CN110642822A - Sesquiterpenoids and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of medicines, and provides a group of sesquiterpene compounds, a preparation method and application thereof.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a group of sesquiterpene compounds, and a preparation method and application thereof.
Background
Inflammation is a defense reaction of living tissues with a vascular system to injury factors, most diseases are accompanied by inflammation, the inflammation can aggravate the occurrence and development of the diseases, and some chronic inflammations can cause the occurrence of tumors, so that the inflammation control and treatment are very important.
Tumor is a new organism formed by local histiocyte proliferation under the action of various tumorigenic factors, is one of the main killers threatening human health at present, and no effective cure medicine exists at present.
Alzheimer's Disease (AD) is a central neurodegenerative disorder with progressive cognitive impairment and memory impairment, with changes in mood and personality, that severely affects the performance and quality of life of patients. Nowadays the incidence of AD is increasing year by year. The main pathological features of AD are that a protein plaque formed by the deposition of extracellular beta-amyloid protein and intracellular microscopic related protein are over-phosphorylated to form neuron fiber tangles, and finally inflammation, oxidative stress, neuron death and the like are caused to cause a series of AD symptoms. Microglia (BV-2) are central immune cells that, in the activated state, inhibit beta-amyloid deposition and aggregation. Numerous studies have shown that various factors contribute to the activation of BV-2 and thus to the neuroinflammatory response, which plays an important role in the development of AD.
Natural drugs, especially those derived from plants, have a wide variety of chemical structures and biological activities, and have been a major source of diseases prevention and treatment in humans. Many drugs applied clinically are directly or indirectly derived from natural products, the natural products can be used not only as semi-synthetic precursors of drugs, but also as templates of chemical synthetic drugs, and provide a new idea for the design of new drugs, and the natural products have become one of the main sources for finding new drugs or lead compounds.
Wedelia trilobata (Wedelia trilobata) is a herb of Wedelia (Wedelia) in Compositae (Compositae), and is mainly used for treating snake bite, fish bite, diarrhea, kidney stone, cold and other diseases. Modern pharmacological experimental research also shows that wedelia trilobata has certain pharmaceutical activity, but specific active ingredients exerting effects, corresponding diseases and pharmacological mechanisms are still in an unknown state.
Disclosure of Invention
The invention aims to provide a group of sesquiterpene compounds and a preparation method and application thereof.
The structural formula of the group of sesquiterpene compounds provided by the invention is as follows:
the invention also provides a preparation method of the sesquiterpenoids, which comprises the following steps:
(1) drying trilobate wedelia chinensis, and extracting with ethanol to obtain an extract;
(2) sequentially extracting the extracting solution by using a low-polarity solvent, a medium-polarity solvent and a high-polarity solvent;
(3) separating the medium-polarity layer by silica gel column chromatography, and performing gradient elution by using a medium-polarity solvent and a high-polarity solvent to obtain dry weight samples A and B of an elution part with the volume ratio of the medium-polarity solvent to the high-polarity solvent being 100:1 and 50: 1;
(4) carrying out silica gel column layer separation, ODS column chromatography separation and semi-preparative reverse phase HPLC separation on the sample A to obtain compounds 1, 2 and 3;
(5) and (3) carrying out silica gel column layer separation on the sample B, wherein the volume ratio of the polar solvent to the high-polar solvent is 50:1, performing ODS column chromatography separation and semi-preparative reverse phase HPLC separation on an elution part to obtain a compound 4;
(6) or carrying out silica gel column layer separation on the sample B, and carrying out the separation on the polar solvent and the high-polar solvent at a volume ratio of 30: ODS column chromatography separation and semi-preparative reverse phase HPLC separation are carried out on the elution part 1 to obtain the compounds 5, 6, 7 and 8.
The low-polarity solvent is at least one selected from hydrocarbon solvents such as cyclohexane, petroleum ether, n-hexane, isooctane, trimethylpentane, cyclopentane and heptane.
Further, the medium-polarity solvent is at least one selected from ethyl acetate, chloroform, dichloromethane, diethyl ether, methyl formate, nitromethane, butyl acetate and isopropyl ether; the high-polarity solvent is selected from at least one of n-butanol, methanol, tert-butanol, propanol, isopropanol, ethanol, acetone, tetrahydrofuran, and pyridine.
Further, the lower alcohol is C1-C6 alkyl alcohol.
Further, the gradient elution sequence of the medium-polarity-high-polarity solvent in the step (3) is as follows: 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, 0: 1.
The invention also provides application of the sesquiterpene compounds 1-4 and tautomers and pharmaceutically acceptable salts thereof in preparation of antitumor drugs.
