A cembranoid diterpenoid compound, and an extraction method and application thereof

Abstract

The application belongs to the technical field of natural medicine, and particularly relates to a cembranoid diterpenoid compound and an extraction method and application thereof. The structure of the compound is shown as formula I, and the compound is extracted through the following steps: S1: mixing Tripterygium wilfordii Hook. f. roots and 95% ethanol solution, heating and refluxing extraction, concentrating the extract, and extracting the concentrated solution with ethyl acetate; S2: performing column chromatography on the extract obtained in S1 with aluminum oxide, and eluting with anhydrous ethanol; concentrating the eluent into an extract, and drying and crushing; S3: extracting the crushed extract obtained in S2 with petroleum ether for multiple times, combining the extract, and concentrating under reduced pressure to obtain an extract; and S4: separating and purifying the extract obtained in S3 to obtain the compound shown as formula I. It is verified through experiments that the compound disclosed in the application has good anti-inflammatory effect, and can be applied to the preparation of anti-inflammatory drugs. The extraction method disclosed in the application is simple, rapid, and has high purity of the compound separated by the method, and the purity is greater than 95%.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a rosinane-type diterpenoid compound, its extraction method, and its application. Background Technology

[0002] Thunder vine ( Tripterygium wilfordii *Tripterygium wilfordii* (Hook.F.) is a plant belonging to the genus *Tripterygium* in the family Celastraceae. Its root is used medicinally and has a history of use in traditional Chinese medicine for hundreds of years. It is believed to have effects such as dispelling wind and dampness, promoting blood circulation, reducing swelling and relieving pain, and killing parasites and detoxifying. Clinically, it is commonly used to treat rheumatoid arthritis, glomerulonephritis, nephrotic syndrome, lupus erythematosus, Sjögren's syndrome, Behçet's disease, eczema, psoriasis, leprosy, scabies, and stubborn tinea. Its preparations, such as Tripterygium wilfordii tablets, Tripterygium wilfordii polyglycoside tablets, and Tripterygium wilfordii total terpenoid tablets, are widely used clinically with significant efficacy.

[0003] Phytochemical studies have shown that the roots of *Tripterygium wilfordii* mainly contain diterpenoids, triterpenoids, and sesquiterpenoid alkaloids. Pharmacological studies have revealed that *Tripterygium wilfordii* roots possess various pharmacological effects, including anti-inflammatory, immunosuppressive, antitumor, antiviral, insecticidal, antioxidant, and neuroprotective properties. Current technology suggests that triptolide (a diterpenoid) and celastrol (a triterpenoid) are the main anti-inflammatory components of *Tripterygium wilfordii* roots and their preparations. However, current technology has not yet achieved effective isolation and identification of substances with anti-inflammatory activity in *Tripterygium wilfordii* roots, and further research is needed on these substances.

[0004] To further explore new anti-inflammatory active substances from Tripterygium wilfordii root, this application conducted in-depth research on the chemical composition of Tripterygium wilfordii root and screened its anti-inflammatory activity, from which a new rosinane-type diterpenoid compound was isolated. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rosinane-type diterpenoid compound, its extraction method and application.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] Technical Topic 1

[0008] A rosinane-type diterpenoid compound, the structure of which is shown in Formula I:

[0009] .

[0010] Technical Theme Two

[0011] A method for extracting the compound described in Technical Subject 1, comprising the following steps:

[0012] S1: Mix Tripterygium wilfordii root with 95% ethanol solution and heat under reflux to extract. Concentrate the extract and extract the concentrate with ethyl acetate.

[0013] S2: The extract obtained in S1 was subjected to alumina column chromatography, eluted with anhydrous ethanol, and the eluent was concentrated into a paste, dried and pulverized.

[0014] S3: Extract the powdered extract from S2 multiple times with petroleum ether, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0015] S4: The extract obtained in S3 was separated and purified to obtain the compound shown in Formula I.

[0016] As a further improvement of the present invention, the mass-to-volume ratio of Tripterygium wilfordii root and ethanol solution in S1 is 1kg:8L-1kg:12.5L; the extraction temperature is 70~80℃, the extraction time is 1-2 h each time, and the extraction is repeated 3 times;

[0017] The extract in S3 was extracted three times with petroleum ether.

[0018] As a further improvement of the present invention, the separation and purification includes the following steps:

[0019] Step 1: Dissolve the extract obtained from S3 in dichloromethane, add an equal mass of 100-200 mesh silica gel and mix well. After evaporating the solvent, the silica gel containing the sample is obtained. Under normal pressure, perform gradient elution with petroleum ether-acetone at a volume ratio of 100:0 to 1:1 to obtain 12 components, denoted as Fr.A ~ Fr.L.

