CN101880308A - New hopane-type triterpene and preparation method and application thereof - Google Patents
New hopane-type triterpene and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical filed of medicines, and relates to the preparation of three new hopane-type triterpenes and applications thereof. The new hopane-type triterpene saponins have the following structure shown in the specification. The new hopane-type triterpene compounds have quinone reductase induced activity, and can be used for development and application of anti-tumor drugs and tumor chemoprevention drugs.
Description
Technical field
The invention belongs to medical technical field, relate to the hopane-type triterpene compounds and separate, prepare and use, be specifically related to three new hopane-type triterpenes, preparation method and the application in antitumor drug thereof.
Background technology
The generation of cancer and evolution and metabolic enzyme are closely related, metabolic enzyme is divided into I phase metabolic enzyme and II metabolic enzyme mutually, and II phase metabolic enzyme comprises reduced glutathion S-transferring enzyme, glucuronyl transferase, quinone reductase NAD (P) H:quinone reductase (QR) etc.I phase metabolic enzyme can not have the activation of carcinogenic precursor under the effect of II phase metabolic enzyme, can be easily has an effect with macromole such as DNA, thereby starts the generation of cancer; And after II phase metabolic enzyme is activated, can catalysis is combined into water-soluble substances by I phase metabolic enzyme activated product and above-mentioned substance such as reduced glutathion etc. and excretes; Perhaps (Quinonereductase under effect QR), changes into the hydrogenation quinone with electrophilic quinone, and the hydrogenation quinone is easy to glucuronic acid and changes into water-soluble substances and excrete, and this process has reduced the generation of oxidizing substance at quinone reductase; Avoid the biomacromolecules such as DNA in activatory carcinogens and the normal cell to act on by said process, play the effect of cancer prevention.Because QR and other II metabolic enzyme expression mutually exist interrelated, that is to say when inducing QR to express, other II phase metabolic enzyme also will be by abduction delivering, so QR can be used as and weighs certain chemical substance (metabolic enzyme inductor) in the cancer chemoprophylaxis and whether have the biomarker that suppresses cancer startup effect.Up to the present, external having reported for work found that from natural product some have the compound of quinone reductase inducing action, as sulfocompound dithiolthiones (oltipraz, oltipraz), the sulphoraphane that from cress, finds; Steroid compound is as withanolides; Flavonoid compound as 7,4 '-dihydroxyl-3 ', 5 '-dimethoxy isoflavones etc.; But up to the present, yet there are no the relevant report of relevant hopane-type triterpene.
Summary of the invention
The purpose of this invention is to provide three new hopane-type triterpenes and its production and application.
The invention provides three new hopane-type triterpenes 1,2 and 3, have following structure:
The invention provides the preparation method of new hopane-type triterpene, prepare by the following method,
(1) dry Eurycorymbus cavaleriei over-ground part adopts ethanol or the methanol extraction of 50%-90% through solvent-extraction process, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract obtains extract suspendible solution with the distilled water suspendible, successively with sherwood oil or hexanaphthene, methylene dichloride or chloroform, ethyl acetate, n-butanol extraction;
(3) step (2) gained methylene dichloride or chloroform extract separate through silica gel column chromatography, with petrol ether/ethyl acetate or sherwood oil/acetone mixed solvent gradient elution;
(4) the gained flow point is through half preparation or preparation HPLC chromatographic separation in the above-mentioned steps (3), and with methanol, acetonitrile/water or methyl alcohol/acetonitrile/water are the moving phase wash-out, obtain new compound 1,2 and 3.
Step (2) among the preparation method of new hopane-type triterpene saponin(e provided by the invention is that crude extract is successively with sherwood oil or hexanaphthene, methylene dichloride or chloroform, ethyl acetate, n-butanol extraction, extraction solvent is 1: 3～3: 1 with extract suspendible liquor capacity ratio, and extraction times is 2-5 time.
Step (3) among the preparation method of new hopane-type triterpene saponin(e provided by the invention is for methylene dichloride or chloroform extract separate through silica gel column chromatography, and eluting solvent is 10: 1～1: 1 petrol ether/ethyl acetate or sherwood oil/acetone.
