CN103086879A - 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 PDF

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CN103086879A
CN103086879A CN2013100155756A CN201310015575A CN103086879A CN 103086879 A CN103086879 A CN 103086879A CN 2013100155756 A CN2013100155756 A CN 2013100155756A CN 201310015575 A CN201310015575 A CN 201310015575A CN 103086879 A CN103086879 A CN 103086879A
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extracting
extraction
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medicinal extract
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CN103086879B (en
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高坤
陈佳
陈艳君
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Ningbo Institute of Technology of ZJU
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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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

Separate from Wedelia trilobata and prepare the method for mapping kaurane-16-alkene-19-acid
Technical field
The present invention relates to active components in medicinal plant extractive technique field, particularly prepare the method for mapping kaurane-16-alkene-19-acid a kind of the separation from Wedelia trilobata.
Background technology
Mapping kaurane-16-alkene-19-acid (ent-KA) is from the traditional antifertility herbal medicine Montanoa tomentosa(Zoapatle of Mexico the earliest) separate and obtain, referring to document: Journal of ethnopharmacology (1986), 18 (1), 89-94.Bibliographical information was once arranged, 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 all seldom.So extract with higher extraction yield the focus that this compound becomes research 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, other weeds are seldom arranged in group, has extremely strong invasion, form harm at many tropical and subtropical regions, be listed in one of " the most harmful Invasive Alien Species in the world ".Natural disposition is strong, and greening can suppress weed growth rapidly, the sheet of often growing up growth.Breeding is easy, and winning the cauline leaf cuttage can survive.Once the chemical composition of South America Herbia Wedeliae was studied both at home and abroad, found that kaurane-16-alkene-19-acid was its main active ingredient in mapping.But the product purity that conventional extracting method obtains is lower, yield is lower, troublesome poeration, the production technique cycle is long and poor repeatability, is unfavorable for scale operation, can not meet the need of market.
Supercritical fluid extraction is to use higher than the fluid of critical temperature, the emergent pressure extraction process as solvent.The fluid that is 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, efficient is high, the advanced technologies of less energy consumption.Supercritical fluid extraction have aspect herbal medicine and natural product extraction separation low temperature, fast, the characteristics 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, efficient is high, prepare the method for mapping kaurane-16-alkene-19-acid separating from Wedelia trilobata of less energy consumption, the product yield of acquisition is high, purity is high.
The technical solution adopted for the present invention to solve the technical problems is: prepare the method for mapping kaurane-16-alkene-19-acid a kind of the separation 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 ℃, 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, the 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 namely gets mapping kaurane-16-alkene-19-acid.
The present invention is directed to the Wedelia trilobata self character, the technique that exploitation is fit to, adopt the critical abstraction technique of carbonic acid gas to extract the effective constituent mapping kaurane of Wedelia trilobata-16-alkene-19-acid, that the solvent for use carbonic acid gas has is nontoxic, tasteless, do not fire, not the burn into low price, the advantage such as be easy to make with extra care.Compare with the conventional solvent extraction method, step is relatively few, and flow process is short, and operating parameters also is 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.By using 60-95 wt% ethanol as entrainment agent, selectivity and efficient that target compound is extracted have been increased in leaching process.Adopt the decolouring of D101 type macroporous adsorbent resin to process the technology of purifying with existing silica gel column chromatography and compare, have a processing step relatively few, the advantage such as flow process is short, and the filler cost is low, and solvent load is few.
When controlling wash-out, aqueous ethanolic solution concentration is 50-60wt%, and yield is high, and it is minimum disturbed by impurity component for the target product under this concentration elutriant (being mapping kaurane-16-alkene-19-acid).
As preferably, the flow velocity of described entrainment agent is 10-12ml/min, and the entrainment agent add-on is 1000-1200ml.Control flow and the add-on of entrainment agent, improved selectivity and efficient 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 also is 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, pass through to use 60-95 wt% ethanol as entrainment agent in leaching process, increased selectivity and efficient that target compound is extracted.
