CN105641078B - From the middle method and its application for extracting active component pointed at both ends - Google Patents

From the middle method and its application for extracting active component pointed at both ends Download PDF

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CN105641078B
CN105641078B CN201410707851.XA CN201410707851A CN105641078B CN 105641078 B CN105641078 B CN 105641078B CN 201410707851 A CN201410707851 A CN 201410707851A CN 105641078 B CN105641078 B CN 105641078B
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active component
pointed
hexane
ethyl alcohol
extracting active
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CN105641078A (en
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张耀洲
唐楚沉
马琳
顾朋嫒
李聪聪
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TIANJIN YAOYU BIOLOGICAL TECHNOLOGY Co Ltd
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TIANJIN YAOYU BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The present invention provides a kind of from the middle method for extracting active component pointed at both ends: will pulverize after drying and dewatering pointed at both ends, ethyl acetate is added to extract, filter to take supernatant, methanol-water solution is added after reduced pressure and carries out ultrasonic dissolution, then it is separated in Silica positive preparation chromatography, using A n-hexane, B ethyl alcohol binary mobile phase, separation condition is 0~7min100%A isocratic elution, 7~27min90%A isocratic elution, 27~40min5%A isocratic elution, 26-28min eluent is collected according to ultra-violet absorption spectrum, it is concentrated under reduced pressure and dries, obtain the object of the invention active component.The separation method is stablized, and repeatability is high, and preparation amount is big, easy to operate time saving, is suitble to industrialized production, and obtained active component can be used for preparing in anti-lung-cancer medicament or anti-tumor health care product.

Description

From the middle method and its application for extracting active component pointed at both ends
Technical field
The present invention relates to technical field of extraction of Chinese traditional medicine, and in particular to a method of from middle extraction active component pointed at both ends, And the obtained active component is preparing the application in anti-lung-cancer medicament.
Background technique
Pointed at both ends also known as Radde Anemone Rhizome is Ranunculaceae Anemone anemone raddeana Regel (AnemoneraddeanaRege1) Dry rhizome.The ground such as three provinces in the northeast of China and Shandong, Hebei, Shanxi are distributed mainly on, it is most with Jilin Province's yield.Medicinal material more summers adopt It digs, removes residual stem and fibrous root, dry.This medicine " Bencao Pinhui Jingyao " written by the Ming Dynasty Liu Wentai.With " dispelling wind and eliminating dampness, The effect of subduing inflammation ".Early in the Qing Dynasty, " determining cancer to dissipate " of Wang Xugao is once with treatment breast cancer pointed at both ends.It is wet to be clinically used for chill now Numbness, brothers' muscular constricture, arthralgia, ulcer sore pain.Mostly with other medicines compatibility, folk prescription is not used as medicine, and is that famous Chinese patent drug is " active The primary raw material of pellet " and " Re-creating Bolus ".According to the literature, a large amount of saponin component is contained in pointed at both ends, also containing lactone, The ingredients such as volatile oil, grease type, alkaloid, amino acid, microelement and carbohydrate.Research shows that pointed at both ends also have anti-swell Tumor, anti-inflammatory, analgesia, calm and anticonvulsant action.
Authorization Notice No. is that the patent of invention of CN1210289C discloses a kind of preparation process of Radde anemone rhizome extract, the work Skill mainly uses ethyl alcohol extraction+silica gel dry chromatography method to be separated, and obtains colorless powder and crystallizes Raddeanin A pointed at both ends, The defect of this method is complex steps, time-consuming, poor repeatability.Authorization Notice No. is that the patent of invention of CN1312170C discloses one The preparation process of kind of Radde anemone rhizome extract, it mainly takes by using alkali carries+and the method extracted of ethyl alcohol extracts to improve purpose The yield and purity of anemonin A in object.Authorization Notice No. is that the patent of invention of CN102584927B discloses one kind from both ends The method of separation and Extraction one kind triterpene compound in point, this method elder generation ethyl alcohol extract, and then successively use petroleum ether extraction, acetic acid Ethyl ester extraction, extracting n-butyl alcohol, then chromatographed through column, efficient liquid phase preparation obtains purpose product.The extracting method complex steps, Time-consuming, stability is poor, is not suitable for industrialized production.
Summary of the invention
The technical problem to be solved by the present invention is to be directed to the above the deficiencies in the prior art, a kind of Radde anemone rhizome extract is provided Preparation method, after this method is to sample preliminary treatment, mainly isolated and purified using high performance liquid chromatography, separation method is steady Fixed, repeatability is high, and preparation amount is big, easy to operate time saving, is suitble to industrialized production.