The invention also provides application of the sesquiterpene compounds 5-8 and tautomers and pharmaceutically acceptable salts thereof in preparation of anti-inflammatory drugs.
The invention also provides application of the sesquiterpene compounds 5-8 and tautomers and pharmaceutically acceptable salts thereof in preparation of medicines for resisting Alzheimer disease.
Further, the medicament for resisting the Alzheimer disease is any one of oral preparation, powder, granules, capsules, pills and tablets.
Compared with the prior art, the invention extracts a group of new sesquiterpene compounds from wedelia trilobata, and identifies the new sesquiterpene compounds through physicochemical constants and modern spectroscopy, thereby defining the physicochemical properties and the chemical structure of the new sesquiterpene compounds. Pharmacodynamic tests show that the sesquiterpene compounds have antitumor and anti-inflammatory activities, can be used for preparing antitumor drugs and anti-inflammatory drugs, have good pharmaceutical activity on inflammation related to Alzheimer disease, and can also be used for preparing anti-Alzheimer disease drugs.
Drawings
FIGS. 1 to 3 are views of Compound 11H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 4 to 6 are views of Compound 21H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 7 to 9 are views of Compound 31H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 10 to 12 are views of Compound 4 in the order named1H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 13 to 15 are views of Compound 5 in sequence1H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 16 to 18 are views of 6 in the order of compounds1H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 19 to 21 are views of Compound 7 in the order named1H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIGS. 22 to 24 are views of Compound 8 in the order named1H-NMR，13C-NMR, HR-ESI-MS spectrum;
FIG. 25 shows the results of Hochests staining experiments for the compounds 1 to 3 in example 2;
FIG. 26 shows the results of the experiments of Compounds 1-3 of example 2 for inhibiting the growth of cancer cells;
FIG. 27 shows the results of Western Blot experiment in example 2;
FIG. 28 shows the results of Western Blot experiment in example 3.
Detailed Description
The technical solution of the present invention will be described below with reference to specific examples.
Example 1
In this example 1, a group of compounds was extracted and isolated from wedelia trilobata and identified.
The extraction and separation process comprises the following steps:
(1) drying trilobate wedelia chinensis, and extracting with ethanol to obtain an extract;
(2) sequentially extracting the extracting solution by using a low-polarity solvent, a medium-polarity solvent and a high-polarity solvent;
(3) separating the medium-polarity layer by silica gel column chromatography, and performing gradient elution by using a medium-polarity solvent and a high-polarity solvent to obtain dry weight samples A and B of an elution part with the volume ratio of the medium-polarity solvent to the high-polarity solvent being 100:1 and 50: 1;
(4) performing silica gel column layer separation, ODS column chromatography separation and semi-preparative reverse phase HPLC separation on a sample A (the volume ratio of the medium-polarity solvent to the high-polarity solvent is 100:1) to obtain compounds 1, 2 and 3;
(5) and (3) carrying out silica gel column layer separation on the sample B, wherein the volume ratio of the polar solvent to the high-polar solvent is 50:1, performing ODS column chromatography separation and semi-preparative reverse phase HPLC separation on an elution part to obtain a compound 4;
(6) or carrying out silica gel column layer separation on the sample B, wherein the volume ratio of the medium-polarity solvent to the high-polarity solvent is 30: ODS column chromatography separation and semi-preparative reverse phase HPLC separation are carried out on the elution part 1 to obtain the compounds 5, 6, 7 and 8.
The isolated compounds 1-8 were identified as follows:
1. identification of compound 1:
HR-ESI-MS m/z 517.2412[M+Na]+(calcd for C26H38O9na, 518.2408), determining the molecular formula of the compound as C26H38O9。
1H-NMR(500MHz，CDCl3) In the low field region deltaH5.45(1H, m, H-1), 5.80(1H, dd, J ═ 10.5, 8.3Hz, H-9) and the hydrogen signals of the four oxygen-linked methines or methylenes of 4.97(1H, dd, J ═ 9.7, 3.2Hz, H-8) and 5.35(1H, d, J ═ 3.2Hz, H-6) coupled to one another; deltaH6.26(1H, d, J ═ 3.2Hz, H-13), 5.58(1H, d, J ═ 3, 2Hz, H-13) has two terminal olefinic hydrogen signals coupled to one another; high field region deltaH3.41(1H, m, H-7) Hydrogen Signal, δ, with a methine groupH 0.90(3H，s，4”’)，0.91(3H，s，5”’)，1.24(3H，s，3”)，1.26(3H，s，4”)，1.29(3H，s，15-CH3)，1.83(3H，s，14-CH3) 1.97(3H, s, 2') has a hydrogen signal for seven methyl groups.