[0020] Step 2: Fr.F was further separated by silica gel column chromatography under normal pressure, with a gradient elution of petroleum ether-dichloromethane at a volume ratio of 5:1 to 1:5, to obtain 9 components, denoted as Fr.Fa ~ Fr.Fj;

[0021] Step 3: Fr.Fd was separated by reversed-phase column chromatography under normal pressure ODS, using a gradient elution of acetone-water at a volume ratio of 60:40 to 100:0 to obtain 5 subfractions, denoted as Fr.Fd-1 ~ Fr.Fd-5;

[0022] Step 4: Fr.Fd-2 was separated by ODS reversed-phase column chromatography at ambient pressure, using a gradient elution of acetone-water at a volume ratio of 70:30 to 100:0 to obtain the compound shown in Formula I.

[0023] Technical Theme 3

[0024] A composition comprising a rosinane-type diterpenoid compound as described in Technical Subject 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0025] Technical Theme 4

[0026] The use of the rosinane-type diterpenoid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof as described in Technical Subject 1 in the preparation of anti-inflammatory drugs.

[0027] The beneficial effects of adopting the above technical solution are as follows:

[0028] The rosinane-type diterpenoid compound provided by this invention is the first to be extracted and isolated. Experimental verification shows that it can significantly inhibit the release of NO in RAW264.7 and significantly inhibit the production of IL-6 in RAW264.7, demonstrating that it has good anti-inflammatory effects. This lays the foundation for the development of new drugs based on this compound or its pharmaceutically acceptable salt or its stereoisomers.

[0029] The extraction method provided by this invention is simple and fast, and the compounds separated by this method have high purity, >95%. Attached Figure Description

[0030] Figure 1 For the compound Triptotin L 1 H-NMR spectrum;

[0031] Figure 2 For the compound Triptotin L 13 C-NMR spectrum;

[0032] Figure 3 The HSQC spectrum of compound Triptotin L;

[0033] Figure 4 The ¹H- of compound Triptotin L 1 H COSY spectrum;

[0034] Figure 5 The HMBC spectrum of compound Triptotin L;

[0035] Figure 6 The NOESY spectrum of compound Triptotin L;

[0036] Figure 7 This is the high-resolution mass spectrum of the compound Triptotin L;

[0037] Figure 8 The ultraviolet spectrum of compound Triptotin L;

[0038] Figure 9 This is a circular dichroism chromatogram of the compound Triptotin L. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0040] The mouse mononuclear macrophage leukemia cells RAW264.7 used in this application were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0041] The NO kit was purchased from Beyotime Biotechnology Co., Ltd., catalog number: S0021S.

[0042] The IL-6 kit was purchased from Linko Biotechnology Co., Ltd., catalog number: EK206HS.

[0043] Example 1 Extraction of the target compound

[0044] S1: Mix the pulverized Tripterygium wilfordii root with 95% ethanol solution and heat under reflux for extraction three times at a temperature of 70-80℃ for 1.5 hours each time. The mass-to-volume ratio of Tripterygium wilfordii root to 95% ethanol solution is 1 kg: 10 L. After the extract is concentrated under reduced pressure, it is extracted with ethyl acetate.

[0045] S2: The extract obtained in S1 was subjected to alumina column chromatography, eluted with anhydrous ethanol, and the eluent was concentrated into a paste, dried and pulverized.

[0046] S3: Extract the pulverized extract obtained in S2 with petroleum ether. Extract three times with equal volumes, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0047] S4: Purify the obtained extract, specifically including the following steps:

[0048] Step 1: Dissolve the extract obtained from S3 in dichloromethane, add an equal mass of 100-200 mesh silica gel and mix well. After evaporating the solvent, the silica gel containing the sample is obtained. Under normal pressure, perform gradient elution with petroleum ether-acetone 100:0→1:1 (v / v). The fractions are identified and combined by thin-layer chromatography to obtain 12 components, denoted as Fr.A ~ Fr.L.

[0049] Step 2: Fr.F was further separated by silica gel column chromatography under normal pressure. Nine fractions were obtained by elution with petroleum ether-dichloromethane (elution buffer v / v, 5:1→1:3), denoted as Fr.Fa ~ Fr.Fj.

[0050] Step 3: Fr.Fd was separated by reversed-phase column chromatography under normal pressure ODS with acetone-water (elution buffer v / v, 60:40→100:0) gradient elution to obtain 5 subfractions, denoted as Fr.Fd-1 ~ Fr.Fd-5.

[0051] Step 4: Fr.Fd-2 was separated by ODS reversed-phase column chromatography at ambient pressure using acetone-water gradient elution (eluent v / v, 70:30→100:0) to obtain the target compound Triptotin L, with a purity greater than 95%.

