CN111057125B - Diterpenoid compound containing triepoxide structure and preparation method and application thereof - Google Patents

Diterpenoid compound containing triepoxide structure and preparation method and application thereof Download PDF

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CN111057125B
CN111057125B CN201911377316.1A CN201911377316A CN111057125B CN 111057125 B CN111057125 B CN 111057125B CN 201911377316 A CN201911377316 A CN 201911377316A CN 111057125 B CN111057125 B CN 111057125B
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张水寒
黄小龙
周融融
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HUNAN ACADEMY OF CHINESE MEDICINE
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Abstract

The invention relates to a diterpenoid compound containing a triepoxide structure, a preparation method and application thereof, wherein the structure of the compound is as follows:
Figure DDA0002341317320000011
the preparation method comprises the following steps: the method comprises the following steps of (1) taking osmyl peony powder as a raw material, carrying out reflux extraction and petroleum ether extraction on the raw material by using an ethanol water solution, and taking a petroleum ether layer to obtain an extract liquid; subjecting the extract to silica gel column chromatography, eluting with petroleum ether-ethyl acetate as mobile phase, collecting eluate, detecting with thin layer chromatography, sequentially obtaining 10 fractions according to the detection result and the elution order, and respectively naming as Fr.A-Fr.J; concentrating fraction Fr.F under reduced pressure, performing silica gel column chromatography, eluting with petroleum ether-acetone as mobile phase, collecting eluate, detecting with thin layer chromatography, sequentially obtaining 6 fractions according to the elution order according to the detection result, and respectively naming the fractions as Fr.01-Fr.06; concentrating the fraction Fr.03 under reduced pressure, eluting the concentrated solution by using methanol-water as a mobile phase through preparative liquid chromatography, collecting the eluent for 10-14 min, concentrating under reduced pressure and drying.

Description

Diterpenoid compound containing triepoxide structure and preparation method and application thereof
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to a diterpenoid compound containing a triepoxide structure, and a preparation method and application thereof.
Background
Clerodendrum bungei (Clerodendrum bungei Steud) is a shrub of the genus Clerodendrum, family Verbenaceae. The plants of the genus Clerodendrum are about 400 plants, which are mainly distributed in tropical zone and subtropical zone, and the minority is distributed in temperate zone, mainly eastern hemisphere; in China, 34 varieties of 6 varieties are distributed in the southwest and south China.
The clerodendron bungei produces north, northwest and southwest China, as well as Jiangsu, Anhui, Zhejiang, Jiangxi, Hunan, Hubei and Guangxi. It is grown in hillside, forest edge, valley, roadside, irrigated land and wet area below 2500 m. Northern India, Vietnam and Malaysia are also distributed. The clerodendron bungei mainly contains diterpene, phenylethanoid glycoside and other chemical components, has the effects of dispelling wind, removing toxicity, relieving swelling and pain by being used as a medicine for roots, stems and leaves, and is also used for treating uterine prolapse recently.
Disclosure of Invention
Based on this, there is a need to provide a diterpenoid compound containing a triepoxide structure.
The invention provides a diterpenoid compound containing a triepoxide structure, which has the following structural formula:
Figure BDA0002341317300000021
another object of the present invention is to provide a method for preparing the above compound, comprising the steps of:
1) crushing clerodendrum bungei, performing reflux extraction by using an ethanol water solution, concentrating an extracting solution under reduced pressure to obtain an extract, dissolving the extract by using water to obtain a suspension, extracting the suspension by using petroleum ether, and taking a petroleum ether layer to obtain an extracting solution;
2) subjecting the extract to silica gel column chromatography, eluting with petroleum ether-ethyl acetate as a mobile phase, collecting eluates, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 10 fractions which are named as Fr.A-Fr.J according to the detection result and the elution sequence;
3) taking fraction Fr.F, concentrating under reduced pressure, carrying out silica gel column chromatography on concentrated solution, eluting by taking petroleum ether-acetone as a mobile phase, collecting eluent, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 6 fractions according to the detection result and the elution sequence, wherein the fractions are respectively named as Fr.01-Fr.06;
4) and concentrating the fraction Fr.03 under reduced pressure, eluting the concentrated solution by using methanol-water as a mobile phase through preparative liquid chromatography, collecting the eluent for 10-14 min, concentrating under reduced pressure, and drying to obtain the diterpenoid compound.