13C-NMR(125MHz，CDCl3) Gives a 26 carbon signal at δC170.9, 43.0, 25.0, 22.5 and 21.9 are ester carbonyl, methine, methylene and two methyl carbon signals, and the combination of the hydrogen spectrum chemical shift value can be used for deducing that the 9-position of the parent nucleus of the compound is connected with a 2-methyl butyl ester group.
The chemical structure of the compound 1 is determined to be 1 alpha-acetoxy-4 alpha-hydroxy-6 alpha-isobutryloxy-9 beta-15 alpha-methylsolvalyloxystannolide, and the structural formula is as follows:
2. identification of compound 2:
HR-ESI-MSm/z 515.2254[M+Na]+(calcdforC26H36O9na, 516.2252), determining the molecular formula of the compound as C26H36O9。
1H-NMR(500MHz，CDCl3) In the low field region deltaH5.46(1H, t, J ═ 8.5Hz, H-1), 5.90(1H, m, H-6) and two δ coupled to one anotherH5.00(1H, dd, J ═ 9.8, 3.1Hz, H-8), 5.43(1H, d, J ═ 3.1Hz, H-9) hydrogen signals from four methine or methylene groups with attached oxygen, deltaH1.73(1H, m, H-5), 3.42(1H, m, H-7) has a hydrogen signal of two methines, deltaH6.73(1H, m, 3') and δ coupled to each otherHThree alkene hydrogen signals of 6.21(1H, d, J ═ 3.2Hz, H-13) and 5.56(1H, d, J ═ 2.8Hz, H-13), δH1.26(3H，s，14-CH3)，1.31(3H，s，15-CH3) Seven methyl hydrogen signals 1.94(3H, s, 2 '), 1.23(3H, s, 3 "), 1.20(3H, s, 4"), 1.75(3H, s, 4 "'), 1.74(3H, s, 5" '). Wherein deltaH1.26(3H，s，14-CH3)，1.31(3H，s，15-CH3) The characteristic hydrogen signals of the 14 and 15 methyl groups on the parent nucleus.
13C-NMR(125MHz，CDCl3) Gives a 26 carbon signal at δC166.2, 127.4, 139.2, 12.3, 14.7 has an ester carbonyl group, two double bonds, two methyl carbon signals, presumably a (trans) 3-methyl-2-butenoate group attached at the 9-position of the compound.
The compound 2 is 1 beta-acetoxyl-4 a-hydroxy-6 beta-isobutroyloxy-9 a-Tiglioyloxyprostatice, and the specific structural formula is as follows:
3. identification of compound 3:
HR-ESI-MS m/z 515.2252[M+Na]+(calcd for C26H36O9na, 515.2252), determining the molecular formula of the compound as C26H36O。
1H-NMR(500MHz，Methanol-d6) In the low field region deltaH5.48(1H, t, J ═ 8.8Hz, H-1), 5.31(1H, d, J ═ 2.8Hz, H-6) and δ coupled to one anotherH5.16(1H, dd, J ═ 9.7, 2.6Hz, H-8), 3.53(1H, tt, J ═ 9.6, 3.0Hz, H-7) have four hydrogen signals with vicinal oxymethylene or methylene groups; deltaH6.84(1H, m, 3') and δ coupled to each otherHThree alkene hydrogen signals of 5.58(1H, d, J ═ 2.8Hz, H-13) and 6.10(1H, d, J ═ 3.2Hz, H-13). High field region deltaH3.53(1H, tt, J ═ 9.6, 3.0Hz, H-7) has a methine hydrogen signal, δH 1.34(3H，s，15-CH3)，1.35(3H，s，14-CH3) 1.21(3H, d, J ═ 6.9Hz, 3 "), 1.26(3H, d, J ═ 7.1Hz, 4"), 1.77(3H, s, 4 "'), 1.79(3H, s, 5" '), 1.89(3H, s, 2 ').
13C-NMR(125MHz，Methanol-d6) Gives a 26 carbon signal, and the difference in comparison with the compound 2 carbon spectrum data is deltaC70.4(C-1), 23.0(C-2), 36.0(C-3), 70.9(C-4), 74.0(C-6), 44.5(C-7), 75.5(C-8), 41.8(C-10), 137.6(C-11), and 171.5 (C-12). Thus, it can be presumed that there is a difference in the relative configuration between the two.
