CN102311481B - Methyl toonapubesate A and preparation method as well as application thereof - Google Patents
Methyl toonapubesate A and preparation method as well as application thereof Download PDFInfo
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- CN102311481B CN102311481B CN 201010216729 CN201010216729A CN102311481B CN 102311481 B CN102311481 B CN 102311481B CN 201010216729 CN201010216729 CN 201010216729 CN 201010216729 A CN201010216729 A CN 201010216729A CN 102311481 B CN102311481 B CN 102311481B
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Abstract
The invention relates to the technical field of medicines, and relates to novel methyl toonapubesate A, a methyl derivative of toonapubesic acid serving as a triterpene compound, which is separated from Chinese toona ciliatavar.pubescens and a preparation method as well as application thereof to preparing medicaments for treating alzheimer's diseases. The methyl toonapubesate A has a structural formula which is shown in the specification. Experiments prove that the compound has an obvious protection effect on SH-SY5Y cells damaged by Abeta or H2O2 and is expected to be developed into a medicament for resisting the alzheimer's diseases.
Description
Technical field
The present invention relates to medical technical field; More specifically; Relate to a kind of from China hair toon (Toona ciliata var.pubescens) extraction separation obtain the novel triterpene compound hair toon acid of skeleton, the sour methyl esters (Methyl toonapubesate A) of compound hair toon that methylates and obtain through diazomethane then.The invention still further relates to the preparation method of this compound.Biological activity test shows: this compound is to A β or H
2O
2The SH-SY5Y cell of damage has the activeconstituents of provide protection, thereby it can be used for preparing the medicine of treating presenile dementia.
Background technology
The hair toon is a Meliaceae Cedrela plant, extensively is distributed in provinces such as Jiangxi, Hubei, Hunan, Guangdong, Guizhou and Yunnan; Be born in the mountain region thick forest or sparse woods of low height above sea level and intermediate altitude.
Degenerative brain disorder and nerve injury have important dependency, and as intending neural cell model, SH-SY5Y clone is widely used in the research of pathogenesis and new drug development of degenerative brain disorder.
Summary of the invention
The present invention is that extraction separation obtains the novel triterpene compound hair toon acid of skeleton from China hair toon, and methylating through diazomethane then obtains compound hair toon acid methyl esters.Show that through pharmacological testing research this compound is to A β or H
2O
2The SH-SY5Y cell of damage has significant provide protection, and then can be used for preparing the medicine of treating degenerative brain disorder.
Therefore, an object of the present invention is to provide new triterpene compound hair toon acid methyl esters.
Another object of the present invention provides the preparation method of said hair toon acid methyl esters.
Further purpose of the present invention provides said hair toon acid methyl esters and is preparing the neuroprotective cell (for example, through A β (amyloid-beta) or H
2O
2The SH-SY5Y cell of damage) medicine or prepare the purposes of the medicine aspect of anti-degenerative brain disorder.
According to first purpose of the present invention, the present invention has found a hair toon acid derivative hair toon acid methyl esters with new skeleton first from the hair toon, and its chemical structure is as follows:
According to second purpose of the present invention; The invention provides mao preparation method of toon acid methyl esters; It is that extraction separation obtains the novel triterpene compound hair toon acid of skeleton from hair toon T.ciliata var.pubescens; Methylate through diazomethane then and obtain compound hair toon acid methyl esters, concrete steps are following:
Extract: China's hair toon bark is used methanol extraction, get CE after the gained extracting solution concentrates; This CE is water-soluble, use chloroform and n-butanol extraction successively after suspendible is even, the gained extraction liquid obtains chloroform medicinal extract and propyl carbinol medicinal extract respectively after concentrating;
Separate: chloroform medicinal extract carries out silica gel column chromatography, with the petroleum ether/ethyl ether gradient elution; Wherein, 50: 50 wash-out parts of petroleum ether/ethyl ether volume ratio through Sephadex LH-20 gel filtration chromatography, with 1: 1 wash-out of chloroform/methanol volume ratio, obtain a mao toon acid;
Methylate: the acid of hair toon is methylated through diazomethane obtains a mao toon acid methyl esters.
In above-mentioned preparation method, in extraction step, the method that said extraction is adopted is extracted for the methyl alcohol diacolation.
In above-mentioned preparation method, in separating step, the concentration of petroleum ether/ethyl ether gradient elution was followed successively by volume ratio 80: 20,70: 30,60: 40 and 50: 50.
In above-mentioned preparation method, in the step that methylates, said methylate to be specially the acid of hair toon is dissolved in the chloroform, add diazomethane and carry out.
According to the 3rd purpose of the present invention, the invention provides mao purposes of toon acid methyl esters.
The present invention adopts mtt assay to test hair toon acid methyl esters antagonism A β or H
2O
2The activity that the neuroblastoma SH-SY5Y cell survival rate that causes descends.Experiment confirm, hair toon acid methyl esters has provide protection to the SH-SY5Y cell injury in the above-mentioned model.