In one embodiment, in step 2), the elution is gradient elution, the number of gradients is 5 to 12, the amount of the mobile phase in each gradient is 3 to 5 column volumes, and each eluent is collected according to 1/6 to 1/4 of the column volumes to obtain 120 eluents;
detecting each eluent by thin-layer chromatography under 366nm and sunlight by using 10% sulfuric acid ethanol as a color developing agent, and combining 1 st to 18 th, 19 th to 26 th, 27 th to 39 th, 40 th to 54 th, 55 th to 67 th, 68 th to 73 th, 74 th to 83 th, 84 th to 93 th, 94 th to 106 th and 107 th to 120 th eluents with similar chromatographic behaviors to obtain fractions Fr.A to Fr.J.
In one embodiment, in the step 2), the number of the gradient elution is 10, and the mobile phase of each gradient is petroleum ether-ethyl acetate in a volume ratio of 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, and 0: 1.
In one embodiment, in step 3), the elution is gradient elution, the number of gradients is 2 to 6, the amount of the mobile phase in each gradient is 3 to 5 column volumes, and each part of eluent is collected according to 1/6 to 1/4 of the column volumes to obtain 60 parts of eluent;
detecting each eluent by thin-layer chromatography under 366nm and sunlight by using 10% sulfuric acid ethanol as a color developing agent, and combining 1 st to 15 th, 16 th to 18 th, 19 th to 26 th, 27 th to 38 th, 39 th to 47 th and 48 th to 60 th eluents with similar chromatographic behaviors to obtain fractions Fr.01 to Fr.06.
In one embodiment, in the step 3), the number of the gradient elution is 4, and the mobile phase of each gradient is petroleum ether-acetone in the volume ratio of 20:1, 15:1, 10:1 and 5: 1.
In one embodiment, in step 4), the chromatographic column of the preparative liquid chromatography is a C18 column, and the mobile phase is methanol-water with a volume concentration of 40% to 60%.
In one embodiment, in the step 4), the chromatographic column of the preparative liquid chromatography is a C18 column, the mobile phase is methanol-water with a volume concentration of 40-60%, and the eluent is collected for 11-12 min.
In one embodiment, in step 2) and step 3), the silica gel used for silica gel column chromatography has a particle size of 80-100 mesh or 200-300 mesh.
In one embodiment, in step 1), the concentration of ethanol in the ethanol aqueous solution is 60% to 95%; the reflux extraction is carried out for 3-5 times, and each time lasts for 1-2 hours.
The invention also aims to provide application of the diterpenoid compound or the diterpenoid compound prepared by the method in preparing a medicament for treating tumor diseases.
Specifically, the diterpenoid compound provided by the invention has an obvious inhibition effect on the expression of liver cancer HepG2 cells and lung cancer A549 cells under the condition of low concentration, embodies good anti-tumor activity, and can be used for preparing medicines for treating or inhibiting tumor diseases.
The invention has the beneficial effects that:
the diterpenoid compound containing the triepoxide structure is extracted and separated from clerodendrum bungei for the first time, and the structure of the diterpenoid compound is identified, so that the preparation process of the diterpenoid compound is determined.
The compound provided by the invention has an inhibition effect on liver cancer HepG2 cells and lung cancer A549 cells, and the low-concentration compound has a good inhibition effect on liver cancer HepG2 cells and lung cancer A549 cells, so that a new direction is provided for preparing a medicament for treating cancer diseases.