The compound 3 is 1 alpha-acetoxy-4 beta-hydroxy-6 alpha-isobutroyloxy-9 beta-15 alpha-methyl Tiglioyloxyprostatic e, and the specific structural formula is as follows:
4. identification of Compound 4
HR-ESI-MS m/z 475.1941[M+Na]+(calcd for C23H32O9Na, 475.1939), determining the molecular formula of the compound as C23H32O9。
1H-NMR (400MHz, DMSO) spectrum, in the low field region deltaH5.26(1H, d, J ═ 4.7Hz, H-9), 4.68(1H, dd, J ═ 8.4, 4.7Hz, H-8) hydrogen signals of two vicinal oxymethylene or methylene groups coupled to one another; deltaH4.92(1H, dd, J ═ 11.3, 4.1Hz, H-1) has a hydrogen signal of methine attached to oxygen.
13C-NMR (100MHz, DMSO) gives a total of 23 carbon signals at deltaC166.8, 127.1, 136.3, 18.5 is an ester carbonyl, two double bonds, a methyl, combined with a hydrogen spectroscopic chemical shift value to form an isopropenyl group attached at position 6.
Finally, the compound 4 is determined to be 1a,9 beta-diacetoxy-4 beta-hydroxy-6 a-methacryloxy-11 beta-methylprostaticide, and the structural formula is as follows:
5. identification of Compound 5
HR-ESI-MS m/z 369.1918[M+Na]+(calcd for C19H29O7369.1913), determination of the compound of formula C19H28O7。
1H-NMR (500MHz, DMSO) at the low field region deltaH5.68(1H, m, H-13), 6.19(1H, d, J ═ 1.4Hz, H-13) two ene hydrogen signals; deltaH4.68(1H, t, J ═ 2.8Hz, H-9), 3.72(1H, m, H-8), 3.44(1H, m, H-1) triple tandem oxymethylene or methylene hydrogen signals, deltaH4.14(1H, td, J ═ 10.7, 4.0Hz, H-6) and high field region δH1.57(1H, d, J ═ 10.9Hz, H-5) are the methine or methylene hydrogen signals of the vicinal oxygens coupled to one another, deltaH 0.84(3H，s，14-CH3)、1.20(3H，s，15-CH3) 1.12(3H, d, J ═ 7.0Hz, 3 '), 1.10(1H, d, J ═ 6.9Hz, 4'), there are four methyl hydrogen signals, δ being the number of methyl hydrogen signalsH0.84(3H, s) and 1.20(3H, s) are eudesmanol glycoside type sesquiterpene lactone compounds 14, 15 methylHydrogen radical signal.
13C-NMR (125MHz, DMSO) has 19 carbon signals in total, and the low field region delta of the carbon spectrumC166.8, 175.1 has two carbonyl carbon signals, and deltaC166.8 is the carbon signal of 12-carbonyl on its mother nucleus, high field region deltaC18.7, 19.0, 33.5 two methyl groups, one methine carbon signal with the exception of the remaining carbon signals being methyl, methylene, methine and quaternary carbon signals on the parent nucleus,. delta.C14.9 and 22.8 are characteristic carbon signals of two methyl groups at positions 14 and 15 on the mother nucleus. DeltaC175.1, 33.5, 19.0, 18.7 one ester carbonyl group, one methine group, two methyl groups, combine the chemical shift value of hydrogen spectrum to form isobutyryl group, so that the compound is presumed to be eudesmanol glycoside type sesquiterpene lactone compound.
The compound 5 is 1a,9 beta, 4a-dihydroxy-6 beta-isobutryloxystannolide, and the structural formula is as follows:
6. identification of Compound 6
HR-ESI-MS m/z 281.1390[M+Na]+(calcd for C15H21O5281.1369), determination of the compound of formula C15H20O5。
1H-NMR (500MHz, DMSO) at the low field region deltaH5.33(1H, d, J ═ 3.8Hz, H-3) and δH5.76(1H, dd, J ═ 2.9, 1.2Hz, H-13), 5.83(1H, dd, J ═ 3.3, 1.2Hz, H-13) two three alkene hydrogen signals coupled to one another, δHFour hydrogen signals are present for vicinal oxymethylene groups in 3.98(1H, m, H-1), 3.84(1H, m, H-9), 3.60(1H, dd, J ═ 5.7, 2.8Hz, H-6) and 4.83(1H, dd, J ═ 9.0, 2.5Hz, H-8), high field regions δH 0.80(3H，s，15-CH3).1.82(3H，s，14-CH3) Two methyl hydrogen signals are the signals of the 14 and 15 methyl hydrogen on the compound parent nucleus.
