CN100402483C - Process of extracting cimitt acid - Google Patents
Process of extracting cimitt acid Download PDFInfo
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- CN100402483C CN100402483C CNB2006100358943A CN200610035894A CN100402483C CN 100402483 C CN100402483 C CN 100402483C CN B2006100358943 A CNB2006100358943 A CN B2006100358943A CN 200610035894 A CN200610035894 A CN 200610035894A CN 100402483 C CN100402483 C CN 100402483C
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- cimitt
- acid
- ethyl acetate
- chloroform
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Abstract
The present invention discloses a method for extracting Ximite acid, which has the working procedures: sponges are immersed in ethanol, and are processed with extraction to obtain an ethanol extract liquid; sponge extracts can be obtained by concentration; the sponge extracts are dispersed in water, and are extracted with ethyl acetate; extracting liquids are combined and concentrated to obtain concentrates of ethyl acetate; the concentrates of ethyl acetate are dispersed in a methanol water solution; the mixtures are extracted and edulcorated with petroleum ether; a residual liquid is diluted with water to obtain chloroform extracts after being extracted, combined and concentrated with chloroform; the chloroform extracts are processed with laminar analysis through silica gel columns and gradient elution by a petroleum ether-ethyl acetate solvent system till a condition that beige Ximite acid constituents are completely eluted; all Ximite acid constituents are combined and concentrated to obtain Ximite acid crude products. The present invention has the characteristics of short production period, low cost, high output rate, etc.
Description
Technical field
The present invention relates to a kind of compound extracting method, refer in particular to a kind of method of from South China Sea sponge Rhabdastrella globostellata, extracting cimitt acid (foreign odor Chinese toon acid compound).
Background technology
Sponge is one of primary multicellular animals, and of a great variety, distributed pole is wide, based on marine products.China South Sea is located in the torrid zone and subtropics, and the marine organisms species are very abundant, and sponge particularly accounts for half of the world total.People have found organic compound such as terpene, steroidal, glycoside, alkaloid, polypeptide and the polyethers etc. of many structure uniquenesses from spongy biological in recent years, wherein many have antitumor, cardiovascular disease resistant, a physiologically active such as antibiotic and antiviral, and many compounds have the potential clinical value.Be guide's thing particularly,, obtain the organic drug that physiologically active is stronger, toxicity is lower, cause that countries in the world more and more pay close attention to through the modification of chemical structure with these compounds.
Foreign odor Chinese toon acid compound involved in the present invention belongs to a kind of triterpenic acid on the structure, therefore also claim foreign odor Chinese toon acid triterpene, is the triterpenes natural product that a class has important physiologically active in the sponge, and we are referred to as cimitt acid.It is the natural product that a class has important physiologically active.The preliminary screening of institute of materia medica, Chinese Academy of Sciences Shanghai finds that cimitt acid demonstrates 100% restraining effect (under the 0.1 μ M concentration) to bone-marrow-derived lymphocyte and the lymphocytic propagation of T; Inhibition test shows that cimitt acid is to ehrlich carcinoma solid tumor, S180 sarcoma, A-549 human lung adenocarcinoma, people's lung giant cells (PG) cancer and people LOVO colorectal carcinoma etc. in the animal body
Foreign odor Chinese toon acid triterpene (cimitt acid)
The kinds of tumors model has higher inhibition proliferation activity, and safety coefficient is bigger.The antitumous effect mechanism of carrying out in south China oncology National Key Laboratory studies show that cimitt acid can be by activating the apoptosis of Caspase-3 enzyme induction HL-60 tumour cell; Gene chip detects test and shows that cimitt acid can increase or reduce the expression of some apoptogenes; Suppressing the PI3K/AKT signal transduction pathway may be its antineoplastic action mechanism.
