CN103086879B - Method for preparing and separating enkaurane-16-alkene-19-acid from wedelia trilobata - Google Patents
Method for preparing and separating enkaurane-16-alkene-19-acid from wedelia trilobata Download PDFInfo
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- CN103086879B CN103086879B CN201310015575.6A CN201310015575A CN103086879B CN 103086879 B CN103086879 B CN 103086879B CN 201310015575 A CN201310015575 A CN 201310015575A CN 103086879 B CN103086879 B CN 103086879B
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Abstract
The invention relates to the technical field of extracting of plant active ingredients and discloses a method for preparing and separating enkaurane-16-alkene-19-acid from wedelia trilobata. The method comprises the following steps of: drying, crushing and sieving the wedelia trilobata; putting the wedelia trilobata powder to an extraction kettle of a supercritical extractor for extracting, wherein an entrainer is alcohol, the extracting pressure is 20MPa-25MPa, the extracting temperature is 40 DEG C to 60 DEG C, the CO2 flow rate is 30L/h to 40L/h, and the extracting time is two hours to three hours; concentrating the extract under reduced pressure to obtain total extract, dissolving the total extract in water, extracting the solution for three times by using ethyl acetate and concentrating the organic phase under reduced pressure to remove ethyl acetate to obtain the extract; loading the extract to D101 macroreticular resin, eluting using alcohol aqueous solution, collecting the eluate, and concentrating under reduced pressure to obtain a crude crystal; and re-crystallizing the crude crystal by petroleum ether to obtain the product. The method disclosed by the invention is high in extracting speed, high in efficiency, less in energy consumption, and high in yield and purity of the obtained product.
Description
Technical field
The present invention relates to active components in medicinal plant extractive technique field, particularly a kind of method of preparing mapping kaurane-16-alkene-19-acid that separates from Wedelia trilobata.
Background technology
Mapping kaurane-16-alkene-19-acid (ent-KA) is the antifertility herbal medicine Montanoa tomentosa(Zoapatle traditional from Mexico the earliest) separate and obtain, referring to document: Journal of ethnopharmacology (1986), 18 (1), 89-94.Once there is bibliographical information, Verbindung nt-KA has very strong toxicity to human body nasopharyngeal carcinoma cell (KB) and stomach cancer cell (BGC-823), referring to document: (1) Phytotherapy Research (2002), 16,387 – 388 and (2) Heilungkiang medicine (2006), 19,26-27.This compound and its derivative have very large antitumor researching value and application prospect.Now had document to disclose and also separablely from some other plant obtained this material, but content is all little.Become the focus of research so extract this compound with higher extraction yield from the strong plant of growth and breeding ability.
Wedelia trilobata is that South America Herbia Wedeliae (Wedelia trilobata) is composite family (Compositae) amphibious crab Chrysanthemum (Wedelia) plant, perennial herb, originate in tropical America, in group, seldom there are other weeds, there is extremely strong invasion, form harm in many tropical and subtropical region, be listed in one of " the most harmful Invasive Alien Species in the world ".Natural disposition is strong, and greening rapidly, can suppress weed growth, the sheet of often growing up growth.Easily, winning cauline leaf cuttage can survive in breeding.Once the chemical composition of South America Herbia Wedeliae was studied both at home and abroad, found that mapping kaurane-16-alkene-19-acid was its main active ingredient.But the product purity that conventional extracting method obtains is lower, yield is lower, troublesome poeration, production technique cycle are long and poor repeatability, is unfavorable for scale operation, can not meet the need of market.
Supercritical fluid extraction is to use the extraction process as solvent higher than the fluid of critical temperature, emergent pressure.Fluid in Near The Critical Point not only has high dissolving power to material, and the solubleness of material changes with the pressure of system or the variation of temperature, thereby just can carry out easily optionally extracting and separating material by pressure or the temperature of regulation system.Supercritical extraction is regarded as environmental friendliness and energy-efficient new chemical separation technology earns widespread respect and develops in a lot of fields.Be better than the extraction of general liquid-liquid and rectifying at supercritical extraction aspect separated from solvent and recovery, be considered to that rate of extraction is fast, efficiency is high, the advanced technologies of less energy consumption.Supercritical fluid extraction aspect herbal medicine and natural product extraction separation, have low temperature, fast, the feature such as high, the product pure natural of yield.This technology has important application prospect to realizing the modernization of Chinese medicine.