3, adopt the decolouring of D101 type macroporous adsorbent resin to process the technology of purifying with existing silica gel column chromatography and compare, have a processing step relatively few, the advantage such as flow process is short, and the 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, the raw material that adopts and equipment etc. all can be buied from market or this area is commonly used.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 supercritical extraction instrument (the supercritical extraction instrument: American I SCO company of packing into, Model 260D, Syringe pump) in extraction kettle, entrainment agent is selected 60wt% ethanol, the 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 ℃, CO 2Flow is 40L/h, extraction time 3h; Then concentrating under reduced pressure is removed ethanol and is obtained approximately 40g of total medicinal extract, total medicinal extract is dissolved in 100ml water, with 100ml ethyl acetate extraction three times, merge organic phase, the organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained approximately 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, have a large amount of white crystals (coarse-grain) to separate out in this concentrated solution, coarse-grain sherwood oil 20ml recrystallization, namely get 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 supercritical extraction instrument (the supercritical extraction instrument: American I SCO company of packing into, Model 260D, Syringe pump) in extraction kettle, entrainment agent is selected 80 wt% ethanol, the 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 ℃, CO 2Flow is 35L/h, extraction time 2.5h; Then concentrating under reduced pressure is removed ethanol and is obtained approximately 45g of total medicinal extract, total medicinal extract is dissolved in 150ml water, with 150ml ethyl acetate extraction three times, merge organic phase, the organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained approximately 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, have a large amount of white crystals (coarse-grain) to separate out in this concentrated solution, coarse-grain sherwood oil 20ml recrystallization, namely get 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 supercritical extraction instrument (the supercritical extraction instrument: American I SCO company of packing into, Model 260D, Syringe pump) in extraction kettle, entrainment agent is selected 95 wt% ethanol, the 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 ℃, CO 2Flow is 30L/h, extraction time 2h; Then concentrating under reduced pressure is removed ethanol and is obtained approximately 50g of total medicinal extract, total medicinal extract is dissolved in 200ml water, with 200ml ethyl acetate extraction three times, merge organic phase, the organic phase concentrating under reduced pressure is removed ethyl acetate and is obtained approximately 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, have a large amount of white crystals (coarse-grain) to separate out in this concentrated solution, coarse-grain sherwood oil 20ml recrystallization, namely get mapping kaurane-16-alkene-19-acid, yield 2%, purity 99.8%.
[mapping kaurane-16-alkene-19-sour (ent-KA)] carries out Structural Identification to above-mentioned product:
Figure BDA0000274074441
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 that occurs in C-NMR and DEPT spectrum C(184.7 C, C-19), δ C(155.9 C, C-16), 103.0 (CH 2, C-17) illustrate that this compound exists a carboxylic carbonyl and one two structural unit that replaces two keys.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 low 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 (the 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 13The C-NMR data see Table 1).
Figure BDA0000274074442
By the product that method of the present invention obtains, the yield that obtains than traditional method high 10 times (traditional extraction yield is 2 ‰, and the method in the present invention is 2% left and right); 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 a kind of better scheme of the present invention, is not that the present invention is done any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim puts down in writing.

Claims (3)

1. one kind is separated from Wedelia trilobata and prepares the method for mapping kaurane-16-alkene-19-acid, 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 60-95 wt% ethanol, and controlling extracting pressure is 20-25 MPa, extraction temperature is 40-60 ℃, 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, the 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 namely gets mapping kaurane-16-alkene-19-acid.
2. according to claim 1, it is characterized in that: the flow velocity of described entrainment agent is 10-12ml/min, and the entrainment agent add-on is 1000-1200ml.
3. method according to claim 1 and 2, it is characterized in that: the mass ratio of medicinal extract and resin is 1:5-10.
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Cited By (1)

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CN107522680A (en) * 2017-09-07 2017-12-29 海南师范大学 South America amphibious crab chrysanthemum antineoplastic extract and its production and use

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN107522680A (en) * 2017-09-07 2017-12-29 海南师范大学 South America amphibious crab chrysanthemum antineoplastic extract and its production and use
CN107522680B (en) * 2017-09-07 2021-01-08 海南师范大学 Anti-tumor extract of south American wedelia chinensis and preparation method and application thereof

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