Sponge Rhabdastrella globostellata resource is a considerable number of at the South Sea, gathers convenient (2～3 meters of the depth of waters), the cimitt acid content in the sponge abundant (account for dry weight 0.02%).But original extraction process (seeing number of patent application " 95116029.X "), this patent disclosure following extraction process: use the ethanol extracting after drying the sponge fragmentation, extract is concentrated into pulpous state, then it is scattered in the suitable quantity of water, again with sherwood oil, ethyl acetate and propyl carbinol carry out extracting and separating successively, with gained ethyl acetate part through 5 silica gel column chromatographies and 2 dextrane gel column chromatographies, make eluent with petroleum ether-ethyl acetate and chloroform-methanol respectively, golden yellow solid will be obtained behind the elutant concentrating under reduced pressure, again this solid is dissolved in an amount of benzene, obtains disk shape crystal, i.e. cimitt acid.Its productive rate is 0.0022%.The shortcoming of this method is more loaded down with trivial details, length consuming time, cost height, need repeatedly, use repeatedly not easy-operating silica gel column chromatography, conversion eluent repeatedly, and need use the more expensive sephadex lh-20 of price and make column chromatography for separation, not only the cycle is long, cost is high, and productive rate is low, can't adapt to fairly large preparation, the production of 1 gram sample 2～3 months consuming time usually, sometimes even reach half a year.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, the object of the present invention is to provide the extracting method of a kind of cimitt acid (foreign odor Chinese toon acid compound).This method has made full use of the physico-chemical property of this compounds, is preliminary extraction agent with industrial alcohol, and continuously with ethyl acetate, chloroform give extraction agent, obtains pure product foreign odor Chinese toon acid compound by steps such as sherwood oil removal of impurities, column chromatography and recrystallizations again.That extracting method of the present invention has is with short production cycle, cost is low and productive rate is high, adapts to the advantages such as requirement of macro preparation.
Purpose of the present invention is achieved through the following technical solutions: the extracting method of a kind of cimitt acid (foreign odor Chinese toon acid compound) comprises following processing step:
(1) sponge (Rhabdastrella sp.) obtains ethanol extract with the alcohol immersion extracting.
(2) ethanol extract obtains sponge medicinal extract through concentrating, and sponge medicinal extract is scattered in the water, uses ethyl acetate extraction, and combining extraction liquid concentrates, and obtains the ethyl acetate enriched material.
(3) the ethyl acetate enriched material being scattered in volumetric concentration is in 80～95% the methanol aqueous solution, and after the petroleum ether extraction removal of impurities, residual solution is diluted with water to 40～70% (volumetric concentrations of nail alcohol), uses chloroform extraction again, merge, concentrate, chloroform extract.
(4) chloroform extract is through silica gel column chromatography, with petroleum ether-ethyl acetate (volume ratio 9: 1 → 8: 2 → 7: 3 → 5: 5) solvent systems gradient elution, until the complete wash-out of cimitt acid component of beige.
(5) merge all cimitt acid components, concentrate, obtain the cimitt acid crude product.
In order to realize the present invention better,, obtain golden yellow powder with the column chromatography removal of impurities of cimitt acid crude product; Recrystallization then obtains the pure product of cimitt acid of orange.
Described ethanol is the industrial alcohol of volumetric concentration 95%.Described sponge is used 3～5 weeks of alcohol immersion, extracting 3 times.Described water is distilled water.The complete wash-out of cimitt acid component of beige in the step (4), available chromatography or TLC (thin-layer chromatography) method is monitored.Described simmer down to concentrating under reduced pressure.
Described column chromatography adopts 8: 2 sherwood oil of volume ratio-acetone column chromatography or 9: 1 chloroform of volume ratio-acetone column chromatography.Described recrystallization adopts chloroform-sherwood oil recrystallization, and chloroform: the volume ratio of sherwood oil is 6: 4.