Drawings
FIG. 1 is a HR-ESI-MS spectrum of a target compound prepared in example 1 of the present invention;
FIG. 2 is a UV spectrum of a target compound prepared in example 1 of the present invention;
FIG. 3 is a Fourier transform infrared (FT-IR) spectrum of a target compound prepared in example 1 of the present invention;
FIG. 4 shows the target compound prepared in example 1 of the present invention13A C-NMR spectrum;
FIG. 5 is a HSQC spectrum of the target compound prepared in example 1 of the present invention;
FIG. 6 shows the target compound prepared in example 1 of the present invention1H-NMR spectrum;
FIG. 7 is a HMBC spectrum of the target compound prepared in example 1 of the present invention;
FIG. 8 is a micrograph of the results of the experiment of inhibiting the growth of HepG2 cells in liver cancer cells by the target compound prepared in example 1 of the present invention, wherein A is an experimental group and C is a control group;
fig. 9 is a micrograph of the experimental result of the lung cancer a549 cell culture inhibition by the target compound prepared in example 1 of the present invention, in which a experimental group a and C are control groups.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
One embodiment of the present invention provides a diterpenoid compound having a triepoxide structure, which has the following structural formula:
Figure BDA0002341317300000051
furthermore, the diterpenoid compound is extracted and separated from clerodendrum bungei for the first time by the applicant. Applicants name the diterpenoid compound as: choumudansu a. The diterpene compound can be synthesized by a chemical synthesis method or can be obtained by a crude drug extraction method.
Another embodiment of the present invention provides a method for preparing the diterpenoid compound, which is a crude drug extraction method, comprising the following steps 1) to 4).
Step 1), crushing clerodendron bungei, performing reflux extraction by using an ethanol water solution, concentrating an extracting solution under reduced pressure to obtain an extract, dissolving the extract by using water to obtain a suspension, extracting the suspension by using petroleum ether, and taking a petroleum ether layer to obtain an extracting solution.
Further, the volume concentration of ethanol in the ethanol water solution is 60-95%, and the reflux extraction times are 3-5 times, and each time lasts for 1-2 hours.
Further, the volume concentration of the ethanol in the ethanol water solution is 70-75%.
Specifically, crushing stems and leaves of clerodendron bungei, performing reflux extraction for 3 times by using an ethanol water solution with the volume concentration of 70% -75%, 1-2 hours each time, combining three extracting solutions, performing reduced pressure concentration to obtain an extract, dissolving the extract by using 8-10 times of water to obtain a suspension, repeatedly extracting for 3-5 times by using petroleum ether, recovering an organic solvent, and combining petroleum ether layers to obtain an extract liquid.
And 2) subjecting the extract liquor obtained in the step 1) to silica gel column chromatography, eluting by using petroleum ether-ethyl acetate as a mobile phase, collecting eluates, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 10 fractions which are named as Fr.A-Fr.J according to the elution sequence according to the detection result.
Furthermore, the particle size of silica gel in a chromatographic column adopted by the silica gel column chromatography is 80-100 meshes or 200-300 meshes.
Further, petroleum ether-ethyl acetate with different volume ratios is sequentially taken as a mobile phase for gradient elution.
Further, the number of the gradients is 5-12, each gradient elutes 3-5 column volumes, the elutes are collected according to 1/6-1/4 column volumes, and 120 bottles of elutes are obtained through collection; detecting each eluent by thin-layer chromatography under 366nm and daylight by using 10% sulfuric acid ethanol as a color developing agent and cyclohexane-ethyl acetate and dichloromethane-methanol as developing agents respectively, merging the eluents with similar chromatographic behaviors, merging the eluents from 1 st to 18 th bottles into a fraction Fr.A, merging the eluents from 19 th to 26 th bottles into a fraction Fr.B, merging the eluents from 27 th to 39 th bottles into a fraction Fr.C, merging the eluents from 40 th to 54 th bottles into a fraction Fr.D, merging the eluents from 55 th to 67 th bottles into a fraction Fr.E, merging the eluents from 68 th to 73 th bottles into a fraction Fr.F, merging the eluents from 74 th to 83 th bottles into a fraction Fr.G, merging the eluents from 84 th to 93 th bottles into a fraction Fr.H, merging the eluents from 94 th to 106 th bottles into a fraction Fr.I, merging the eluents from 107 th to 120 th bottles into a fraction Fr.J, and finally obtaining 10 fractions Fr.A to Fr.
Furthermore, gradient elution is carried out by setting 10 concentration gradients, and the adopted mobile phases are petroleum ether-ethyl acetate with the volume ratio of 100:0, 100:1, 80:1, 50:1, 30:1, 20:1 and 10:1, 5:1, 2:1 and 0:1 respectively.
