A guaiacane-type sesquiterpene dimer compound and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and discloses a guaiacyl sesquiterpene dimer compound, a preparation method and application thereof. The guaiacyl sesquiterpene dimer compound of the present invention is compound 1 or compound 2 separated from lilac, lilac is extracted with alcohol, extracted with dichloromethane and ethyl acetate, the dichloromethane extraction part is sequentially subjected to two silica gel column chromatography separations and preparative liquid chromatography separations, and compound 1 and compound 2 are obtained at different retention times. The preparation method of the present invention is simple to operate, and the guaiacyl 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 guaiacyl sesquiterpene dimer compound and a preparation method and application thereof. Background Art

[0002] Guaiane-type sesquiterpene dimers are a special class of natural compounds, which use a [5,7] ring structure substituted by 4,10-dimethyl-7-isopropyl composed of three isoprene units as the basic mother nucleus. According to the differences in functional groups and degree of polymerization, these compounds can be further subdivided into simple guaiacane type, guaiac lactone type, dimers and trimers. They are widely distributed in plants, fungi and marine organisms, especially in plants such as Asteraceae, Zingiberaceae, Thymelaeaceae, Lamiaceae and Alismataceae. Guaiane-type sesquiterpene dimers have attracted much attention due to their diverse biological activities, including antibacterial, anti-inflammatory, anti-tumor and neuroprotective. In recent years, researchers have conducted in-depth studies on the new isolation and identification, chemical structure and biological activity of this type of compounds, in order to provide references for their further research and development and utilization. For example, guaiacyl sesquiterpenoids extracted from Lactarius were found to be able to effectively inhibit the activity of Mycobacterium tuberculosis, especially against drug-resistant Mycobacterium tuberculosis or multidrug-resistant Mycobacterium tuberculosis. Therefore, these compounds have the potential to be used in the treatment of tuberculosis, especially drug-resistant tuberculosis. In addition, some studies have also focused on the anti-inflammatory activity of guaiacyl sesquiterpenoid dimers. For example, nine new guaiacyl sesquiterpenoids isolated from the roots of Aconitifolia lappa and two known compounds were tested for in vitro anti-inflammatory activity, and some of them showed effective inhibitory effects. In general, guaiacyl sesquiterpenoid dimers have become an important area in natural product research and drug development due to their unique chemical structure and diverse biological activities.

[0003] Lilac (Latin name: Syringaoblata Lindl.), a deciduous tree belonging to the genus Syringa of the family Oleaceae, is a common warming herb. It is mild in nature and spicy in taste. It mainly acts on the spleen, stomach and lung meridians. It has antibacterial, anti-inflammatory, antiviral, liver-protecting and choleretic effects. According to the Xinhua Materia Medica Compendium, lilac can be used to treat "acute icteric hepatitis". The Changbai Mountain Plant Medicinal Records also mentions that it can be used to treat diarrhea and hepatitis. In view of these pharmacological effects, the extraction and development of anti-inflammatory drugs from plants has become a hot topic in the current field of drug research. 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 guaiacyl sesquiterpene dimer compound in lilac and a preparation method and application thereof, and the research finds that the two guaiacyl sesquiterpene dimer compounds of the present invention have anti-inflammatory effects.

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

[0006] The two guaiacyl sesquiterpene dimer compounds extracted and separated from lilac have the following structural formulas:

[0007] .

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

[0009] (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;

[0010] (2) The dichloromethane extraction fraction obtained in step (1) was dissolved with methanol, mixed with silica gel by dry method, loaded onto a silica gel column for column chromatography, and then gradient eluted with petroleum ether-ethyl acetate in 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;

[0011] (3) The fraction 7 obtained in step (2) was dissolved in methanol, mixed with silica gel dry method, loaded onto a silica gel column for column chromatography, and then eluted with a gradient of petroleum ether-ethyl acetate with a volume ratio of 20:1, 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 c 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 42:58. R = 40 min to obtain the target compound 1. R =43 min to obtain the target compound 2.

[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 present invention also provides the use of the guaiacyl 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 present invention also provides a pharmaceutical composition comprising the guaiacane-type sesquiterpene dimer compound.