[0052] The compound Triptotin L extracted in this embodiment data, The data is shown in Table 1. See the spectrum Figure 1 , See the spectrum Figure 2 HSQC spectrum (see) Figure 3 , See the spectrum Figure 4 HMBC spectrum (see) Figure 5 See NOESY spectrum Figure 6 High-resolution mass spectra are shown below. Figure 7 Ultraviolet spectra are shown Figure 8 Circular dichroism chromatogram Figure 9 Through the analysis using the above technical methods, the structure of the compound was confirmed as follows, and it was named Triptotin L.

[0053]

[0054] Table 1. Compound Triptotin L 1 H and 13 C-NMR data (CDCl3)

[0055]

[0056] Example 1: Evaluation of the anti-inflammatory activity of compound Triptotin L

[0057] Mouse mononuclear macrophage leukemia cells RAW264.7 were cultured in DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin, and were routinely cultured at 37ºC with 5% CO2.

[0058] RAW264.7 cells in the logarithmic growth phase were cultured at 1×10⁻⁶. 5Each well was seeded with one sample of the same substance. After incubation for 24 h in 0.5 ml of DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin / streptomycin, the medium was discarded. Three replicates were then created by adding 0.5 ml of DMEM medium containing 20, 15, 10, 5, and 2.5 μmol / L Triptotin L, respectively, in a mixture of 10% fetal bovine serum and 1% penicillin / streptomycin. After 4 h of incubation, 10 μL of lipopolysaccharide (LPS) solution was added to each well to a final concentration of 1 μg / mL to induce NO production. After another 20 h of incubation, the medium was collected, centrifuged at 3000 r / min for 20 min, and the supernatant was used.

[0059] 1) Follow the instructions for the NO kit and calculate the NO concentration in each sample based on the standard curve and absorbance values ​​of each group. Experimental results show that Triptotin L significantly inhibits NO release from RAW264.7, as detailed in Table 2.

[0060] 2) Follow the instructions for the IL-6 kit and calculate the concentration of IL-6 in each sample based on the standard curve and absorbance values ​​of each group. Experimental results show that Triptotin L significantly inhibits the production of IL-6 in RAW264.7, as detailed in Table 2.

[0061] Table 2 Anti-inflammatory activities of compounds

[0062]

[0063] The EC of the compound Triptotin L,NO disclosed in this application is... 50 The EC50 of IL-6 was 10.04 ± 0.49. 50 The value was 14.92 ± 2.76, indicating that the compound has good anti-inflammatory activity.

[0064] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cembranoid diterpene compound, characterized by, The structure is shown as formula I: 。 2. A method of extracting the compound of claim 1, characterized in that, It comprises the following steps: S1: mixing the root of Tripterygium wilfordii Hook.f. with 95% ethanol solution and heating to reflux extraction, concentrating the extract, and extracting the concentrated solution with ethyl acetate; S2: performing column chromatography on the extract obtained in S1 with alumina, eluting with anhydrous ethanol, concentrating the eluate into an extract, and drying and crushing; S3: extracting the crushed extract in S2 with petroleum ether for multiple times, combining the extract, and concentrating under reduced pressure to obtain an extract; S4: obtaining the compound shown as formula I after separating and purifying the extract obtained in S3; In S1, the mass-volume ratio of the root of Tripterygium wilfordii Hook.f. and the ethanol solution is 1 kg:8 L-1 kg:12.5 L; the extraction temperature is 70-80 DEG C; the extraction time is 1-2 h each time; and the extraction is repeated for 3 times; In S3, the extract is extracted with petroleum ether for 3 times; The separating and purifying comprises the following steps: Step 1: dissolving the extract obtained in S3 with dichloromethane, mixing with silica gel of 100-200 mesh in an amount equal to that of the extract, drying the solvent to obtain silica gel mixed with the sample, and performing gradient elution with petroleum ether-acetone (volume ratio 100:0→1:1) under normal pressure to obtain 12 components, denoted as Fr.A ~ Fr.L; Step 2: further separating Fr.F with normal pressure silica gel column chromatography, performing gradient elution with petroleum ether-dichloromethane (volume ratio 5:1→1:5) to obtain 9 components, denoted as Fr.Fa ~ Fr.Fj; Step 3: separating Fr.Fd with normal pressure ODS reverse phase column chromatography, performing gradient elution with acetone-water (volume ratio 60:40→100:0) to obtain 5 sub-components, denoted as Fr.Fd-1 ~ Fr.Fd-5; Step 4: separating Fr.Fd-2 with normal pressure ODS reverse phase column chromatography, performing gradient elution with acetone-water (volume ratio 70:30→100:0) to obtain the compound shown as formula I.

3. A composition containing the abietane diterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1.

4. Use of the abietane diterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-inflammatory drug.

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