For example, the volume of the chromatographic column is 3L (the volume of the silica gel packing is about 2L), gradient elution is sequentially performed by using petroleum ether-ethyl acetate as mobile phases with the volume ratios of 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1 and 0:1, the dosage of each gradient mobile phase is 6L, and the eluent is collected by 500mL in one bottle to obtain 120 bottles of eluent, and the eluent is sequentially marked as 1-120 according to the sequence of collection.
And 3) taking the fraction Fr.F, concentrating under reduced pressure, carrying out silica gel column chromatography on the concentrated solution, eluting by using petroleum ether-acetone as a mobile phase, collecting the eluent, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 10 fractions which are named as Fr.A-Fr.J according to the detection result and the elution sequence.
Furthermore, the particle size of the adopted silica gel is 80-100 meshes or 200-300 meshes.
Further, petroleum ether-acetone with different volume ratios is adopted as a mobile phase for gradient elution; the mobile phase is petroleum ether-acetone with the volume ratio of (0-20): 1.
Further, gradient elution is carried out by taking petroleum ether-acetone with different volume ratios as a mobile phase, 2-6 concentration gradients are set, the using amount of each gradient eluent is 3-5 column volumes, the eluates are collected according to 1/6-1/5 column volumes, and 60 bottles of eluates are collected in total. Detecting each eluent by thin layer chromatography under 366nm and daylight with 10% sulphuric acid ethanol as developer and dichloromethane-methanol as developing agent, combining fractions with similar chromatographic behavior, combining 1-15 bottles of eluent into fraction Fr.01, combining 16-18 bottles of eluent into fraction F02, combining 19-26 bottles of eluent into fraction Fr.03, combining 27-38 bottles of eluent into fraction Fr.04, combining 39-47 bottles of eluent into fraction Fr.05, and combining 48-60 bottles of eluent into fraction Fr.06 for total 6 fractions.
Further, the mobile phase is petroleum ether-acetone with the volume ratio of (5-20): 1.
Further, elution is carried out by setting 4 concentration gradients, and the mobile phases adopted by the gradient elution are petroleum ether-acetone with the volume ratio of 20:1, 15:1, 10:1 and 5:1 respectively.
Specifically, the volume of the chromatographic column is 0.3L, elution is sequentially carried out by using petroleum ether-acetone as mobile phases with the volume ratios of 20:1, 15:1, 10:1 and 5:1 respectively, the dosage of the mobile phase of each gradient is 0.9L, and 60 bottles of eluent are collected according to 60 mL.
It is understood that the elution in step 2) and step 3) may be performed by a single volume ratio of mobile phase, or may be performed by sequentially performing gradient elution with mobile phases having different volume ratios, and then combining the eluates, so as to reduce the loss of the target compound caused by elution with a single ratio of mobile phase.
In addition, when gradient elution is performed, the volumes of the mobile phases used in the respective stages may be different from each other, or may be the same, and elution is performed using the mobile phases in the above-described ratios, respectively, which is more advantageous in obtaining the target compound.
And 4) concentrating the fraction Fr.03 obtained in the step 3) under reduced pressure, eluting the concentrated solution by using methanol-water as a mobile phase through preparative liquid chromatography, collecting the eluent for 10-14 min, concentrating under reduced pressure, and drying to obtain the target diterpenoid compound.
Specifically, the eluent is collected for 10min to 14min according to chromatographic peaks.
Further, a chromatographic column for preparing the liquid chromatogram is a C18 column, a mobile phase is a methanol water solution with the volume concentration of 40-60%, and the flow rate of the mobile phase is 1.5-3 mL/min; collecting the eluent for 11-12 min.
Further, the mobile phase was 56% by volume methanol-water, the flow rate was 2mL/min, and the column temperature of the column was 35 ℃.
The following are specific examples
EXAMPLE 1 preparation of the object Compound choumudansu A
1. Materials: clerodendrum bungei plant
2. Chromatographic column conditions:
the silica gel particle size of the silica gel column 1 is 80-100 meshes, the silica gel particle size of the silica gel column 2 is 200-300 meshes, and the silica gel is subjected to C18 chromatographic column.