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

[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 can separate and obtain the guaiacane type sesquiterpene dimer compound in lilac.

[0028] Experiments show that the compound of the present invention can significantly reduce the NO content in LPS-induced RAW264.7 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 1is the chemical structural formula of the guaiacane-type sesquiterpene dimer compound prepared by the present invention;

[0030] Figure 2 The mass spectrum of compound 1 prepared in the present invention;

[0031] Figure 3 The mass spectrum of compound 2 prepared in the present invention;

[0032] Figure 4 Compound 1 prepared by the present invention 1 H NMR spectrum;

[0033] Figure 5 Compound 1 prepared by the present invention 13 C NMR spectrum;

[0034] Figure 6 DEPT NMR spectrum of compound 1 prepared in the present invention;

[0035] Figure 7 HSQC spectrum of compound 1 prepared in the present invention;

[0036] Figure 8 is the HMBC spectrum of compound 1 prepared in the present invention;

[0037] Fig. 9 Compound 1 prepared by the present invention 1 H- 1 H COSY spectrum;

[0038] Fig.10 Compound 2 prepared by the present invention 1 H NMR spectrum;

[0039] Fig.11 Compound 2 prepared by the present invention 13 C NMR spectrum;

[0040] Fig.12 DEPT NMR spectrum of compound 2 prepared in the present invention;

[0041] Fig.13 HSQC spectrum of compound 2 prepared in the present invention;

[0042] Fig.14 This is the HMBC spectrum of compound 2 prepared in the present invention;

[0043] Fig.15 Compound 2 prepared by the present invention 1 H- 1 H COSY spectrum;

[0044] Fig.16The effect of the guaiacyl sesquiterpene dimer compound prepared by the present invention on the cytotoxicity of RAW264.7 cells;

[0045] Fig.17 The invention discloses the effect of the guaiacyl sesquiterpene dimer compound prepared by the present invention on NO in RAW264.7 cells induced by LPS. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] Example 1 Preparation of Compounds

[0049] This embodiment provides a method for preparing a guaiacyl sesquiterpene dimer compound, the steps being:

[0050] (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;

[0051] (2) Take the dichloromethane extraction fraction obtained in step (1), dissolve it with methanol, dry-mix it with silica gel (100-200 mesh, twice the mass of the dichloromethane extraction fraction), load it onto a silica gel (100-200 mesh) column for 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 to obtain 12 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0052] (3) Dissolve the fraction 7 obtained in step (2) in methanol, dry-mix the sample with silica gel (200-300 mesh), load the sample onto a silica gel column (200-300 mesh) for column chromatography, and then gradiently elute with petroleum ether-ethyl acetate in a volume ratio of 20:1, 10:1, 5:1, 1:1, and 0:100 to obtain 6 fractions a, b, c, d, e, and f;

[0053] (4) Take the fraction c obtained in step (3) and dissolve it in methanol, then separate it by preparative liquid chromatography, the mobile phase is CH3CN:H2O, the volume ratio is 42:58, the flow rate is 10 ml / min, and at the retention time t R = 40 min to obtain the target compound 1. R =43 min to obtain the target compound 2.

[0054] 1. Structural analysis and identification of compounds

[0055] The structural analysis of compound 1 and compound 2 was mainly carried out by using spectroscopy techniques, including mass spectrometry and nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR, 2D-NMR) to identify its structure. The specific spectrum is shown in the attached Figure 2 to Figure 15 As shown, the spectral data and analysis process are as follows:

[0056] (1) Compound 1 is a light yellow powder. High resolution mass spectrum HR-ESI-MS: 505.2253 [M+H] + (Theoretical value C 30 H 33 O7: 505.2221). Compound 2 is a light yellow powder, high resolution mass spectrum HR-ESI-MS: 489.2295 [M+H] + (Theoretical value C 30 H 33 O6:489.2272).