Step (4) among the preparation method of new hopane-type triterpene saponin(e provided by the invention is separated through high performance liquid chromatography for step (3) wash-out flow point, moving phase is methanol, acetonitrile/water or methyl alcohol/acetonitrile/water, is that 7: 3～1: 0, acetonitrile/water mixed solvent ratio are to obtain compound 1,2 and 34: 6～1: 0 the flow point from methanol mixed solvent ratio.
We from the plant Eurycorymbus cavaleriei isolation identification three kinds of new what uncle alkane triterpene saponin compounds 1,2 and 3, these three new compounds have the quinone reductase induced activity.The present invention has embodied the preparation method and the application in antitumor, chemoprevention of cancer medicine thereof of these three new hopane-type triterpenes.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Embodiment 1
(1) dry Eurycorymbus cavaleriei spray 15kg is through 95% alcohol reflux three times, and each 2 hours, the reclaim under reduced pressure extracting solution got the ethanol crude extract;
(2) ethanol extraction is suspended in 8L distilled water, uses isopyknic sherwood oil, methylene dichloride, ethyl acetate and n-butanol extraction three times respectively;
(3) step (2) gained dichloromethane extract separates through silica gel column chromatography, with sherwood oil: ethyl acetate 8: 1,5: 1,3: 1,2: 1 wash-outs,
(4) gained sherwood oil in the above-mentioned steps (3): 5: 1 flow points of ethyl acetate are through partly preparing the HPLC chromatographic separation, with the acetonitrile/water gradient elution, acetonitrile/water 50: 50～100: 0, elution time 50 minutes, flow velocity 8mL/min, obtained compound 1 in 25 minutes in retention time, retention time obtained compound 2 in 44 minutes.
The sherwood oil that is able in the above-mentioned steps (3): 8: 1 flow points of ethyl acetate are through partly preparing the HPLC chromatographic separation, with the acetonitrile/water gradient elution, acetonitrile/water 50: 50～90: 10, elution time 50 minutes, flow velocity 8mL/min obtained compound 3 in 40 minutes in retention time.
Compound 1,2 and 3 structure appraising datum are as follows:
Compound 1: white powder (methyl alcohol); [α]
D+ 70.6 ° (c 0.5, methyl alcohol); IR v
Max3373,2942,2832,1450,1390cm
-1 1H NMR (Table 1);
13C NMR (Table 1); HRESIMS m/z463.2536[M-H
2O+Na]
+(calcd for C
29H
44O
3Na, 463.3188).
Compound 2: white powder (methyl alcohol); [α]
D+ 20.7 ° (c 1.1, methyl alcohol); IR v
Max3444,2946,2832,1643,1450,1390cm
-1 1H NMR (Table 1);
13C NMR (Table 1); HRESIMS m/z627.3703[M+Na]
+(calcd for C
38H
52O
6Na, 627.3662).
Compound 3: yellow powder (methyl alcohol); [α]
D+ 21 ° (c 0.5, methyl alcohol); IR v
Max3400,3392,2831,1454,1410cm
-1 1H NMR (Table 1);
13C NMR (Table 1); HRESIMS m/z 621.4129[M+Na]
+(calcd for C
37H
58O
6, 621.4131).
Table 1 compound 1,2 and 3 NMR data
a)Measured?in?DMSO-d
6at?500MHz.
b)Measured?in?CDCl
3?at?500MHz.
Embodiment 2:
(1) dry Eurycorymbus cavaleriei over-ground part (10kg) is that solvent refluxing extracts with 75% ethanol, and the reclaim under reduced pressure extracting solution gets the ethanol crude extract;
(2) ethanol extraction is suspended in 5L distilled water, uses twice of isopyknic sherwood oil, methylene dichloride, ethyl acetate and n-butanol extraction respectively;
(3) step (2) gained dichloromethane extract separates through silica gel column chromatography, with sherwood oil: acetone 8: 1,6: 1,4: 1,3: 1 wash-outs,
(4) sherwood oil that is able in the above-mentioned steps (3): 4: 1 flow points of acetone are through partly preparing the HPLC chromatographic separation, with the methanol gradient elution, methanol 75: 25～100: 0, elution time 60 minutes, flow velocity 8mL/min, obtained compound 1 in 33 minutes in retention time, retention time obtained compound 2 in 54 minutes.