3. The preparation method of choumudansu A comprises the following specific steps:
1) and (3) extracting: taking 6Kg of air-dried whole clerodendrum bungei plant, crushing the plant in a crusher, performing reflux extraction for 3 times by using ethanol with the concentration of 70%, using 40L of ethanol each time, sequentially performing extraction for 2h, 2h and 1h, combining extracting solutions, performing reduced pressure concentration to obtain 600g of extract, and dissolving the extract with 10 times of distilled water to obtain suspension; the suspension was repeatedly extracted with petroleum ether 3 times, and the organic solvent was recovered to obtain 80g of a petroleum ether extract.
2) Silica gel column chromatography: loading silica gel of 80 meshes to 100 meshes into a chromatographic column, mixing the petroleum ether extract obtained in the step 1) with the silica gel, adding the obtained sample into the chromatographic column, wherein the total column loading amount is two thirds (2L), and performing gradient elution by using petroleum ether (60 ℃ -90 ℃) and ethyl acetate mixed solvent as mobile phases in a volume ratio of 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1 and 0:1 in sequence, wherein the dosage of the mobile phase of each gradient is 6L; collecting 120 bottles of eluent according to 500mL bottles, taking 10% sulfuric acid ethanol as a color developing agent and cyclohexane-ethyl acetate and dichloromethane-methanol as developing agents, performing thin-layer chromatography detection on each eluent under 366nm and sunlight, merging fractions similar to the chromatography industry according to the detection result of the thin-layer chromatography, merging fractions from 1 bottle to 18 bottles of the eluent into fraction Fr.A, merging fractions from 19 bottles to 26 bottles of the eluent into fraction Fr.B, merging fractions from 27 bottles to 39 bottles of the eluent into fraction Fr.C, merging fractions from 40 bottles to 54 bottles of the eluent into fraction Fr.D, merging fractions from 55 bottles to 67 bottles of the eluent into fraction Fr.E, merging fractions from 68 bottles to 73 bottles of the eluent into fraction Fr.F, merging fractions from 74 bottles to 83 bottles of the eluent into fraction Fr.G, merging fractions from 84 bottles to 93 bottles of the eluent into fraction Fr.H, merging fractions from 94 bottles to 106 bottles of the eluent into fraction Fr.I, merging fractions from 107 bottles to 120 bottles of the eluent into fraction Fr.J, and obtaining 10 fractions from Fr.A.
3) Silica gel column chromatography: concentrating fraction Frs.F under reduced pressure, mixing with 200-300 mesh silica gel, loading the sample with fraction F (1.3g) on silica gel column, gradient eluting with petroleum ether-acetone mixed solution at volume ratio of 20:1, 15:1, 10:1 and 5:1 as mobile phase, wherein the amount of each gradient mobile phase is 900mL, collecting eluate in 60mL bottles, and collecting to obtain 60 bottles of eluate. Detecting each eluent by using dichloromethane-methanol as a developing agent and 10% ethanol sulfate as a color developing agent under 366nm and daylight by using thin layer chromatography, merging fractions with similar chromatographic behaviors according to the detection result of silica gel thin layer chromatography, merging 1-15 bottles of eluent into fraction Fr.01, merging 15-18 bottles of eluent into fraction Fr.02, merging 19-26 bottles of eluent into fraction Fr.03, merging 27-38 bottles of eluent into fraction Fr.04, merging 39-47 bottles of eluent into fraction Fr.05, merging 48-60 bottles of eluent into fraction Fr.06, and combining 6 fractions.
4) And liquid phase separation and enrichment:
the fraction fr.03 was collected, concentrated under reduced pressure, and then separated from 80mg of the F03 fraction by preparative HPLC (preparation conditions: column C18, 56% methanol-water solution by volume concentration as mobile phase, flow rate 2mL/min, column temperature 35 ℃, sample size 100uL, chromatographic peak Rt 11.5min, and collection of eluate for 11 to 12 minutes) to obtain 4.0mg of the target compound.