[0057] Comprehensive analysis 1 H NMR, 13 C NMR and HSQC spectra show that the two compounds have extremely similar NMR spectra, so it can be preliminarily determined that they are of the same structure. There are four carbonyl carbons in the structure of compound 1, which are [ δ C 186.0,185.3, 179.1, 179.4], with 6 methyl groups [ δ C : 22.8, 10.2, 23.1, 22.2, 10.2, 26.0] respectively corresponding to the hydrogen proton signals [ δ H: 1.21, 1.98, 1.42, 1.27, 1.94, 1.25], 6 pairs of unsaturated double bond carbons [ δ C : 133.9, 144.8, 124.4, 157.4, 169.5, 123.2, 133.8, 144.6, 124.1, 157.0, 134.9, 161.5], three olefinic hydrogen proton signals [ δ H :6.59, 6.57, 5.76]. HMBC correlation showed that H-15 ( δ H 1.42) and C-7 ( δ C 169.5), C-14 ( δ C 32.8)、C-14′( δ C 30.7) related, H-15′ ( δ H 1.25) and C-14′ ( δ C 30.7)、C-8′( δ C 161.5) related, H-14 ( δ H 1.83) and C-14′ ( δ C 30.7)、C-13( δ C 42.7)、C-7′( δ C 134.9)、C-8′( δ C 161.5), indicating that this compound is a sesquiterpene dimer. The difference between compound 1 and compound 2 is that the C-13′ position of compound 1 is hydroxylated, while compound 2 has no substituent. Finally, the planar structures of compounds 1 and 2 were determined by combining two-dimensional nuclear magnetic resonance spectroscopy data such as HMBC, DEPT and HSQC spectra. The structural formulas of the two compounds are as follows: Figure 1 .

[0058] (2) The spectral data of compound 1 are as follows:

[0059] 1 H NMR (600 MHz, CDCl3) δ H 6.59 (s, 1H, H-5), 6.57 (s, 1H, H-5′), 5.76 (s, 1H, H-8), 3.30 (d,1H, J= 14.7 Hz, Ha-9′), 2.78 (d,1H, J = 12.7 Hz, H a -9),2.49 (d,1H, J = 12.7 Hz, H b -9), 2.47 (d,1H, J = 14.7 Hz, H b -9′), 1.98 (s, 3H, H-12), 1.94 (s, 3H, H-12′), 1.83 (m, 2H, H-14′), 1.77 (m, 1H, H a -14), 1.58 (m,1H, H b -14), 1.42 (s, 3H, H-15), 1.27 (s, 3H, H-11′), 1.25 (s, 3H, H-15′), 1.21 (s, 3H, H-11).

[0060] 13 C NMR (175 MHz, CDCl3) δ C 186.0 (C-3), 185.3 (C-3′), 179.4 (C-4′), 179.1 (C-4), 169.5 (C-7), 161.5 (C-8′), 157.4 (C-6), 157.0 (C-6′), 144.8 (C-2), 144.6 (C-2′), 134.9 (C-7′), 133.9 (C-1), 133.8 (C-1′), 124.4 (C-5), 124.1(C-5′), 123.2 (C-8), 67.9 (C-13′), 43.1 (C-10′), 42.7 (C-13), 42.2 (C-10), 36.0 (C-9), 33.9 (C-9′), 32.8 (C-14), 30.7 (C-14′), 26.0 (C-15′), 23.1 (C-15), 22.8 (C-11), 22.2 (C-11′), 10.2 (C-12′), 10.2 (C-12).

[0061] The spectral data of compound 2 are as follows:

[0062] 1 H NMR (600 MHz, CDCl3) δ H6.77 (s, 1H, H-5), 6.74 (s, 1H, H-5′), 5.92(s, 1H, H-8), 2.92 (d,1H, J = 17.5 Hz, H a -9′), 2.85 (d,1H, J = 17.6 Hz, H a -9),2.70 (d,1H, J = 17.5 Hz, H b -9′), 2.60 (d,1H, J = 17.5 Hz, H b -9), 2.57 (m, 1H, H-13′), 2.08 (s, 3H, H-12′), 2.03 (s, 3H, H-12), 2.00 (m, 1H, H a -14), 1.95 (m,1H, H a -14′), 1.68 (m, 1H, H b -14), 1.53 (s, 3H, H-15), 1.43 (m, 1H, H b -14′),1.40 (s, 3H, H-11′), 1.33 (s, 3H, H-11), 1.22 (d, 3H, J = 7.1 Hz, H-15′)。