Summary of the invention
The object of the present invention is to provide that a kind of rate of extraction is fast, efficiency is high, less energy consumption from Wedelia trilobata, separate the method for preparing mapping kaurane-16-alkene-19-acid, the product yield of acquisition is high, purity is high.
The technical solution adopted for the present invention to solve the technical problems is: a kind of method of preparing mapping kaurane-16-alkene-19-acid that separates from Wedelia trilobata, Wedelia trilobata is dry, pulverize, sieve, take Wedelia trilobata powder 200-300g and pack in the extraction kettle of supercritical extraction instrument, entrainment agent is selected 60-95 wt% ethanol, and controlling extracting pressure is 20-25 MPa, extraction temperature is 40-60 DEG C, CO
2flow is 30-40L/h, extraction time 2-3h; Then concentrating under reduced pressure is removed ethanol and is obtained total medicinal extract (40-50g), total medicinal extract is dissolved in 100-200ml water, with 100-200ml ethyl acetate extraction three times, merge organic phase, organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained medicinal extract (20-30g), D101 type macroporous adsorbent resin on medicinal extract, with 50-60wt% aqueous ethanolic solution wash-out, collect elutriant, concentrating under reduced pressure obtains coarse-grain, coarse-grain sherwood oil recrystallization, obtains mapping kaurane-16-alkene-19-acid.
The present invention is directed to Wedelia trilobata self character, develop applicable technique, the effective constituent mapping kaurane-16-alkene-19-acid that adopts carbonic acid gas critical abstraction technique to extract Wedelia trilobata, that solvent for use carbonic acid gas has is nontoxic, tasteless, do not fire, not burn into low price, the advantage such as be easy to refine.Compared with conventional solvent extraction method, step is relatively few, and flow process is short, and operating parameters is also easy to control, and is constant product quality and residual without hazardous solvent; Not only extraction efficiency is high, energy consumption is low, and extract impurity reduces, effective constituent is highly enriched.In leaching process, by using 60-95 wt% ethanol as entrainment agent, selectivity and efficiency that target compound is extracted are increased.The advantages such as the decolouring of employing D101 type macroporous adsorbent resin is processed compared with the technology of purifying with existing silica gel column chromatography, has processing step relatively few, and flow process is short, and filler cost is low, and solvent load is few.
While controlling wash-out, aqueous ethanolic solution concentration is 50-60wt%, and target product (the being mapping kaurane-16-alkene-19-acid) yield under this concentration elutriant is high, and it is minimum disturbed by impurity component.
As preferably, the flow velocity of described entrainment agent is 10-12ml/min, and entrainment agent add-on is 1000-1200ml.Control flow and the add-on of entrainment agent, improved selectivity and efficiency that target compound is extracted.
As preferably, the mass ratio of medicinal extract and resin is 1:5-10.Separation and purification is effective like this.
The invention has the beneficial effects as follows:
1, step is relatively few, and flow process is short, and operating parameters is also easy to control, and is constant product quality and residual without hazardous solvent; Not only extraction efficiency is high, energy consumption is low, and extract impurity reduces, effective constituent is highly enriched.
2, in leaching process, pass through to use 60-95 wt% ethanol as entrainment agent, increased selectivity and efficiency that target compound is extracted.
The advantages such as 3, the decolouring of employing D101 type macroporous adsorbent resin is processed compared with the technology of purifying with existing silica gel column chromatography, has processing step relatively few, and flow process is short, and filler cost is low, and solvent load is few.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, raw material and the equipment etc. adopting all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Embodiment 1:
Wedelia trilobata is dry, pulverize, cross 30 mesh sieves, take Wedelia trilobata powder 200g and pack supercritical extraction instrument (supercritical extraction instrument: American I SCO company into, Model 260D, Syringe pump) extraction kettle in, entrainment agent is selected 60wt% ethanol, entrainment agent add-on is 1200ml, and the flow velocity of entrainment agent is 12ml/min; Controlling extracting pressure is 25 MPa, and extraction temperature is 60 DEG C, CO
2flow is 40L/h, extraction time 3h; Then concentrating under reduced pressure is removed ethanol and is obtained the about 40g of total medicinal extract, total medicinal extract is dissolved in 100ml water, with 100ml ethyl acetate extraction three times, merge organic phase, organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained the about 20g of medicinal extract, 100g D101 type macroporous adsorbent resin on medicinal extract, with 10L 60wt% aqueous ethanolic solution wash-out, collect elutriant, be evaporated to 50ml, in this concentrated solution, have a large amount of white crystals (coarse-grain) to separate out, sherwood oil 20ml recrystallization for coarse-grain, obtain mapping kaurane-16-alkene-19-acid, yield 1.9%, purity 99.4%.