Therefore hair toon acid methyl esters of the present invention can be used as preparation neuroprotective cell (for example, through A β or H
2O
2The SH-SY5Y cell of damage) medicine, so can be used as preparation treatment degenerative brain disorder (Alzheimer ' s disease, medicine AD).
In addition, hair toon acid methyl esters of the present invention can make up with pharmaceutically acceptable carrier, vehicle or auxiliary material, can further research and develop into the lead compound or the pharmaceutical composition of treatment degenerative brain disorder.
After hair toon acid methyl esters of the present invention can obtain mao toon acid through extraction separation from plant, the process diazomethane methylated and obtains; Also can be through the synthetic acquisition of chemical modification method well known to those skilled in the art.
Description of drawings
Figure 1A and Figure 1B be respectively mao toon acid methyl esters under different concns to A β and H
2O
2The cell survival rate histogram of the SH-SY5Y cell injury of bringing out.
Embodiment
The chemical structural formula (Arabic numeral in the structural formula are marks of carbon atom in the chemical structure) of the hair toon acid methyl esters of indication in following embodiment:
The preparation of 1 mao of toon acid of embodiment methyl esters
Extract: China hair toon bark (picking up from the Xinfeng County, Jiangxi Province) 4kg is carried out diacolation with methyl alcohol (5L) extract three times, get CE after the gained extracting solution concentrates; This CE is water-soluble, use chloroform (1L) and propyl carbinol (1L) to extract respectively successively three times after suspendible is even, the gained extraction liquid obtains chloroform medicinal extract (64g) and propyl carbinol medicinal extract (40g) respectively after concentrating
Separate: chloroform medicinal extract carries out silica gel column chromatography, and with the petroleum ether/ethyl ether gradient elution, the concentration of gradient elution was followed successively by volume ratio 80: 20,70: 30,60: 40 and 50: 50; Wherein, 50: 50 wash-out parts of petroleum ether/ethyl ether volume ratio through Sephadex LH-20 gel filtration chromatography, with 1: 1 wash-out of chloroform/methanol volume ratio, obtain a mao toon acid (21mg).
Hair toon acid (Toonapubesic acids A): white powder; [α]
25 D+ 111.4 (c 0.070, CH
3OH); CD (CH
3OH) λ
Max(Δ ε) 192 (+16.54), 209 (0.01), 232 (+16.57), 337 (3.83) nm;
1H NMR (CDCl
3, 300MHz) δ 7.16 (d, J=9.9Hz, H-1), 5.95 (d, J=9.9Hz, H-2), 2.49 (d, J=12.6Hz, H-5); 5.34 (dd, J=12.6,2.7Hz, H-6), 5.01 (d, J=2.7Hz, H-7), 2.56 (dd, J=12.6,3.6Hz; H-9), 3.38 (br s, H-15), 2.39 (dd, J=10.5,6.6Hz H-17), 1.18 (s, Me-18), 1.20 (s, Me-19); 1.25 (s, Me-28), 1.16 (s, Me-29), 1.22 (s, Me-30), 2.01 (s, OAc-6), 2.11 (s, OAc-7); ESIMS m/z (rel int) 487.2 [M-H]
-(100).
Methylate: the acid of above-mentioned hair toon is dissolved in a spot of chloroform, adds diazomethane (2mL) reaction and obtain a mao toon acid methyl esters (21mg).
Hair toon acid methyl esters, colourless crystallization, molecular formula is C
28H
38O
8 1H reaches
13C NMR data are seen table 1.
Table 1 mao toon acid methyl esters
1H with
13C NMR (ppm in CDCl
3)
The test of embodiment 2 anti-presenile dementia related activity
1, laboratory sample and experimental technique
The preparation of sample solution: specimen is the pure article compound hair toon acid methyl esters of preparation in the foregoing description 1.Accurately take by weighing an amount of sample, the solution with DMSO is mixed with desired concn supplies the pharmacologically active test.
The succeeding transfer culture of clone and cell: active testing adopts SH-SY5Y clone (available from U.S. ATCC (American type culture collection)).With the DMEM substratum that contains 10%FBS, succeeding transfer culture in the incubator of 37 ℃ of feeding 5% carbonic acid gas.
Cytoactive testing method (mtt assay): the present invention adopts mtt assay, test evaluation sample to be tested to A β or H
2O
2The provide protection that causes the SH-SY5Y cell viability to descend.In the viable cell plastosome desaturase can metabolism reduction xanchromatic bromination 3-(4, the 5-dimethylthiazole)-2,5-phenylbenzene tetrazole is a hepatic water-fast first hairpin (formazan), what of first hairpin can be measured its optical density through ELIASA and try to achieve.Because the amount of formazan is directly proportional with viable cell, thus can obtain the number of viable cell according to optical density, thus understand the influence of sample pair cell.