The sherwood oil that is able in the above-mentioned steps (3): 9: 1 flow points of acetone are through partly preparing the HPLC chromatographic separation, with the methanol gradient elution, and methanol 70: 30～100: 0, elution time 60 minutes, flow velocity 9mL/min obtained compound 3 in 45 minutes in retention time.
Compound 1-3 structure authentication method is with embodiment 1.
Embodiment 3 compounds 1,2 and the test of 3 quinone reductase induced activity
Experiment material:
(1) main agents and major equipment: calf serum (available from Hyclone); Foetal calf serum (TBD company); Tetramethyl-azo azoles salt (MTT) U.S. Sigma (St.Louis, MO); Glucose-6-phosphate dehydrogenase (G6PD) (worker is given birth in Shanghai); 96 porocyte culture plates (Costar company); Cell strain: mouse hepatoma cell strain Hepa 1c1c7 experimental technique:
(1) cultivation of cell:
Every hole kind 10
4Individual cell, growth is 24 hours in the nutrient solution that comprises 10% (v/v) foetal calf serum, 0.01% penicillin G, 0.15% sodium bicarbonate, 0.01% Vetstrep, and envrionment conditions is 37 ℃, contain 5%CO
2Damp atmosphere.
(2) preparation of medicine
Testing compound uses the DMSO dissolving, is made into the mother liquor that concentration is 50mmol/L, is stored in-20 ℃.
(3) the Viola crystallina method is determined cell survival rate
The testing compound of concentration known is dissolved in adds each hole among the DMSO and kept 24 hours.Should guarantee final concentration≤0.5% (v/v) of DMSO behind every hole adding DMSO.Discard nutrient solution afterwards, every hole adds 200 μ L, 0.2 μ M Viola crystallina (2% ethanolic soln).Normal temperature is placed dyeing about 10 minutes down, discards crystal violet solution, and water washs rapidly 3 times, water is dried and dry up with blower.Every hole adds 200 μ L 0.5%SDS (50% ethanolic soln), and normal temperature vibrated 5～10 minutes down, and 595nm measures absorbancy down.The absorbancy that records is proofreaied and correct through blank group (hole of gutless cell), and represents cell survival rate with the percentage that is equivalent to control group (cell in the hole that does not have to handle) absorbancy.Every group of experiment should independently at least repeat 3 times and average as end-result.
Mean value * 100% of the mean value of cell survival rate %=administration group OD value/blank breast group OD value
(4) the quinone reductase induced activity is measured
It is that G-6-P can be reduced by glucose-6-phosphate dehydrogenase (G6PD) under the condition that cofactor NADP exists that QR induces the ultimate principle of mensuration, at this moment can produce NADPH, NADPH is in case formation just makes vitamin k4 be reduced to menadiol as a kind of electron donor, menadiol can make MTT be reduced to the Jia Za, but last Dui Jia Za carries out the mensuration of absorbancy.NADP and vitamin k4 are renewable in this catalytic cycle process, so need not replenish it in the experiment.The QR inductor can increase the generation of menadiol, thereby more Jia Za is formed.This experiment in, the compound concentrations that adopts to pass through Hepa 1c1c7 cell screening in advance, its concentration should satisfy makes cell survival rate 〉=55%.
Active being determined on 96 orifice plates of quinone reductase carried out with the mouse liver cancer cell, and process is as described below: after the cell cultures testing compound of concentration known is dissolved in and adds each hole among the DMSO and kept 24 hours.Should guarantee final concentration≤0.5% (v/v) of DMSO behind every hole adding DMSO.After this, nutrient solution poured out and add contain 0.8% (w/v) digitonin and 2mM EDTA, stir 10 minutes with peptic cell.Add " complete reaction mixes liquid " 200 μ l in every hole.Should mixed liquid should face and use preceding configuration, collocation method is as follows: the solution that G-6-P, the NADP of 90 μ L 50mM, 300 unit glucose-6-phosphate dehydrogenase (G6PD)s, the 45mg MTT of FAD, the 1mL 150mM of the polysorbas20 of the Tris-Cl (pH=7.4) of 7.5mL 0.5M, 100mg calf serum, 1mL 1.5%, 0.1mL 7.5mM is configured to 150mL with deionized water.Before adding mixed liquid, add 0.2 μ L vitamin k4 (50mM is dissolved in the acetonitrile).96 orifice plates jolting 5 minutes gently after to be added the finishing.The 0.3mM temparin is dissolved in 0.5%DMSO and the 5mM potassium primary phosphate (pH=7.4) to be drawn 50 μ L and adds on the entering plate in every hole with termination reaction.Under the 590nm wavelength, measure absorbancy.Do not contain cell in the blank group, control group is Hepa 1c1c7 cell and contains 0.5%DMSO matrix liquid but do not contain testing compound.The method of calculation of the QR induced activity of testing compound: earlier the absorbancy of administration group and control group is deducted the absorbancy of blank group, refer to that with the absorbancy of administration group absorbance (IR) than last control group is as the index of QR induced activity then.Every group of experiment should independently at least repeat 3 times.