Embodiment 2:
Wedelia trilobata is dry, pulverize, cross 30 mesh sieves, take Wedelia trilobata powder 250g and pack supercritical extraction instrument (supercritical extraction instrument: American I SCO company into, Model 260D, Syringe pump) extraction kettle in, entrainment agent is selected 80 wt% ethanol, entrainment agent add-on is 1000ml, and the flow velocity of entrainment agent is 10ml/min; Controlling extracting pressure is 22 MPa, and extraction temperature is 50 DEG C, CO
2flow is 35L/h, extraction time 2.5h; Then concentrating under reduced pressure is removed ethanol and is obtained the about 45g of total medicinal extract, total medicinal extract is dissolved in 150ml water, with 150ml ethyl acetate extraction three times, merge organic phase, organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained the about 25g of medicinal extract, 200g D101 type macroporous adsorbent resin on medicinal extract, with 10L 55wt% aqueous ethanolic solution wash-out, collect elutriant, be evaporated to 50ml, in this concentrated solution, have a large amount of white crystals (coarse-grain) to separate out, sherwood oil 20ml recrystallization for coarse-grain, obtain mapping kaurane-16-alkene-19-acid, yield 1.8%, purity 99.2%.
Embodiment 3:
Wedelia trilobata is dry, pulverize, cross 30 mesh sieves, take Wedelia trilobata powder 300g and pack supercritical extraction instrument (supercritical extraction instrument: American I SCO company into, Model 260D, Syringe pump) extraction kettle in, entrainment agent is selected 95 wt% ethanol, entrainment agent add-on is 1200ml, and the flow velocity of entrainment agent is 10ml/min; Controlling extracting pressure is 20 MPa, and extraction temperature is 40 DEG C, CO
2flow is 30L/h, extraction time 2h; Then concentrating under reduced pressure is removed ethanol and is obtained the about 50g of total medicinal extract, total medicinal extract is dissolved in 200ml water, with 200ml ethyl acetate extraction three times, merge organic phase, organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained the about 30g of medicinal extract, 300g D101 type macroporous adsorbent resin on medicinal extract, with 10L 50wt% aqueous ethanolic solution wash-out, collect elutriant, be evaporated to 50ml, in this concentrated solution, have a large amount of white crystals (coarse-grain) to separate out, sherwood oil 20ml recrystallization for coarse-grain, obtain mapping kaurane-16-alkene-19-acid, yield 2%, purity 99.8%.
To above-mentioned product [mapping kaurane-16-alkene-19-acid (ent-KA)] carry out Structural Identification:
product is colorless prismatic crystal.Mass spectrum EI-MS provides its molecular ion peak [M]
+m/z=302, in conjunction with
13c-NMR and DEPT, the molecular formula that can determine this compound is C
20h
30o
2, degree of unsaturation 6.This compound
1320 skeleton carbon signals that C-NMR and DEPT spectrum show, wherein have CH
3× 2, CH
2× 9, CH × 3, C × 4.Further analyze it
13the fignal center δ occurring in C-NMR and DEPT spectrum
c184.7 (C, C-19), δ
c155.9 (C, C-16), 103.0 (CH
2, C-17) and illustrate that this compound exists the structural unit of a carboxylic carbonyl and one the two two keys of replacement.Remove above 2 degrees of unsaturation, also remaining 4 degrees of unsaturation, infer that this compound is a tetracyclic diterpene.In addition, can also observe two unimodal methyl [δ
h0.95 (s), 1.24 (s)], the wide unimodal methyne [δ of lower
h2.64 (brs)], infer that thus this compound is a Kaurane diterpine.Further By consulting literatures is found spectral data and mapping kaurane-16-alkene-19-acid (ent-kaur-16-en-19-oic acid of this compound; Ent-KA) spectral data is in full accord.So far, the structure of product determined (colorless prismatic crystal,
1h-NMR and
13c-NMR data are in table 1).