13C-NMR (125MHz, DMSO) has a total of 15 carbon signals,13C-NMR low field region at deltaC 1707 has a carbonyl carbon signal, δC117.5, 120.9, 133.4, 140.1 have four double bond carbon signals, δCFour continuous oxygen methine carbon signals exist at 64.6, 73.0, 73.1 and 75.9, and the high field region deltaC16.9, 25.9 have two methyl carbon signals, the remaining carbon signals being methylene, methine and quaternary carbon signals on the parent nucleus of the compound.
Compound 6 was identified as 1a,6a,9 β -trihydroxy-4,10a-dimethyl-5aH,7aH,8 aH-endsm-3-en-8, 12-olide and has the formula:
7. identification of Compound 7
HR-ESI-MS m/z 369.1908[M+Na]+(calcd for C19H29O7369.1913), determination of the compound of formula C19H28O7。
1H-NMR (500MHz, DMSO) at the low field region deltaH6.34(1H, s, H-13) and 5.64(1H, s, H-13) are the two terminal alkene hydrogen signals, deltaHFour oxymethylene or oxymethylene hydrogen signals exist in 3.54(1H, m, H-1), 5.27(1H, m, H-6), 4.09(1H, s, H-8) and 4.35(1H, s, H-9), and the high field region deltaH 1.13(3H，s，14-CH3)、1.12(3H，s，15-CH3) 1.10(3H, s, 3 '), 1.14(3H, s, 4') are the four methyl hydrogen signals.
13C-NMR (125MHz, DMSO) with a total of 19 carbon signals, the compound was one 2-methacryloyl group less at the 8 position, giving 7 (. delta.) (delta.) of the compoundC 45.6)、8(δC 60.3)、9(δC84.2) bit chemical shift. Compound low field region deltaC163.7, 175.1 has two ester carbonyl carbon signals, where deltaC163.7 is the ester carbonyl carbon signal at the 12-position on the parent nucleus of the compound. DeltaCThe 130.6 and 134.5 have two double bond carbon signals which are the 11 and 13 carbon signals on the compound parent nucleus. DeltaC18.9, 18.4, 14.5, 24.6 are four methyl carbon signals.
Finally, the chemical structure of the compound is determined to be 1 alpha, 4 beta, 9 beta-trihydroxy-6 alpha-isobutryloxyyeudeman-9, 12-olide, and the structural formula is as follows:
8. identification of Compound 8
HR-ESI-MSm/z 369.1902[M+Na]+(calcdforC19H29O7369.1913), determination of the compound of formula C19H28O7。
1H-NMR in the low field region deltaH5.70(1H, m, 3 '), 6.02(1H, s, 3') are the two terminal olefinic hydrogen signals, δH4.23(1H, dd, J ═ 7.9, 2.3Hz, H-1), 5.51(1H, d, J ═ 2.9Hz, H-6), 3.71(1H, dd, J ═ 5.7, 4.2Hz, H-8), 4.67(1H, dd, J ═ 6.9, 3.9Hz, H-9) are four oxymethylene or oxymethylene hydrogen signals, high δ field regionsH1.07(3H，s，15-CH3)、1.10(3H，s，13-CH3) 1.87(3H, s, 4') are the three methyl hydrogen signals.
13C-NMR has a total of 19 carbon signals at deltaC18.1, 125.9, 136.4, 165.8 (one methyl, two double bonds, one carbonyl) combined hydrogen spectrum δH5.70(1H, m), 6.02(1H, s) and 1.90(3H, s) can be presumed to be a 2-methacryloyl group and the rest 15 carbon signals are presumed to be a eudesmanol glycoside type sesquiterpene compound mother nucleus skeleton and a low field region deltaC176.7 is a carbonyl carbon signal, 12 ester carbonyl carbon signal, deltaC13.1, 25.6 are the signals for the methyl carbons at the 13, 15 positions.
Finally, the chemical structure of the compound 8 is determined to be 1 alpha, 9 beta-dihydroxy-4 beta-hydroxymethy-6 beta-methyloxy-13 beta-methylethenomalide:
TABLE 1 preparation of the compounds 1 to 4 of the present invention1H-NMR spectrum and13C-NMR spectral data
The solvent of the compound a is deuterated chloroform, and the solvent of the compound b is deuterated methanol.
The compound 1-3 is 500MHz and 125MHz, and the compound 4 is 400MHz and 100 MHz.
TABLE 2 preparation of compounds 5 to 8 of the present invention1H-NMR spectrum and13C-NMR spectrum data.