Example 2
Example 2 is substantially the same as example 1 except that in step 2), the mobile phase used in the gradient elution of step 2) is petroleum ether-ethyl acetate in the volume ratio of 100:0, 50:1, 20:1, 10:1, 5:1 and 2:1, the amount of the mobile phase used in each gradient is 10L, and the mobile phase is collected in a 500mL bottle.
The final isolated weight of the title compound was 3.88 mg.
Example 3
Example 3 is substantially the same as example 1 except that step 3), step 3) and gradient elution are performed using petroleum ether-acetone in a volume ratio of 15:1 and 10:1, respectively, and the amount of each gradient mobile phase is 1800 mL.
The final isolated weight of the title compound was 3.92 mg.
Experimental example 1
The target compound prepared in example 1 was identified as follows:
1) and property: white amorphous powder (dimethylsulfoxide).
2) The molecular formula is as follows: c20H24O6。
3) As shown in figure 1, the HR-ESI-MS spectrogram parameter of the target compound is M/z [ M + H ]]+The molecular weight was determined to be 361.1637 according to HR-ESI-MS spectrum.
4) As shown in FIG. 2, the ultraviolet spectrum of the objective compound showed a maximum absorption peak at 217 nm.
5) As shown in FIG. 3, the infrared spectrum of the objective compound showed the presence of a hydroxyl group (3455 cm)-1) And carbonyl (1768 cm)-1) Absorption peak of functional group.
6) As shown in FIG. 4, is of the target Compound13C-NMR (100MHz, DMSO-d6) spectrum, and13the C-NMR spectrum was analyzed and the data are shown in Table 1 below, showing a 20 carbon signal.
As shown in fig. 5, the HSQC (400MHz) spectrum of the target compound was classified by HSQC spectrum to show 7 quaternary carbons, 4 oxygen-containing tertiary carbons, 3 methyl groups, 3 methylene groups, 2 methine groups, and 1 carbonyl carbon at δ c 173.6.
As shown in FIG. 6, is of the target compound1H-NMR (400MHz, DMSO-d6) spectra and analyses thereof were performed with data shown in Table 1 below showing 3 methyl singlet at δ 0.77(d, J ═ 6.9Hz, 3H, H-17), δ 0.90(d, J ═ 6.9Hz, 3H, H-16), δ 00.96(s, 3H, H-20), four oxygen-containing methine signals at δ 3.35(m, 1H, H-6), δ 3.36(m, 1H, H-7), δ 3.55(d, J ═ 2.7Hz, 1H, H-11), δ 3.88(d, J ═ 3.2Hz, 1H, H-12), the 3 methylene signals are the hydroxyl signals at δ 1.27(m, 2H, H-1), δ 2.18(m, 12H, H-2), δ 4.81(m, 2H, H-19) and one at δ 4.68(d, J ═ 7.6Hz, 1H, -OH).
As shown in fig. 7, the HMCB (400MHz) spectrum of the target compound was obtained and its main relevant signals were analyzed, and the data are shown in table 1 below.
TABLE 1 of the target Compounds1H-NMR and13C-NMR Nuclear magnetic data and the principal signals of HMBC
Position δH(J in Hz) δC Position δH(J in Hz) δC
1 1.27(2H,m) 29.5 11 3.55(d,J＝2.7Hz,1H) 54.6
2 2.18(2H) 23.1 12 3.88(d,J＝3.2Hz,1H) 55.7
3 123.5 13 65.1
4 162.9 14 17.1
5 2.62(1H) 40.1 15 2.13(1H,m) 27.9
6 3.35(1H,m) 71.7 16 δ0.90(d,J＝6.9Hz,3H) 18.1
7 3.36(1H,m) 60.3 17 0.77(d,J＝6.9Hz,3H) 17.1
8 61.4 18 173.6
9 64.7 19 4.81(2H,dd) 70.7
10 35.7 20 δ0.96(s,3H) 14.2
OH δ4.68(d,J＝7.6Hz,1H).
These data indicate that the target compound is a diterpenoid triepoxide type compound closely related to triptolide. It differs from triptolide in that the hydroxyl group is attached at position C6, rather than C14, which is supported by HMBC-related signals, ranging from δ H4.68(3H, s, H-10) to δ C71.7 (C-6). And further analysis of 2D NMR spectrum data shows that other parts of the target compound are the same as triptolide, the structure of the compound is determined as shown in the specification and named as choumudansu A,
Figure BDA0002341317300000131
experimental example 2
1. Experimental materials:
1) choumudansu A, obtained in example 1.