The present invention compared with prior art has following advantage and beneficial effect:
Productive rate of the present invention is brought up to present 0.03% by 0.0024% of former technology, increase about 12 times; Production cycle foreshortened to 1～2 month by original 2～3 months; Simplified control is easy to grasp; Cost reduces, and the present invention makes full use of the physico-chemical property of product, and the solvent of utilization opposed polarity repeatedly, fractional extraction, replaces the repeatedly silica gel column chromatography and the gel filtration chromatography of former technology, the consumption of having saved a large amount of silica gel and gel.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
Embodiment 1
With after 2.0 kilograms of (dry weight) South Sea sponge Rhabdastrella globostellata fragmentations with 1 week of industrial alcohol soaking at room temperature of 95%, filter is done the back residue and is soaked 2 times again, in each 1 week, extracting is 3 times altogether, obtains ethanol extract.To be evaporated to black soup compound (sponge medicinal extract) after the ethanol extract merging.This soup compound is dispersed in the 500ml distilled water, and with ethyl acetate (500ml) extraction 3 times, combining extraction liquid and concentrating under reduced pressure get ethyl acetate enriched material 64 grams.The ethyl acetate enriched material is dispersed in 300 milliliter 93% the methanol aqueous solution, with 3 removal of impurities of sherwood oil (300ml) extraction.The residual solution water layer with chloroform (500ml) extraction 3 times, combining extraction liquid, concentrates to such an extent that chloroform extract 20 restrains again with distilled water diluting to 70% (volumetric concentration of nail alcohol).This extract is through silica gel column chromatography, with petroleum ether-ethyl acetate (volume ratio 9: 1 → 8: 2 → 7: 3 → 5: 5) solvent systems gradient elution, remove the low-polarity constituents of green, black successively, to the cimitt acid component that beige occurs, with color interface monitoring (being chromatography), elute fully until the cimitt acid component.The cimitt acid chromatogram layer that merges all beiges, concentrating under reduced pressure gets cimitt acid crude product 10 grams.This crude product with 8: 2 sherwood oil of volume ratio-further removal of impurities of acetone column chromatography, is got golden yellow powder 2 grams.Use chloroform-sherwood oil (volume ratio 6: 4) recrystallization at last, get pure product 0.35 gram of cimitt acid of orange.Productive rate 0.0175%.
The affirmation of product chemical structure: infrared data v
Max(cm
-1): 3427,2939,1711,1691,1579,1542,1422,1391,1289,1184,1160,974,803; Nuclear magnetic data δ
1H (CDCl
3): 7.42 (1H, d), 7.06 (1H, dd), 6.76 (1H, d), 6.676 (1H), 6.659 (1H), 6.44 (1H, d), 2.76 (1H, m), 2.44 (1H, m), 2.37 (3H, s), 2.25 (2H, dd), 2.21 (2H, m), 2.07 (3H, s), 2.05 (3H, s), 1.90 (1H, dd), 1.64 (1H, m), 1.54 (2H, m), 1.46 (3H, s), 1.15 (3H, s), 1.08 (3H, s), 0.88 (3H, s).
Embodiment 2
With after 2.0 kilograms of (dry weight) South Sea sponge Rhabdastrella globostellata fragmentations with 2 weeks of industrial alcohol soaking at room temperature of 95%, filter is done the back residue and is soaked 2 times again, in each 1.5 weeks, extracting is 3 times altogether, obtains ethanol extract.With being evaporated to sponge medicinal extract (soup compound) after the ethanol extract merging, this sponge medicinal extract is dispersed in the 500ml distilled water, with ethyl acetate (500ml) extraction 3 times, combining extraction liquid and concentrating under reduced pressure get ethyl acetate enriched material 80 grams.The ethyl acetate enriched material is dispersed in 300 milliliter 80% the methanol aqueous solution, with 3 removal of impurities of sherwood oil (300ml) extraction, the residual solution water layer is again with distilled water diluting to 40% (volumetric concentration of nail alcohol), with chloroform (500ml) extraction 3 times, combining extraction liquid concentrates to such an extent that chloroform extract 35 restrains.This extract is through silica gel column chromatography, with petroleum ether-ethyl acetate (volume ratio 9: 1 → 8: 2 → 7: 3 → 5: 5) solvent systems gradient elution, remove the low-polarity constituents of green, black successively, to the cimitt acid component that beige occurs, monitor with the TLC method, elute fully until the cimitt acid component.Merge all TLC spots and the approaching component of cimitt acid standard model, concentrating under reduced pressure gets cimitt acid crude product 20 grams.This crude product with 9: 1 chloroform of volume ratio-further removal of impurities of acetone column chromatography, is got golden yellow powder 4 grams.Use chloroform-sherwood oil (volume ratio 6: 4) recrystallization at last, get pure product 0.6 gram of cimitt acid, productive rate 0.03%.