The solvent of the compound 5-8 is DMSO, and is 500MHz and 125 MHz.
Example 2
In this example 2, the cytotoxic activities of the compounds 1 to 4 in example 1 on four human tumor cells, namely HeLa, HepG2, SGC-7901 and HUVEC, were studied, specifically as follows:
(1) cell culture and preparation of test solution
Culturing human tumor cells such as HeLa, HepG2, SGC-7901, HUVEC, etc. in RPMI-1640 culture medium, adding 10% fetal calf serum, 1% antibiotic, and adding 5% CO2And cultured at 37 ℃. The cells were maintained in logarithmic growth phase by subculture every two days.
DMSO solutions of the compounds 1-4 are respectively prepared to be used as test sample solutions, stored at 4 ℃ for later use, and diluted to corresponding concentrations by using complete culture media when in use. MTT was dissolved in PBS (phosphate buffer solution) to a concentration of 5mg/mL, stored at 4 ℃ in the dark and diluted to 0.5mg/mL using basal medium.
(2) Activity test (MTT method)
100 μ L of tumor cells (approximately 5000 cells) grown well in step (1) were added to each well of a 96-well plate in 5% CO2Culturing at 37 deg.C for 12 hr, addingThe test solution or the anticancer drug cis-dichlodiammineplatinum (II) (cisplatin) is provided with 3 compound holes in parallel in each group. A blank control group (cell culture solution without cells and without test solution) and a negative control group (cell culture solution without test solution) were also included.
At 5% CO2After culturing at 37 ℃ for 48 hours, the culture medium was discarded by centrifugation, 100. mu.L of MTT solution (0.5mg/mL) was added to each well, the culture was continued for 4 hours, 100. mu.L of SDS (sodium dodecyl sulfate) was added, and after 6 hours, the OD value was measured at 546nm using a microplate reader, and the cell proliferation inhibition rate [ cell proliferation inhibition rate ═ average (negative control OD value average-sample OD value average) ÷ (negative control OD value average-blank control OD value average) × 100%)]And half inhibitory concentration IC50The results are shown in Table 3.
TABLE 3 cytostatic Activity of Compounds 1-4 on tumor cells
The data in Table 3 show that the compounds 1-4 have good antiproliferative effect on four tumor cells, namely HeLa, SGC-7901, HepG2 and HUVEC.
(3) Experiment for observing nuclear morphology by Hochest staining
Tumor cells were seeded in 48-well plates at approximately 8X 10 per well3And incubated for 12 hours. After the cells adhere to the wall, DMSO solutions of compounds 1-3 with different concentrations are added respectively, and the DMSO solutions act for 48 hours to stimulate apoptosis. The cells were then photographed 3 times with a fluorescence microscope objective, the supernatant was discarded, the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 20 minutes, and then washed three times with PBS. Finally, after adding 200. mu.L of dye from Hochest to each well plate to dilute four times, washing was performed three times with PBS and degradation of cell nuclei was observed under a fluorescence microscope, and the results are shown in FIG. 25.
FIG. 25 shows that, as the concentration of the compounds 1 to 3 is gradually increased, the cell nuclei of the tumor cells are gradually changed from a normal form to a kidney form, and then are cracked into fragments, which further proves that the compounds 1 to 3 have an inhibitory effect on the tumor cells.
(4) Experiment for inhibiting cancer cell growth
And (3) inoculating 80 tumor cells to a 24-well plate, culturing for 12h, and after the cells adhere to the wall, respectively setting the compounds 1-3 into a low-dose group, a medium-dose group and a high-dose group to act on the tumor cells. After 48h of drug treatment, the supernatant was removed and complete medium (1640+ 10% FBS + 1% double antibody) was added, and the solution was changed once every three days. After four fluid changes, the cell morphology was observed. And then removing the supernatant, washing with PBS for three times, fixing the cells with 4% paraformaldehyde for 20 minutes, washing with PBS for three times, adding 0.2% crystal violet after volatilizing, dyeing for 20 minutes, washing each hole with distilled water, inverting the 24-hole plate with absorbent paper, drying, and photographing and counting.
As shown in FIG. 26, compounds 1 to 3 inhibited tumor cell proliferation, and the inhibition was stronger as the number of cells was gradually decreased with increasing concentration.