2) HepG2 cell line, purchased from Shanghai cell Bank of Chinese academy of sciences.
3) Calf serum, purchased from Hangzhou ilex bioengineering materials, Inc.
4) Inverted fluorescence microscope, purchased from olympus, inc.
5) Enzyme-linked plate reader, available from Saimer Feishel, USA.
2. Experimental methods
1) And (3) cell culture: the liver cancer HepG2 cells were inoculated in DMEM medium containing 10% calf serum and cultured at 37 ℃ under 5% carbon dioxide and saturated humidity for 48 hours.
2) MTS detection: HepG2 cells were seeded in 96-well plates in logarithmic growth phase at 1X 10 cells/well4After 24h of culture, 3.125. mu.g/mL choumudansu A was added to each cell, and after 24h, 48h and 72h of culture, 10. mu.l of MTS reagent was added, incubated at 37 ℃ for 0-4 h, and absorbance was measured at 490nm wavelength with a microplate reader every 30min (A490 nm). The control group was prepared without choumudansu A, and the blank group was prepared with medium alone and without cells.
3) And calculating the cell inhibition rate: tumor cell growth Inhibition (IR) ═ 1-experimental a value/cytogroup a value × 100%.
3. Experimental result of inhibitory activity of choumudansu A on liver cancer HepG2 cells
The effect of choumudansu a on the proliferation of liver cancer HepG2 cells is shown in table 2.
TABLE 2 Effect of choumudansu A on HepG2 cell proliferation
Group of Concentration of Cell survivalRate of change
Control group —— 100％
choumudansu A 3.125ug/ml 0.13％
From the above results, it is understood that 3.125ug/ml of choumudansu A has a significant inhibitory effect on HepG2 cells, and that the inhibitory rate of low concentration (3.125ug/ml) of choumudansu on HepG2 cells reaches 99.87%. The inhibitory effect after the culture was photographed microscopically as shown in FIG. 8, in which A in FIG. 8 is an experimental group to which Choumudansu A was added at 3.125ug/ml and C is a control group. The results show that choumudansu A has good anti-liver cancer activity.
Experimental example 3
1. Experimental Material
1) Choumudansu A, obtained in example 1.
2) Lung cancer cell a549, purchased from the cell bank of the chinese academy of sciences.
2. Experimental methods
1) And (3) cell culture: lung cancer A549 cells were cultured at 37 deg.C and 5% CO2Under the conditions, the cells were cultured in DMEM medium containing 10% FBS, penicillin (100U/mL) and streptomycin (100g/L) for 48 hours. Cells were digested by 0.25% trypsin-EDTA for passaging. Cells in logarithmic growth phase were used throughout the experiment.
2) And MST method detection: a549 cells (5X 10) in logarithmic growth phase4cells/mL) were plated in 96-well microplates (100. mu.L per well), incubated overnight, 3.125ug/mL choumudansu A was added, incubation was continued for 24, 48 and 72h, then 100. mu.L of fresh medium was replaced, 20. mu.L of LMTS (5g/L) was added, and after 4h of incubation, the Optical Density (OD) at 490nm was measured using a microplate reader as an indication of cell growth.
3) Calculation of cytostatic rate: inhibition (IR) × (1-OD value of experimental group/OD value of control group) × 100%.
3. The results of the test on the inhibitory activity of the compounds on lung cancer A549 cells are shown in Table 3 below.
TABLE 3 Effect of choumudansu A on A549 cell proliferation
Group of Concentration of Cell survival rate
Control group —— 100％
choumudansu A 3.125ug/ml 0.01％
The results show that 3.125ug/ml of choumudansu A has obvious inhibition effect on lung cancer A549 cells, and has obvious inhibition effect on A549 cell proliferation at low concentration (3.125ug/ml), and the cell inhibition rate reaches 99.99%. And micrographs were taken of the inhibitory effect after the culture, as shown in FIG. 9, in which A in FIG. 9 was an experimental group to which Choumudansu A was added at 3.125ug/ml and C was a control group.
