A sesquiterpene dimer compound and its preparation method and application

Abstract

The invention belongs to the field of medical technology, and discloses a sesquiterpene dimer compound and a preparation method and application thereof. The compound of the invention is separated from lilac, and lilac is extracted with alcohol, and extracted with dichloromethane and ethyl acetate. The dichloromethane extraction part is first separated by silica gel column chromatography, and then separated by silica gel column chromatography and preparative liquid chromatography in sequence. The preparation method of the invention is simple to operate, and the sesquiterpene dimer compound can be separated and obtained. Experiments show that the compound can significantly reduce the content of NO in LPS-induced RAW264.7 cells, has little toxic side effects, and can be used for the preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a sesquiterpene dimer compound and a preparation method and application thereof. Background Art

[0002] Inflammation is the body's natural defense response to infection, injury or other stimuli, characterized by local redness, swelling, heat, pain and dysfunction, as well as possible systemic reactions such as fever and changes in white blood cell count. The causes of inflammation are diverse, including biological factors (such as bacteria, viruses), physical factors (such as high temperature, low temperature), chemical factors, necrotic tissue and immune response. Inflammation can be divided into acute and chronic, among which chronic inflammation is associated with a variety of diseases such as cardiovascular disease, malignant tumors, diabetes, chronic kidney disease, etc. Traditional Chinese medicine has unique advantages in the treatment of inflammation, and its treatment concepts and methods are increasingly valued in modern medicine. The anti-inflammatory effect of traditional Chinese medicine is mainly achieved through the following mechanisms: 1. Regulating the immune system: Various ingredients in traditional Chinese medicine can affect the activity of immune cells and regulate the intensity and direction of immune responses, such as promoting lymphocyte proliferation or inhibiting excessive immune responses. 2. Improving blood circulation: The blood-activating and stasis-removing ingredients in traditional Chinese medicine can dilate blood vessels, increase blood flow, improve microcirculation, and thus relieve inflammatory symptoms. 3. Inhibit inflammatory mediators: Chinese herbal ingredients can inhibit the expression of inflammatory cytokines, reduce the activation and aggregation of inflammatory cells, and reduce inflammatory responses through antioxidant effects. Therefore, discovering and developing new anti-inflammatory drugs from Chinese herbal medicine has become one of the hot topics in current drug research.

[0003] lilac Syringaoblata Lindl. is a deciduous tree of the genus Syringa in the family Oleaceae. It is warm in nature, pungent in taste, and enters the spleen, stomach, and lungs. It has antibacterial, anti-inflammatory, antiviral, and liver-protecting and choleretic effects. The Xinhua Compendium of Materia Medica and the Changbai Mountain Plant Medicinal Records respectively record that it has the pharmacological activity of treating "acute icteric hepatitis" and "treating diarrhea and hepatitis". Therefore, it is of great significance to discover new anti-inflammatory active natural products from lilac plants. Summary of the invention

[0004] The purpose of the present invention is to conduct in-depth research on the chemical components of lilac stems, provide a sesquiterpene dimer compound and a preparation method and application thereof, and the research finds that the compound of the present invention has anti-inflammatory effect.

[0005] The technical solution of the present invention is as follows:

[0006] The sesquiterpene dimer compound extracted and separated from lilac has the following structural formula:

[0007] .

[0008] The present invention also provides a method for preparing a sesquiterpene dimer compound, comprising the following steps:

[0009] (1) Take the stem of Syringa japonica, add ethanol solution to extract, filter the extract, combine the extracts, concentrate until there is no alcohol taste, and then extract with dichloromethane and ethyl acetate in sequence to obtain dichloromethane extraction part and ethyl acetate extraction part respectively;

[0010] (2) Take the extract of the dichloromethane extraction part obtained in step (1), dissolve it in methanol, dry-mix it with silica gel, load it into a silica gel column for column chromatography separation, and then use a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 100:0, 50:1, 25:1, 10:1, 5:1, 1:1, and 0:100, and use silica gel GF 254 After inspection of the thin layer plate, fractions containing similar spots were combined to obtain 12 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12;

[0011] (3) Dissolve the fraction 3 obtained in step (2) in ethyl acetate, dry-mix the sample with silica gel, load the sample onto a silica gel column for column chromatography, and then gradiently elute with petroleum ether-ethyl acetate in a volume ratio of 25:1, 10:1, 5:1, 1:1, and 0:100 to obtain 6 fractions a, b, c, d, e, and f;

[0012] (4) Take the fraction d obtained in step (3) and dissolve it in methanol, and then separate it by preparative liquid chromatography, with the mobile phase being CH 3 CN and H 2 O, at retention time t R = 43 min to obtain the target compound.