(5) Western Blot experiment
Planting the tumor cells in exponential growth phase in a 96-well plate, and respectively adding compounds 1-2 (10, 20 and 40 mu mol. L) with different concentrations-1) And compound 3(5, 10, 20. mu. mol. L)-1) And extracting the total proteins of each group at corresponding time points, performing electrophoresis by using 10% polyacrylamide gel, performing Western blot experiment according to a conventional operation method, and finally developing and imaging by using an ECL kit. Each set of experiments was repeated 3 times, and the results are shown in fig. 27, wherein "+" in the figure indicates the significance of protein expression,*p＜0.05，***p＜0.01，***p＜0.001)。
as can be seen from FIG. 27, the concentration was 10. mu. mol.L-1The compounds 1-3 can obviously down-regulate the protein expression of PARP-1 and BCL-2 of tumor cells and up-regulate the expression of BAX protein, and the expression effect on the three proteins is more and more obvious along with the increase of the drug concentration, which shows that the compounds 1-3 can play an anti-proliferation role on the cells through the expression of the PARP-1, the BCL-2 and the BAX protein.
The experiments prove that the compounds 1-4 have good inhibition effect on the proliferation of tumor cells, so that the compounds can be used for preparing antitumor drugs, and the tautomers and the pharmaceutically acceptable salts thereof can also be used for preparing antitumor drugs.
When preparing the tumor medicament, the compounds 1-4 or the tautomers thereof, the pharmaceutically acceptable salts thereof and the pharmaceutically acceptable carriers, diluents, excipients, stabilizers and antioxidants can be combined to prepare oral preparations, injections, powders, granules, capsules, pills, tablets, suppositories, films, aerosols, sprays, powder cloud agents, sustained-release and controlled-release agents, targeted preparations or powders. Wherein the pharmaceutically acceptable carrier can be selected from starch, chitosan, alginic acid, agar, fibrin, collagen, polyphosphate, polyurethanes, polyanhydrides, liposome, polyethylene glycol, mannose, galactose, polyvidone, etc., the diluent can be selected from microcrystalline cellulose, lactose, mannitol, starch, saccharin, etc., the excipient can be selected from mannose, glycine, lactose, sodium chloride, glucose, etc., the stabilizer can be selected from albumin, collagen, cyclodextrin and its derivatives, polyethylene glycol, tween, span, dextran, mannitol, etc., and the antioxidant can be selected from VC, VE, benzoic acid, citric acid and its salt, sorbic acid, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, etc.
Example 3
In this example 3, LPS (lipopolysaccharide) induced BV-2 (mouse microglia) is used to establish an in vitro inflammation model, MTT and Griess experiments are used, compounds 5 to 8 are used as test samples, and an anti-inflammatory drug Minocycline (Minocycline) is used as a positive control, so as to investigate the anti-inflammatory activity of the compounds 5 to 8, specifically as follows:
(1) MTT assay
BV-2 cells induced by LPS are inoculated in a 96-well plate, and after 24 hours of culture, a test sample or minocycline solution is added. After further culturing for 24h, determining the BV-2 proliferation inhibition rate and half Inhibition Concentration (IC) of the sample by using MTT method50) The results are shown in Table 5.
(2) Griess experiment
BV-2 cells were plated in 96-well plates and cultured for 24 hours, and then LPS was added to induce inflammation. Then the test article or minocycline solution is added. After further culturing for 24h, each was aspiratedAdding 50 mu L of Griess A reagent and 50 mu L of Griess B reagent into 50 mu L of well culture solution, mixing uniformly, measuring OD value at 546nm by using an enzyme-labeling instrument, and calculating the inhibition rate to NO [ NO inhibition rate ═ between (model control group OD value average value-sample group OD value average value) ÷ (model control group OD value average value-negative control group OD value average value) × 100%, wherein the model control group is BV2 cells induced by LPS, and the negative control group is normal BV2 cells]And calculating the half Inhibitory Concentration (IC) of the tested sample by using Calcusyn software50) The results are shown in Table 4.
TABLE 4 anti-inflammatory Activity of Compounds 5-8 on BV-2 cells
As can be seen from Table 4, compounds 5 to 8 have anti-inflammatory effects on BV-2 cells, and compound 6 has a lower half inhibitory concentration on BV-2 cells than minocycline, and has better anti-inflammatory effects.
(3) Western Blot experiment
The BV-2 cells in exponential growth phase were seeded in 96-well plates and different concentrations of Compound 6(10, 20, 40. mu. mol. L) were added separately-1) And extracting the total proteins of each group at corresponding time points, performing electrophoresis by using 10% polyacrylamide gel, performing Western blot experiment according to a conventional operation method, and finally developing and imaging by using an ECL kit. Each set of experiments was repeated 3 times, and the results are shown in FIG. 28, wherein "#" in the figure represents the significance of LPS relative to normal cells (Control), "#" represents the significance of sesquiterpene compounds relative to LPS,*p＜0.05，**p＜0.01，***p＜0.001)。
as can be seen in FIG. 28, Compound 6 down-regulates NO biosynthesis by inhibiting the expression of both COX-2 and iNOS proteins.