The affirmation of product chemical structure: infrared data v
Max(cm
-1): 3427,2939,1710,1691,1579,1541,1422,1391,1289,1183,1160,974,803; Nuclear magnetic data δ
1H (CDCl
3): 7.42 (1H, d), 7.06 (1H, dd), 6.76 (1H, d), 6.676 (1H), 6.659 (1H), 6.44 (1H, d), 2.76 (1H, m), 2.44 (1H, m), 2.37 (3H, s), 2.25 (2H, dd), 2.21 (2H, m), 2.07 (3H, s), 2.05 (3H, s), 1.90 (1H, dd), 1.64 (1H, m), 1.54 (2H, m), 1.46 (3H, s), 1.15 (3H, s), 1.08 (3H, s), 0.88 (3H, s).
Embodiment 3
With after 2.0 kilograms of (dry weight) South Sea sponge Rhabdastrella globostellata fragmentations with 2 weeks of industrial alcohol soaking at room temperature of 95%, filter is done the back residue and is soaked 2 times again, in each 1 week, extracting is 3 times altogether, obtains ethanol extract.To be evaporated to black soup compound (sponge medicinal extract) after the ethanol extract merging.This soup compound is dispersed in the 500ml distilled water, and with ethyl acetate (500ml) extraction 3 times, combining extraction liquid and concentrating under reduced pressure get ethyl acetate enriched material 78 grams.The ethyl acetate enriched material is dispersed in 300 milliliter 87% the methanol aqueous solution, with 3 removal of impurities of sherwood oil (300ml) extraction.The residual solution water layer with chloroform (500ml) extraction 3 times, combining extraction liquid, concentrates to such an extent that chloroform extract 33 restrains again with distilled water diluting to 55% (volumetric concentration of nail alcohol).This extract is through silica gel column chromatography, with petroleum ether-ethyl acetate (volume ratio 9: 1 → 8: 2 → 7: 3 → 5: 5) solvent systems gradient elution, remove the low-polarity constituents of green, black successively, to the cimitt acid component that beige occurs, with color interface monitoring (being chromatography), elute fully until the cimitt acid component.The cimitt acid chromatogram layer that merges all beiges, concentrating under reduced pressure gets cimitt acid crude product 18 grams.This crude product with 8: 2 sherwood oil of volume ratio-further removal of impurities of acetone column chromatography, is got golden yellow powder 3.5 grams.Use chloroform-sherwood oil (volume ratio 6: 4) recrystallization at last, get pure product 0.55 gram of cimitt acid of orange.Productive rate 0.0275%.
The affirmation of product chemical structure: infrared data v
Max(cm
-1): 3428,2940,1711,1692,1579,1542,1422,1391,1289,1186,1160,973,803; Nuclear magnetic data δ
1H (CDCl
3): 7.42 (1H, d), 7.06 (1H, dd), 6.76 (1H, d), 6.676 (1H), 6.659 (1H), 6.44 (1H, d), 2.76 (1H, m), 2.44 (1H, m), 2.37 (3H, s), 2.25 (2H, dd), 2.21 (2H, m), 2.07 (3H, s), 2.05 (3H, s), 1.90 (1H, dd), 1.64 (1H, m), 1.54 (2H, m), 1.46 (3H, s), 1.15 (3H, s), 1.08 (3H, s), 0.88 (3H, s).