[0013] Preferably, the extraction method in step (1) is cold soaking, percolation, microwave extraction, ultrasonic extraction or reflux extraction. More preferably, reflux extraction is performed twice, each time for 2 hours, and the extraction temperature is 85°C.

[0014] Preferably, the ethanol solution used in step (1) is an ethanol aqueous solution with a volume concentration of 5% to 95%, more preferably a 70% ethanol aqueous solution. The amount of ethanol aqueous solution added during extraction is 3 to 50 times the volume of the medicinal material, more preferably 10 times.

[0015] Preferably, in step (2), the mass ratio of the dichloromethane extraction part to the silica gel used for sample mixing is 1:2.

[0016] Preferably, in step (2), the silica gel used for sample mixing is 100-200 mesh, and the silica gel used in the silica gel column is 100-200 mesh.

[0017] Preferably, in step (3), the silica gel used for sample mixing is 200-300 mesh, and the silica gel used in the silica gel column is 200-300 mesh.

[0018] Preferably, in step (4), the conditions for preparative liquid chromatography are: flow rate 10 ml / min.

[0019] The invention also provides the use of the sesquiterpene dimer compound in the preparation of anti-inflammatory drugs.

[0020] Preferably, the anti-inflammatory model is an LPS-induced RAW264.7 cell model.

[0021] The invention also provides a pharmaceutical composition comprising the sesquiterpene dimer compound.

[0022] The present invention also provides a pharmaceutical preparation, comprising a therapeutically effective amount of a sesquiterpene dimer compound and a pharmaceutically acceptable carrier or excipient.

[0023] Those skilled in the art can directly or indirectly add the compound to various commonly used pharmaceutically acceptable excipients required for preparing different dosage forms, such as fillers, disintegrants, lubricants, adhesives, etc., and prepare commonly used oral preparations or injection preparations using conventional pharmaceutical preparation methods.

[0024] Preferably, the oral preparation is a tablet, a capsule, a granule, a fat emulsion, a microcapsule, or a pill.

[0025] Preferably, the injection preparation is an injection solution or a powder injection.

[0026] The beneficial effects of the present invention are:

[0027] The preparation method of the compound of the invention is simple to operate, and the sesquiterpene dimer compound can be separated from lilac.

[0028] Experiments show that the compound of the present invention can significantly reduce the NO content in LPS-induced RAW246.7 cells, has good anti-inflammatory effect, has little toxic and side effects, and can be used for the preparation of anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The chemical structural formula of the compound prepared by the present invention;

[0030] Figure 2 Mass spectra of compounds prepared according to the present invention;

[0031] Figure 3 The compounds prepared by the present invention 1 H NMR spectrum;

[0032] Figure 4 The compounds prepared by the present invention 13 C NMR spectrum;

[0033] Figure 5 DEPT NMR spectrum of the compound prepared by the present invention;

[0034] Figure 6 HSQC spectrum of the compound prepared by the present invention;

[0035] Figure 7 HMBC spectrum of the compound prepared by the present invention;

[0036] Figure 8 The compounds prepared by the present invention 1 H- 1 H COSY spectrum;

[0037] Fig. 9 The effects of the compounds prepared in the present invention on the cytotoxicity of RAW264.7 cells;

[0038] Fig.10 This is the effect of the compound prepared in the present invention on LPS-induced NO in RAW264.7 cells. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0041] Example 1 Preparation of Compounds

[0042] This embodiment provides a method for preparing a sesquiterpene dimer compound, the steps of which are:

[0043] (1) Take 50 kg of lilac stem medicinal material, add 10 times the volume of 70% ethanol aqueous solution, reflux extract twice at 85°C, each extraction for 2 hours, filter after extraction, combine the extracts, and concentrate until there is no alcohol taste to obtain an extract concentrate; take the extract concentrate, first extract it three times with 3 times the volume of the concentrate each time with dichloromethane, then extract it three times with 3 times the volume of the concentrate with ethyl acetate, respectively, combine the dichloromethane extract and ethyl acetate extract, concentrate under reduced pressure at 40°C, and dry under reduced pressure at 60°C to obtain a dichloromethane extract fraction and an ethyl acetate extract fraction;

[0044] (2) Take the extract of the dichloromethane extraction part obtained in step (1), dissolve it in methanol, dry-mix it with silica gel (100-200 mesh, twice the mass of the dichloromethane extraction part), load it into a silica gel (100-200 mesh) column, perform column chromatography separation, and then use a petroleum ether-ethyl acetate gradient elution with a volume ratio of 100:0, 50:1, 25:1, 10:1, 5:1, 1:1, 0:100, and use silica gel GF 254 After inspection of the thin layer plate, fractions containing similar spots were combined to obtain 12 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12;

[0045] (3) Dissolve the fraction 3 obtained in step (2) in ethyl acetate, dry-mix the sample with silica gel (200-300 mesh), load the sample onto a silica gel (200-300 mesh) column for column chromatography, and then gradiently elute with petroleum ether-ethyl acetate in a volume ratio of 25:1, 10:1, 5:1, 1:1, and 0:100 to obtain 6 fractions a, b, c, d, e, and f;

[0046] (4) Take the fraction d obtained in step (3) and dissolve it in methanol, and then separate it by preparative liquid chromatography with the mobile phase being CH 3 CN:H 2 O, volume ratio of 48:52, flow rate of 10 ml / min, at retention time t R = 43 min to obtain the target compound.

[0047] 1. Structural analysis and identification of compounds

[0048] Structural analysis of compounds: mainly using spectroscopy techniques, including mass spectrometry, nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR, 2D-NMR) to identify its structure, the specific spectrum is as follows Figure 2 to Figure 8 As shown, the spectral data and analysis process are as follows:

[0049] (1) The compound is a white amorphous powder. High-resolution mass spectrum HR-ESI-MS: 485.3248 [M+H] + (Theoretical value C 30 H 45 O 5 :485.3262).

[0050] The compound formula is C 30 H 44 O 5 The exact molecular weight is 484.3189 and the degree of unsaturation is 9.

[0051] The compound 1H NMR showed the presence of five olefinic hydrogen atoms [ δ H 6.45 (s, 1H, H-3), 4.79 (s, 2H, H-12′), 4.63 (d, 1H, J =2.3 Hz, H a -15′) and 4.70 (d, 1H, J =2.3 Hz, H b -15′)], and six methyl groups [ δ H 1.96 (s, 3H, H-15), 1.74 (s, 3H, H-13′), 1.33 (s, 3H, H-14), 1.02 (s, 3H, H-14′), 0.87 (s, 3H, H-12) and 0.83 (s, 3H, H-13)]. 13 CNMR revealed 30 carbon resonance signals, which were assigned to the two carbonyl carbons [ δ C 201.2 (C-10') and 179.8 (C-2)], four groups of olefins [ δ C 144.1 (C-1) and 135.7 (C-10), 124.8 (C-3) and 159.0 (C-4), 146.9 (C-11') and 135.7 (C-12'), 145.9 (C-4') and 110.3 (C-15')], and two oxygen-containing carbons [ δ C 74.5 (C-5) and 70.5 (C-1')]. These data indicate that the compound is a sesquiterpene dimer compound. In the HMBC spectrum, correlations were observed between H-3 and C-1 / C-4 / C-9, H-15 and C-1 / C-9 / C-10, H-6 / H-7 and C-5, H-14 and C-4 / C-8, H-14' and C-1' / C-2' / C-6', and H-15' and C-3' / C-5', indicating that the compound has three six-membered rings. H-15 is associated with C-4 / C-9, and H-14 is associated with C-4 / C-9, proving that the two six-membered rings are bound through C-4 and C-9. Based on the above data, the structural formula of the compound was established as follows Figure 1 shown.