During active testing, the SH-SY5Y cell in the vegetative period of taking the logarithm, using fresh DMEM substratum to be mixed with density is every milliliter 5 * 10
4The cell suspension of individual cell is inoculated in 96 orifice plates by every hole 100 μ L, adds soup 10 μ L/ holes behind the cultivation 24h, and each concentration is all established six multiple holes, and other establishes blank and model group.Cell is cultivated 2h under 37 ℃, 5% carbon dioxide conditions after, except that the blank group, every hole adds A β (ultimate density is 1 μ M) or H
2O
2(ultimate density 100 μ M) damaging cells, 37 ℃, 5%CO
2After cultivating 24h under the condition, every hole adds MTT solution 10 μ L (5mg/mL), after continuing to cultivate 4h, carefully removes supernatant, adds DMSO 100 μ L/ holes, surveys OD with ELIASA then
570/630Value.Calculate the provide protection of analyte pair cell by following formula.
Evaluation of result: the survival rate of sample group is high more, and the provide protection of specimen pair cell is strong more.
2, experimental result
The neurocyte protection activity of hair toon acid methyl esters the results are shown in Figure 1.Wherein, Figure 1A and Figure 1B are respectively mao toon acid methyl esters to A β and H
2O
2The provide protection of the SH-SY5Y cell injury of bringing out.
##P＜0.01 is than the blank group,
*P＜0.01 is than model group): hair toon acid methyl esters is at 5 μ M, and the SH-SY5Y cell injury that during 10 μ M concentration A β is caused has obvious provide protection, and when 10 μ M concentration to H
2O
2The SH-SY5Y cell injury that causes has obvious provide protection.
3, conclusion
Hair toon acid methyl esters has the significant protection effect to neuroblastoma cell under lower concentration.Therefore, compound of the present invention is expected to be developed further into the medicine of anti-degenerative brain disorder.
Claims (7)
1. the following triterpene compound hair toon of chemical structural formula acid methyl esters:
2. the preparation method of the described triterpene compound hair of claim 1 toon acid methyl esters is characterized in that this method may further comprise the steps:
Extract: China's hair toon bark is used methanol extraction, get CE after the gained extracting solution concentrates; This CE is water-soluble, use chloroform and n-butanol extraction successively after suspendible is even, the gained extraction liquid obtains chloroform medicinal extract and propyl carbinol medicinal extract respectively after concentrating;
Separate: chloroform medicinal extract carries out silica gel column chromatography, with the petroleum ether/ethyl ether gradient elution; Wherein, 50: 50 wash-out parts of petroleum ether/ethyl ether volume ratio through Sephadex LH-20 gel filtration chromatography, with 1: 1 wash-out of chloroform/methanol volume ratio, obtain a mao toon acid;
Methylate: the acid of hair toon is methylated through diazomethane obtains a mao toon acid methyl esters.
3. the preparation method of triterpene compound hair toon acid methyl esters according to claim 2 is characterized in that, in extraction step, the method that said extraction is adopted is extracted for the methyl alcohol diacolation.
4. the preparation method of triterpene compound hair toon acid methyl esters according to claim 2 is characterized in that in separating step, the concentration of petroleum ether/ethyl ether gradient elution was followed successively by volume ratio 80: 20,70: 30,60: 40 and 50: 50.
5. the preparation method of triterpene compound hair toon acid methyl esters according to claim 2 is characterized in that in the step that methylates, said methyl turns to the acid of hair toon is dissolved in the chloroform, adds diazomethane and carries out.
6. hair toon acid methyl esters as claimed in claim 1 is protected through A β or H in preparation
2O
2Purposes in the medicine of the SH-SY5Y cell of damage.
7. the purposes of hair toon acid methyl esters as claimed in claim 1 in the medicine of preparation treatment degenerative brain disorder.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1212966A (en) * | 1997-09-26 | 1999-04-07 | 中国人民解放军军事医学科学院放射医学研究所 | Usage of steroi saponin for preventing and curing senile dementia and new steroid saponin |
| WO2009146218A2 (en) * | 2008-04-18 | 2009-12-03 | Reata Pharmaceuticals, Inc. | Compounds including an anti-inflammatory pharmacore and methods of use |
-
2010
- 2010-07-01 CN CN 201010216729 patent/CN102311481B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1212966A (en) * | 1997-09-26 | 1999-04-07 | 中国人民解放军军事医学科学院放射医学研究所 | Usage of steroi saponin for preventing and curing senile dementia and new steroid saponin |
| WO2009146218A2 (en) * | 2008-04-18 | 2009-12-03 | Reata Pharmaceuticals, Inc. | Compounds including an anti-inflammatory pharmacore and methods of use |
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Granted publication date: 20121205 Termination date: 20130701 |