The product obtaining by method of the present invention, the yield high 10 times (traditional extraction yield is 2 ‰, and the method in the present invention is 2% left and right) obtaining than traditional method; Obtain purity high 14 percentage points (it is 85% that tradition is extracted compound purity, and the method in the present invention is 99.2-99.8%) than traditional method.
Above-described embodiment is preferably scheme of one of the present invention, not the present invention is done to any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim records.
Claims (1)
1. one kind separates the method for preparing mapping kaurane-16-alkene-19-acid from Wedelia trilobata, it is characterized in that: Wedelia trilobata is dry, pulverize, sieve, take Wedelia trilobata powder 200-300g and pack in the extraction kettle of supercritical extraction instrument, entrainment agent is selected 60-95wt% ethanol, and control extracting pressure is 20-25MPa, extraction temperature is 40-60 DEG C, CO
2flow is 30-40L/h, extraction time 2-3h; Then concentrating under reduced pressure is removed ethanol and is obtained total medicinal extract, total medicinal extract is dissolved in 100-200mL water, with 100-200mL ethyl acetate extraction three times, merge organic phase, organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained medicinal extract, D101 type macroporous adsorbent resin on medicinal extract, with 50-60wt% aqueous ethanolic solution wash-out, collect elutriant, concentrating under reduced pressure obtains coarse-grain, coarse-grain sherwood oil recrystallization, obtains mapping kaurane-16-alkene-19-acid; The flow velocity of described entrainment agent is 10-12mL/min, and entrainment agent add-on is 1000-1200mL; The mass ratio of medicinal extract and resin is 1:5-10.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4491593A (en) * | 1983-01-27 | 1985-01-01 | Gallegos Alfredo J | Compositions and methods for fertility control |
| CN1431187A (en) * | 2003-01-17 | 2003-07-23 | 南京中医药大学 | Kaurane type diterpene compound and its preparing method as well as application in anticancer drugs |
| BRPI1002115A2 (en) * | 2010-04-30 | 2012-05-22 | Farmaceutico Elofar Ltda Lab | biomarker isolation and purification process; process of obtaining a standardized extract with antinociceptive and antiinflammatory properties, topical pharmaceutical compositions, preparation process thereof |
-
2013
- 2013-01-16 CN CN201310015575.6A patent/CN103086879B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4491593A (en) * | 1983-01-27 | 1985-01-01 | Gallegos Alfredo J | Compositions and methods for fertility control |
| CN1431187A (en) * | 2003-01-17 | 2003-07-23 | 南京中医药大学 | Kaurane type diterpene compound and its preparing method as well as application in anticancer drugs |
| BRPI1002115A2 (en) * | 2010-04-30 | 2012-05-22 | Farmaceutico Elofar Ltda Lab | biomarker isolation and purification process; process of obtaining a standardized extract with antinociceptive and antiinflammatory properties, topical pharmaceutical compositions, preparation process thereof |
Non-Patent Citations (6)
| Title |
|---|
| Neelam Balekar 等.Wound healing activity of ent-kaura-9(11),16-dien-19-oic acid isolated from Wedelia trilobata (L.) leaves.《Phytomedicine》.2012,第19卷(第13期), * |
| Wound healing activity of ent-kaura-9(11),16-dien-19-oic acid isolated from Wedelia trilobata (L.) leaves;Neelam Balekar 等;《Phytomedicine》;20121015;第19卷(第13期);第1178-1184页 * |
| 二萜类化合物的提取与纯化工艺研究进展;韦玮 等;《中国实验方剂学杂志》;20071231;第13卷(第12期);第66-70页 * |
| 半边旗抗肿瘤活性成分5F 对照品的制备和鉴定;吕应年;《化学与生物工程》;20090525;第26卷(第5期);第72-74页 * |
| 吕应年.半边旗抗肿瘤活性成分5F 对照品的制备和鉴定.《化学与生物工程》.2009,第26卷(第5期), * |
| 韦玮 等.二萜类化合物的提取与纯化工艺研究进展.《中国实验方剂学杂志》.2007,第13卷(第12期), * |
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