The technical scheme adopted by the invention is as follows:
A kind of preparation method of Radde anemone rhizome extract, method includes the following steps:
(1) with pointed at both ends for raw material, room temperature is dried in the shade after processing, then drying and dewatering at a temperature of 40~60 DEG C is surpassed Crushing of Ultrafine processing, obtains Ultramicro-powder;
(2) ethyl acetate is added by the solid-liquid ratio of 1g ︰ (4~12) mL in step (1) resulting Ultramicro-powder, extracts 1~6h, Every 0.5~1h stirring is primary, filters after the static 1~5h of room temperature, collects filtered solution, filter residue is taken to repeat 1~3 time, merges on gained Clear liquid is concentrated under reduced pressure into paste, obtains small molecular extract pointed at both ends;
(3) 5~10 times of n-hexane-ethyl alcohol binary mixed solvent is added by mass volume ratio (g/mL), it is molten to carry out ultrasound Solution, excessively organic filter membrane, obtains the sample solution of small molecular extract pointed at both ends after dissolution;
(4) sample solution that step (3) obtain is carried out in the preparation chromatography that filler is positive separation material Silica It isolates and purifies, using A is the binary-mobile phase system of ethyl alcohol, the condition isolated and purified are as follows: 0~7min100%A for n-hexane, B Isocratic elution, 7~27min90%A-10%B (match by percent by volume 90%A, 10%B, under similarly) is isocratic to be washed De-, 27~40min5%A-95%B isocratic elution collects 26-28min eluent (LTJ-AC-7 according to ultra-violet absorption spectrum Peak), it is concentrated under reduced pressure and dries, obtain the object of the invention active component.
It is carried out at a temperature of -20~4 DEG C preferably, being pulverized in the step (1), the time is 5~20min.
Preferably, the Extracting temperature of ethyl acetate is 40~65 DEG C, further preferably 60 DEG C in the step (2).
Preferably, n-hexane-n-hexane of ethyl alcohol binary mixed solvent and the volume ratio of ethyl alcohol are in the step (3) 4 ︰ 1.
Preferably, the aperture of organic filter membrane is 0.22~0.45 μm in the step (3)
Preferably, in the step (4) diameter of reverse phase separation chromatographic column be 150mm, column length 250mm, positive point Partial size from material Silica is 8~10 μm.
Preferably, Detection wavelength when isolating and purifying in the step (4) is 210nm, sample volume 5g, flow velocity is 600mL/min。
It is carried out preferably, being concentrated under reduced pressure in the step (2), (4) using Rotary Evaporators, process conditions are temperature 65 DEG C, vacuum degree 0.08Mpa.
The active component pointed at both ends obtained the present invention further provides said extracted is preparing anti-lung-cancer medicament or anti-lung cancer Application in health food.The active component pointed at both ends is as effective component, according to a conventional method such as oral, injection, and pharmaceutically Acceptable carriers or excipient, the dosage form for being suitble to the administration modes such as oral, injection is made, such as: tablet, capsule, injection.
According to the understanding of those of ordinary skill in the art, the dosage of the active component pointed at both ends should be according to treatment object The factors such as age, weight, disease severity depending on, generally can be 15-50mg/ days.
Compared with prior art, the present invention has the following advantages that and remarkable result:
1, first Ultramicro-powder pointed at both ends is extracted using ethyl acetate, small polar substances therein is extracted, are reached Initial gross separation effect is conducive to the extraction of purpose component, and the recyclable recycling of ethyl acetate, has both saved extraction cost, Low toxicity is pollution-free again.
2, reuse the separation that high performance liquid chromatography carries out purpose active component: ethyl acetate extract polarity is smaller, warp Overtesting is groped, and Zheng Ji Wan ︰ ethyl alcohol=4 ︰ 1 mixed solvent is fine to its dissolubility;Silica chromatographic column for nonpolarity and Middle polar substances have preferable separating effect, 8 components can be got in 40min, and separating degree also compares between each component It is ideal;And under elution requirement of the invention, it is ensured that sample volume and flow velocity all have well when changing in normal ranges Repeatability, the component batch-to-batch consistency for obtaining the method for the present invention is high, and stability is good, can operational degree it is strong.
3, the small molecular extract containing active component is isolated and purified using preparation chromatography, relative to traditional molten Agent extraction, the preparation method stability is good, and reproducible, preparation amount is big, and reagent consumption is small, obtained material composition batch Between consistency it is high, the time cycle for isolating and purifying process is short.
