissolving the methanol extract again by using a mixed solution of water and methanol in a volume ratio of 9:1, then adding ethyl acetate for extraction, and concentrating an ethyl acetate part under reduced pressure to obtain an ethyl acetate extract;

s4, carrying out normal phase silica gel column chromatography on the ethyl acetate extract, and carrying out gradient elution to obtain A-N14 components;

s5, performing medium-pressure reverse phase column chromatography on the obtained K components, and performing gradient elution to finally obtain K-1-K-1414 components;

s6, separating the K-10 component by semi-preparative high performance liquid chromatography to obtain the compound;

the conditions of the gradient elution of the ethyl acetate extract are as follows: petroleum ether-ethyl acetate is used as an eluent, gradient elution is carried out according to the volume ratio of 20:1, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 0:1, and similar components are detected and combined by TLC, wherein the developing agent is a mixed solution of chloroform-toluene-ethyl acetate-methanol;

the conditions for the gradient elution of the component K are as follows: performing gradient elution by using water-methanol as an eluent according to the volume ratio of 90:10, 70:30, 50:50, 30:70 and 0:100, and detecting and combining similar components by TLC, wherein the developing agent is a mixed solution of chloroform-toluene-ethyl acetate-methanol;

the semi-preparative high performance liquid chromatography conditions of the K-10 component are as follows: the mixed solvent of acetonitrile and water is used as eluent, and the following steps are carried out according to acetonitrile: the water volume ratio was 45:55 for isocratic elution with a flow rate of 3mL/min.

4. Use of a 21, 24-cyclized lanostane-type triterpene compound according to claim 1 or 2 for preparing a medicament for inhibiting NO production.

5. Use of a 21, 24-cyclized lanostane-type triterpene compound according to claim 1 or 2 for the preparation of a medicament for inhibiting the expression level of an inflammatory factor mRNA.

6. Use of 21, 24-cyclized lanostane-type triterpenes according to claim 1 or 2 for the preparation of anti-inflammatory medicaments.