The results show that the sesquiterpene compound 5-8 has a good inhibition effect on the inflammation mediator NO, and can be used for preparing anti-inflammatory drugs.
Because BV-2 cell inflammation and Alzheimer's Disease (AD) development have important influence, the compounds 5-8 with anti-inflammatory activity on BV-2 cells can also be used for preparing the medicine for resisting Alzheimer's disease.
In addition, the tautomer and the pharmaceutically acceptable salt of the compound 5-8 can be used for preparing anti-inflammatory drugs and drugs for treating Alzheimer's disease, and can be combined with pharmaceutically acceptable carriers, diluents, excipients, stabilizers and antioxidants to be prepared into oral agents, injections, powders, granules, capsules, pills, tablets, suppositories, films, aerosols, sprays, powder aerosols, sustained-release and controlled-release agents, targeted preparations or powders.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A group of sesquiterpene compounds and tautomers and pharmaceutically acceptable salts thereof have the following structural formula:
compound 1:compound 2:
compound 3:
compound 4:
compound 5:
compound 6:
compound 7:
compound 8:
2. a process for the preparation of a set of sesquiterpenes according to claim 1, characterized in that: the method comprises the following steps:
(1) drying trilobate wedelia chinensis, and extracting with ethanol to obtain an extract;
(2) sequentially extracting the extracting solution by using a low-polarity solvent, a medium-polarity solvent and a high-polarity solvent;
(3) separating the medium-polarity layer by silica gel column chromatography, and performing gradient elution by using a medium-polarity-high-polarity solvent to obtain dry weight samples A and B of elution parts with a volume ratio of the medium-polarity solvent to the high-polarity solvent of 100:1 and 50: 1;
(4) carrying out silica gel column layer separation, ODS column chromatography separation and semi-preparative reverse phase HPLC separation on the sample A to obtain compounds 1, 2 and 3;
(5) and (3) carrying out silica gel column layer separation on the sample B, wherein the volume ratio of the polar solvent to the high-polar solvent is 50:1, performing ODS column chromatography separation and semi-preparative reverse phase HPLC separation on an elution part to obtain a compound 4;
(6) or carrying out silica gel column layer separation on the sample B, wherein the volume ratio of the medium-polarity solvent to the high-polarity solvent is 30: ODS column chromatography separation and semi-preparative reverse phase HPLC separation are carried out on the elution part 1 to obtain the compounds 5, 6, 7 and 8.
3. The production method according to claim 2, characterized in that: the low-polarity solvent is independently selected from at least one of hydrocarbon solvents such as cyclohexane, petroleum ether, n-hexane, isooctane, trimethylpentane, cyclopentane, heptane and the like.
4. The production method according to claim 2, characterized in that: the medium-polarity solvent is independently selected from at least one of ethyl acetate, chloroform, dichloromethane, diethyl ether, methyl formate, nitromethane, butyl acetate and isopropyl ether; the high-polarity solvent is at least one selected from n-butanol, methanol, tert-butanol, propanol, isopropanol, ethanol, acetone, tetrahydrofuran and pyridine.
5. The production method according to claim 2, characterized in that: the lower alcohol is C1-C6 alkyl alcohol.
6. The production method according to claim 2, characterized in that: the gradient elution sequence of the medium-polarity and high-polarity solvents in the step (3) is as follows: 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, 0: 1.
7. The use of the sesquiterpene compounds 1-4 according to claim 1, tautomers thereof and pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.
8. Use of the sesquiterpene compounds 5-8 according to claim 1 in the preparation of anti-inflammatory drugs, tautomers and pharmaceutically acceptable salts thereof.
9. The use of the sesquiterpene compounds 5-8 according to claim 1 in the preparation of anti-Alzheimer drugs, and tautomers and pharmaceutically acceptable salts thereof.
10. Use according to claim 9, characterized in that: the medicament for resisting the Alzheimer disease is any one of oral preparation, powder, granules, capsules, pills and tablets.
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| CN115806538A (en) * | 2022-11-21 | 2023-03-17 | 诺斯贝尔化妆品股份有限公司 | Purslane terpene composition, preparation method and application thereof |
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| CN115806538A (en) * | 2022-11-21 | 2023-03-17 | 诺斯贝尔化妆品股份有限公司 | Purslane terpene composition, preparation method and application thereof |
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