[0052] (2) The spectral data of the compound are as follows:

[0053] 1 H NMR (600 MHz, CDCl 3 ) δ H6.45 (s, 1H, H-3), 4.79 (s, 2H, H-12′), 4.70(d, 1H, J = 2.3 Hz, Ha-15′), 4.63 (d, 1H, J = 2.3 Hz, Hb-15′),3.04 (m,1H, H-8′),2.92 (dd, 1H, J = 12.2, 16.9 Hz, Ha-9′),2.46 (m, 1H, Ha-5), 2.45 (dd, 1H, J =12.2, 16.9 Hz, Hb-9′), 2.27 (m, 1H, Ha-3′), 2.07 (m, 3H, Ha-8), 1.96 (m, 1H,Hb-8), 1.92 (s, 3H, H-15), 1.85 (m, 1H, Hb-3′), 1.82 (m, 1H, Hb-5′), 1.80 (m,1H, Ha-7′), 1.74 (s, 3H, H-13), 1.64 (m, 1H, Ha-2′), 1.62 (m, 1H, Hb-7′),1.35, (m, 1H, Hb-2′), 1.33 (s, 3H, H-14), 1.23 (m, 1H, H-6′), 1.02 (s, 3H, H-14′), 1.00 (m, 2H, H-6), 0.93 (m, 1H, H-7), 0.87 (s, 3H, H-12), 0.83 (s, 3H,H-13)。

[0054] 13 C NMR (150 MHz, CDCl 3 ) δ C201.2 (C-10′), 179.8 (C-2), 159.0 (C-4), 146.9 (C-11′), 145.9 (C-4′), 144.1 (C-1) 135.7 (C-10), 124.8 (C-3), 110.8 (C-12′), 110.3 (C-15′), 74.5 (C-5), 70.5 (C-1′), 54.2 (C-6′), 51.7 (C-7), 46.3(C-5′), 43.0 (C-2′), 42.2 (C-9′), 41.4 (C-9), 38.9 (C-8′), 38.0 (C-8), 34.7(C-3′), 28.6 (C-14), 24.5 (C-11), 24.3 (C-12), 23.0 (C-14′), 21.5 (C-7′), 20.2 (C-13′), 19.3 (C-6), 18.6 (C-13), 11.5 (C-15).

[0055] 2. Experimental study on drug efficacy

[0056] 1. Experimental materials and instruments

[0057] 1. Drugs and reagents

[0058] The compound prepared in Example 1 (self-extracted, purity above 98%); NO detection kit (Shanghai Bio-Tech Biotechnology Co., Ltd.), DMEM culture medium (Beijing Solebow Technology Co., Ltd.), fetal bovine serum (Wuhan Pronosai Biotechnology Co., Ltd.), lipopolysaccharide (LPS) (Beijing Solebow Technology Co., Ltd.).

[0059] 2. Experimental Instruments

[0060] Microplate reader (Molecular Devices: SpectraMax i3, USA), MCV-B161S (T) clean bench (SANYO, Japan), MCO-20ACI CO 2 Constant temperature incubator (Thermo Corporation, USA), BS125S 1 / 10,000 balance (Mettler-Toledo, Switzerland), TDL-5C desktop microcentrifuge (Eppendorf, Germany).

[0061] 2. Experimental methods

[0062] 1. Cytotoxicity test of the compound prepared in Example 1

[0063] The cells were cultured in an incubator at 37 degrees Celsius, 100% humidity and 5% carbon dioxide concentration using DMEM medium containing 10% fetal bovine serum. RAW264.7 cells in the logarithmic growth phase were seeded in a 96-well plate, with a cell count of 8,000 per well. After 24 hours of incubation, compounds (compounds prepared in Example 1) at different concentrations of 25 μM, 12.5 μM, and 6.25 μM were added to the other groups except the control group, and blank wells were set. After another 24 hours of incubation, the cell viability of each group was measured using the CCK-8 method.