[0063] 13 C NMR (150 MHz, CDCl3) δ C187.6 (C-3), 185.5 (C-3′), 180.6 (C-4), 180.4 (C-4′), 171.8 (C-7), 164.3 (C-8′), 158.8 (C-6), 158.4 (C-6′), 145.7 (C-2), 145.6 (C-2′), 136.8 (C-7′), 134.3 (C-1′), 134.3 (C-1), 125.0 (C-8), 124.7(C-5), 124.6 (C-4′), 44.0 (C-10′), 43.5 (C-13), 43.4 (C-10), 40.4 (C-9′), 37.5 (C-13′), 37.2 (C-9), 33.9 (C-14), 26.0 (C-14′), 23.3 (C-15), 22.9 (C-11), 22.1 (C-11′), 17.7 (C-15′), 9.6 (C-12), 9.5 (C-12′).

[0064] 2. Experimental study on drug efficacy

[0065] 1. Experimental materials and instruments

[0066] 1. Drugs and reagents

[0067] The compound prepared in Example 1 (self-extracted, purity above 98%); NO detection kit (Shanghai Bio-Tech Co., Ltd.), DMEM culture medium (Beijing Solebold Technology Co., Ltd.), fetal bovine serum (Wuhan Pronosai Biotechnology Co., Ltd.), PBS buffer (Beijing Solebold Technology Co., Ltd.), lipopolysaccharide (LPS) (Beijing Solebold Technology Co., Ltd.).

[0068] 2. Experimental Instruments

[0069] Microplate reader (Molecular Devices: SpectraMaxi3, USA), MCV-B161S (T) clean bench (SANYO, Japan), MCO-20ACI CO2 constant temperature incubator (Thermo, USA), BS125S 1 / 10,000 balance (Mettler-Toledo, Switzerland), TDL-5C desktop microcentrifuge (Eppendorf, Germany).

[0070] 2. Experimental methods

[0071] 1. Cytotoxicity test of the compound prepared in Example 1

[0072] 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 (Compound 1 and Compound 2 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.

[0073] 2. Study on the anti-inflammatory efficacy of the compound prepared in Example 1

[0074] 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 (Compound 1 and Compound 2 prepared in Example 1). 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.

[0075] 3. Statistical methods

[0076] 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.

[0077] (III) Experimental results

[0078] 1. The cytotoxicity test results of the compounds are shown in Tables 1 and Fig.16 , 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.

[0079] Table 1 Results of cytotoxicity test of compounds on RAW264.7 cells

[0080]

[0081] 2. The anti-inflammatory efficacy results of the compounds are shown in Tables 2 and Fig.17 (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.

[0082] Table 2 Effects of compounds on NO content in the supernatant of RAW264.7 cells induced by LPS

[0083]

[0084] 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 guaiacyl sesquiterpene dimer compound, characterized in that: The guaiacyl sesquiterpene dimer compound is compound 1 or compound 2, and its chemical structure is as follows: 。 2. A method for preparing the guaiacyl 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) The dichloromethane extraction fraction obtained in step (1) was dissolved with methanol, mixed with silica gel by dry method, loaded onto a silica gel column for column chromatography, and then gradient eluted with petroleum ether-ethyl acetate in 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) The fraction 7 obtained in step (2) was dissolved in methanol, mixed with silica gel dry method, loaded onto a silica gel column for column chromatography, and then eluted with a gradient of petroleum ether-ethyl acetate with a volume ratio of 20:1, 10:1, 5:1, 1:1, and 0:100 to obtain 6 fractions a, b, c, d, e, and f; (4) Take the fraction c 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 42:

58. R = 40 min to obtain the target compound 1. R =43 min to obtain the target compound 2.

3. The method for preparing the guaiacyl 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 the guaiacyl 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 the guaiacyl 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 guaiacyl sesquiterpene dimer compound according to claim 1 in the preparation of anti-inflammatory drugs.

7. A pharmaceutical composition, characterized in that: It comprises the guaiacane-type sesquiterpene dimer compound as described in claim 1.

8. A pharmaceutical preparation, characterized in that: The invention comprises a therapeutically effective amount of the guaiacyl 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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