The above shows that low concentrations of choumudansu a have good anti-lung cancer activity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A preparation method of a diterpenoid compound containing a triepoxide structure is characterized by comprising the following steps:
1) crushing clerodendrum bungei, and performing reflux extraction by using an ethanol water solution to obtain an extracting solution; concentrating the extracting solution to obtain an extract, dissolving the extract in water to obtain a suspension, extracting the suspension with petroleum ether, and taking a petroleum ether layer to obtain an extracting solution;
2) subjecting the extract liquor to silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate as mobile phases in volume ratios of 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1 and 0:1, collecting eluates, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 10 fractions which are named as Fr.A-Fr.J according to the detection result and the elution sequence;
3) taking fraction Fr.F, concentrating under reduced pressure, carrying out silica gel column chromatography on concentrated solution, eluting by using petroleum ether-acetone with the volume ratio of (0-20): 1 as a mobile phase, collecting eluent, detecting each eluent by using thin-layer chromatography, and sequentially obtaining 6 fractions according to the detection result and the elution sequence, wherein the fractions are respectively named as Fr.01-Fr.06;
4) concentrating the fraction Fr.03 under reduced pressure, eluting the concentrated solution by using methanol-water as a mobile phase through preparative liquid chromatography, collecting the eluent for 10-14 min, concentrating under reduced pressure, and drying to obtain the diterpenoid compound;
the diterpenoid compounds have the following structural formula:
Figure FDA0003001694100000011
2. the method of claim 1, wherein in step 2), the mobile phase is used in an amount of 3 to 5 column volumes per gradient, and each fraction of eluate is collected in an amount of 1/6 to 1/4 of the column volumes, resulting in 120 fractions of eluate;
detecting each eluent by thin-layer chromatography under 366nm and sunlight by using 10% sulfuric acid ethanol as a color developing agent, and combining 1 st to 18 th, 19 th to 26 th, 27 th to 39 th, 40 th to 54 th, 55 th to 67 th, 68 th to 73 th, 74 th to 83 th, 84 th to 93 th, 94 th to 106 th and 107 th to 120 th eluents with similar chromatographic behaviors to obtain fractions Fr.A to Fr.J.
3. The method according to claim 1, wherein in step 3), the elution is gradient elution, the number of gradients is 2 to 6, the amount of the mobile phase in each gradient is 3 to 5 column volumes, and each eluent is collected at 1/6 to 1/4 of the column volumes to obtain 60 eluents;
detecting each eluent by thin-layer chromatography under 366nm and sunlight by using 10% sulfuric acid ethanol as a color developing agent, and combining 1 st to 15 th, 16 th to 18 th, 19 th to 26 th, 27 th to 38 th, 39 th to 47 th and 48 th to 60 th eluents with similar chromatographic behaviors to obtain fractions Fr.01 to Fr.06.
4. The preparation method according to claim 3, wherein in the step 3), the mobile phase is petroleum ether-acetone in a volume ratio of (5-20): 1.
5. The preparation method according to claim 4, wherein in the step 3), the number of the gradient elution is 4, and the mobile phase of each gradient is petroleum ether-acetone in a volume ratio of 20:1, 15:1, 10:1 and 5: 1.
6. The preparation method according to claim 1, wherein in the step 4), the chromatographic column of the preparative liquid chromatography is a C18 column, the mobile phase is methanol-water with a volume concentration of 40-60%, and the eluent is collected for 11-12 min.
7. The method according to claim 6, wherein the flow rate of the mobile phase is 1.5 to 3 mL/min.
8. The method according to any one of claims 1 to 7, wherein the silica gel used in step 2) has a particle size of 80 to 100 mesh, and the silica gel used in step 3) has a particle size of 200 to 300 mesh.
9. The preparation method according to any one of claims 1 to 7, wherein in the step 1), the volume concentration of ethanol in the ethanol aqueous solution is 60 to 95 percent; the reflux extraction is carried out for 3-5 times, and each time lasts for 1-2 hours.
10. The method according to claim 9, wherein the volume concentration of ethanol in the ethanol aqueous solution is 70 to 75%.
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