[0064] 2. Study on the anti-inflammatory efficacy of the compound prepared in Example 1

[0065] Cells were cultured in an incubator at 37 degrees Celsius, 100% humidity and 5% carbon dioxide concentration using DMEM medium containing 10% fetal bovine serum. RAW264.7 cells in the logarithmic growth phase were inoculated in a 24-well plate, and the cell count in each well was 1×10^5. After incubation for 24 hours, they were divided into a model group and a compound group. The compound group added 0.5 ml of compounds with concentrations of 25 μM, 12.5 μM, and 6.25 μM (compounds prepared in Example 1), and LPS was also added to the model group and the compound group to make the final concentration reach 1 μg / ml. The blank group contained an equal amount of DMEM medium. After 24 hours of induction, the NO content in the cell supernatant was measured at a wavelength of 540 nm using an NO detection kit using an ELISA reader.

[0066] 3. Statistical methods

[0067] The experimental data are The data were compared using t-test using SPSS 19.0 statistical software, and P < 0.05 was considered statistically significant.

[0068] (III) Experimental results

[0069] 1. The cytotoxicity test results of the compounds are shown in Tables 1 and Fig. 9 , indicating that the compound prepared in Example 1 had no obvious cytotoxicity to RAW264.7 cells at concentrations of 25 μM, 12.5 μM, and 6.25 μM.

[0070] Table 1 Results of cytotoxicity test of compounds on RAW264.7 cells

[0071]

[0072] 2. The anti-inflammatory efficacy results of the compounds are shown in Tables 2 and Fig.10 (Compared with the blank control group, ## P<0.01; compared with the model group, ), indicating that the compound prepared in Example 1 can significantly reduce the content of NO in the supernatant of RAW264.7 cells induced by LPS.

[0073] Table 2 Effects of compounds on NO content in the supernatant of RAW264.7 cells induced by LPS

[0074]

[0075] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sesquiterpene dimer compound, characterized in that: The chemical structural formula of the compound is as follows: 。 2. A method for preparing a sesquiterpene dimer compound according to claim 1, characterized in that: The following steps are involved: (1) Take the stem of Syringa japonica, add ethanol solution to extract, concentrate the obtained extract, and then extract with dichloromethane and ethyl acetate in sequence to obtain dichloromethane extraction part and ethyl acetate extraction part respectively; (2) taking the dichloromethane extraction fraction obtained in step (1), dissolving it with methanol, mixing it with silica gel dry method, loading it onto a silica gel column for column chromatography separation, and then eluting it with a gradient of petroleum ether-ethyl acetate with a volume ratio of 100:0, 50:1, 25:1, 10:1, 5:1, 1:1, 0:100 to obtain 12 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; (3) Dissolve the fraction 3 obtained in step (2) in ethyl acetate, dry-mix the sample with silica gel, load the sample onto a silica gel column for column chromatography, and then gradiently elute with petroleum ether-ethyl acetate in a volume ratio of 25:1, 10:1, 5:1, 1:1, and 0:100 to obtain 6 fractions a, b, c, d, e, and f; (4) Take the fraction d obtained in step (3) and dissolve it in methanol, and then separate it by preparative liquid chromatography. The mobile phase is CH3CN and H2O in a volume ratio of 48:

52. R = 43 min to obtain the target compound.

3. The method for preparing a sesquiterpene dimer compound according to claim 2, characterized in that: In step (2), the silica gel used for sample mixing is 100-200 mesh, and the silica gel used in the silica gel column is 100-200 mesh.

4. The method for preparing a sesquiterpene dimer compound according to claim 2, characterized in that: In step (3), the silica gel used for sample mixing is 200-300 mesh, and the silica gel used in the silica gel column is 200-300 mesh.

5. The method for preparing a sesquiterpene dimer compound according to claim 2, characterized in that: In step (4), the conditions for preparative liquid chromatography are: flow rate 10 ml / min.

6. Use of the sesquiterpene dimer compound according to claim 1 in the preparation of anti-inflammatory drugs.

7. A pharmaceutical composition, characterized in that: Including the sesquiterpene dimer compound described in claim 1.

8. A pharmaceutical preparation, characterized in that: The invention comprises a therapeutically effective amount of the sesquiterpene dimer compound according to claim 1 and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical preparation according to claim 8, characterized in that: The pharmaceutical preparation is an oral preparation or an injection preparation.

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