Claims (9)
1. the extracting method of a cimitt acid comprises following processing step:
(1) sponge obtains ethanol extract with the alcohol immersion extracting;
(2) ethanol extract obtains sponge medicinal extract through concentrating, and sponge medicinal extract is scattered in the water, uses ethyl acetate extraction, and combining extraction liquid concentrates, and obtains the ethyl acetate enriched material;
(3) the ethyl acetate enriched material being scattered in volumetric concentration is in 80～95% the methanol aqueous solution, and after the petroleum ether extraction removal of impurities, residual solution is diluted with water to 40～70%, uses chloroform extraction again, merge, concentrate, chloroform extract;
(4) chloroform extract is through silica gel column chromatography, with the gradient elution of petroleum ether-ethyl acetate solvent systems, until the complete wash-out of cimitt acid component of beige;
(5) merge all cimitt acid components, concentrate, obtain the cimitt acid crude product;
Described petroleum ether-ethyl acetate solvent systems is with 9: 1 → 8: 2 → 7: 3 → 5: 5 petroleum ether-ethyl acetate solvent systems of volume ratio.
2. the extracting method of cimitt acid according to claim 1, it is characterized in that: described cimitt acid crude product column chromatography removal of impurities obtains golden yellow powder; Recrystallization then obtains the pure product of cimitt acid of orange.
3. the extracting method of cimitt acid according to claim 1, it is characterized in that: described ethanol is the industrial alcohol of volumetric concentration 95%.
4. the extracting method of cimitt acid according to claim 1 is characterized in that: described sponge usefulness 3～5 weeks of alcohol immersion, extracting 3 times.
5. the extracting method of cimitt acid according to claim 1, it is characterized in that: described water is distilled water.
6. the extracting method of cimitt acid according to claim 1 is characterized in that: the complete wash-out of cimitt acid component of beige in the step (4), monitor with chromatography or tlc.
7. the extracting method of cimitt acid according to claim 1 is characterized in that: described simmer down to concentrating under reduced pressure.
8. the extracting method of cimitt acid according to claim 2 is characterized in that: 8: 2 sherwood oil of described column chromatography employing volume ratio-acetone column chromatography or 9: 1 chloroform of volume ratio-acetone column chromatography.
9. the extracting method of cimitt acid according to claim 2 is characterized in that: described recrystallization adopts chloroform-sherwood oil recrystallization, and chloroform: the volume ratio of sherwood oil is 6: 4.
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| CNB2006100358943A CN100402483C (en) | 2006-06-12 | 2006-06-12 | Process of extracting cimitt acid |
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| CNB2006100358943A CN100402483C (en) | 2006-06-12 | 2006-06-12 | Process of extracting cimitt acid |
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| CN100402483C true CN100402483C (en) | 2008-07-16 |
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| CN108218944B (en) * | 2018-03-05 | 2020-10-02 | 上海诗丹德标准技术服务有限公司 | Preparation method of cyasterone |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1050596C (en) * | 1995-10-07 | 2000-03-22 | 中国科学院广州化学研究所 | Triterpenoid of iso-ailanthic acids and extracting process thereof |
| CN1067052C (en) * | 1998-07-16 | 2001-06-13 | 中国科学院广州化学研究所 | Isocedrela acid methyl ester and method for preparation |
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2006
- 2006-06-12 CN CNB2006100358943A patent/CN100402483C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1050596C (en) * | 1995-10-07 | 2000-03-22 | 中国科学院广州化学研究所 | Triterpenoid of iso-ailanthic acids and extracting process thereof |
| CN1067052C (en) * | 1998-07-16 | 2001-06-13 | 中国科学院广州化学研究所 | Isocedrela acid methyl ester and method for preparation |
Non-Patent Citations (2)
| Title |
|---|
| 南海海绵Rhadbastrella globostellata化学成分的研究。. 邓松之等.中国海洋药物，第4期. 1999 * |
| 异臭椿酸（isomalabaricane)三萜研究进展。. 田春雷等.广州化学，第2期. 1997 * |
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