Test-results:
Compound 1,2 and 3 QR induced activity test result
| ??Sample?name | ??C(μg/ml) | ??Cell?viability(％) | ??IR |
| ??1 | ??20 | ??66％ | ??2.2 |
| ??Sample?name | ??C(μg/ml) | ??Cell?viability(％) | ??IR |
| ??2 | ??20 | ??78％ | ??3.1 |
| ??3 | ??20 | ??89％ | ??2.4 |
Guaranteeing that cell survival rate is higher than under 55% the situation, IR shows promptly that greater than 2 specimen has the QR inducing action, and by last table result as can be known, compound 1,2 has certain QR induced activity, and compound 3 has quinone reductase induced activity preferably.
Claims (6)
1. new hopane-type triterpene is characterized in that, it has following structure:
2. the preparation method of a new according to claim 1 hopane-type triterpene, it is characterized in that: it comprises the steps:
(1) dry Eurycorymbus cavaleriei (Eurycorymbus cavaleriei) over-ground part adopts ethanol or the methanol extraction of 50%-90% through solvent-extraction process, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract gets extract suspendible solution with the distilled water suspendible, uses extraction solvent sherwood oil or hexanaphthene, methylene dichloride or chloroform, ethyl acetate, n-butanol extraction successively;
(3) step (2) gained methylene dichloride or chloroform extract separate through silica gel column chromatography, with eluting solvent petrol ether/ethyl acetate or sherwood oil/acetone mixed solvent gradient elution;
(4) the gained flow point is through half preparation or preparation HPLC chromatographic separation in the above-mentioned steps (3), and with methanol, acetonitrile/water or methyl alcohol/acetonitrile/water are the moving phase wash-out, obtain new compound 1,2 and 3.
3. preparation method according to claim 2 is characterized in that: extraction solvent is 1: 3～3: 1 with extract suspendible liquor capacity ratio, and extraction times is 2-5 time.
4. preparation method according to claim 2 is characterized in that: eluting solvent is 10: 1～1: 1 petrol ether/ethyl acetate or sherwood oil/acetone.
5. preparation method according to claim 2 is characterized in that: from methanol mixed solvent ratio is that 7: 3～1: 0, acetonitrile/water mixed solvent ratio are to obtain compound 1,2 and 34: 6～1: 0 the flow point.
6. the application of the described new hopane-type triterpene compound 1,2 of claim 1 and 3 in antitumor, the chemoprevention of cancer medicine of preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110407907A (en) * | 2019-08-14 | 2019-11-05 | 济南大学 | Sago cuckoo glycol and its preparation method and application |
-
2010
- 2010-07-01 CN CN 201010214754 patent/CN101880308B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《Journal of Chromatography A》 20090419 Lin Cheng, et al Characterization of chemopreventive agents from the dichloromethane extract of Eurycorymbus cavaleriei by liquid chromatography-ion trap mass spectrometry 4859-4867 1-6 第1216卷, * |
| LIN CHENG, ET AL: "A Novel Meroterpene from Eurycorymbus cavaleriei", 《HELVETICA CHIMICA ACTA》 * |
| LIN CHENG, ET AL: "Characterization of chemopreventive agents from the dichloromethane extract of Eurycorymbus cavaleriei by liquid chromatography–ion trap mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 何轶 等: "伞花木化学成分研究", 《中草药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110407907A (en) * | 2019-08-14 | 2019-11-05 | 济南大学 | Sago cuckoo glycol and its preparation method and application |
| CN110407907B (en) * | 2019-08-14 | 2022-03-29 | 济南大学 | Rhododendron simsii diol and preparation method and application thereof |
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Granted publication date: 20130213 Termination date: 20200701 |