4, this method passes through ultramicro grinding, solvent extraction, impurity screening, prepares the series of steps such as chromatographic separation and purification Afterwards, the isolated active component from ethyl acetate extract pointed at both ends, simple process is easy to implement, have preparation amount it is big, It is stably and controllable, be suitable for industrialization large-scale production the features such as, gained active component can be used for research and development prepare anti-lung-cancer medicament or Anti-lung cancer health food.
5, the present invention has expanded the raw material sources of anti-lung-cancer medicament or anti-lung cancer health food, expands application pointed at both ends Range makes the active principle raw material pointed at both ends for becoming anti-lung-cancer medicament or anti-lung cancer health food, significantly improves pointed at both ends Added value.
Detailed description of the invention
It is shown in FIG. 1 be various embodiments of the present invention Radde anemone rhizome extract preparation method in chromatographic fractionation figure;
Shown in Fig. 2 is the real-time monitoring figure of the anti-lung cancer activity of each extraction components in the embodiment of the present invention 1.
Specific embodiment
The present invention is further described in detail with reference to embodiments.It is noted that detailed description below is all to illustrate Property, it is intended to further instruction is provided to the present invention.Unless otherwise indicated, all scientific and technical terms that the present invention uses With with the normally understood identical meanings of the technical field of the invention personnel.
1, raw material, material:
Picking pointed at both ends is from Changchun City and by identification;A549 cell is by blood disease hospital, the Chinese Academy of Medical Sciences It provides.
2, reagent:
Ultrapure water is produced by Mi Libo pure water meter;Analysis level ethyl alcohol is purchased from Tianjin Concord Technology Co., Ltd.;Analysis Level ethyl acetate (95% ethyl acetate) is purchased from Tianjin Concord Technology Co., Ltd.;Chromatographic grade n-hexane, ethyl alcohol are purchased from Honeywell company;DMSO is purchased from Beijing Suo Laibao Science and Technology Ltd;F-12K culture medium is purchased from Gibco company.
3, instrument and equipment:
Micronizer is purchased from Sanqing Stainless Steel Apparatus Co., Ltd., Shandong;Tabletop refrigerated centrifuge is public purchased from Thermo Department;Constant temperature blast drying oven is purchased from Shanghai Yiheng Scientific Instruments Co., Ltd;Rotary Evaporators Shanghai Ya Rong biochemical instrument factory;Just Phase Silica prepares chromatographic column and is purchased from Tianjin Bonaaijieer Technology Co., Ltd;Preparative high performance liquid chromatography instrument is purchased from Jiangsu Han Bang Science and Technology Ltd.;Cell real-time monitor is purchased from ACEA Biosciences Inc (Essen Biology scientific company);Carefully Born of the same parents' incubator is purchased from Britain RS Biotech company.
Embodiment 1:
Method of the present embodiment from middle extraction active component pointed at both ends, comprising the following steps:
(1) room temperature pointed at both ends is dried in the shade after processing, in 60 DEG C of drying and dewaterings, then in -20 DEG C of ultramicro grinding 5min to get two The sharp Ultramicro-powder of head.
(2) above-mentioned Ultramicro-powder 1Kg pointed at both ends is taken, 12.0L analysis ethyl acetate is added in 60 DEG C of extraction 6h, every 0.5h stirring Once.It is filtered after the static 5h of room temperature, collects filtered solution, filter residue is taken to be repeated 3 times, merged gained filtered solution and be concentrated under reduced pressure into paste simultaneously It is dried in 65 DEG C of baking oven, that is, obtains ethyl acetate extract pointed at both ends.
(3) chromatographic grade n-hexane-chromatographic grade ethyl alcohol binary mixed solvent that 10 times of volumes are added is resulting small by step (2) The ratio of molecule extractive progress ultrasonic dissolution, n-hexane and ethyl alcohol is 4:1.0.22 μm of organic filter membrane is crossed after dissolution to get arriving The sample solution of ethyl acetate extract pointed at both ends.
(4) by the sample solution of the ethyl acetate extract pointed at both ends of acquisition using filler as positive separation material Silica It is isolated and purified in the preparation chromatography of (8 μm of partial size), the separation chromatography column diameter used is adopted for 150mm, column length 250mm It is n-hexane with A, B is that ethyl alcohol binary-mobile phase system is isolated and purified;The condition isolated and purified are as follows: 0~7min, 100% A, 0%B;7~27min, 90%A, 10%B, 27~40min, 5%A, 95%B elute sample according to gradient;Detection Wavelength: 210nm;Sample volume: 5g;Flow velocity: 600mL/min.It is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (a), receives Collect at 26-28min eluent (peak LTJ-AC-7), is concentrated under reduced pressure and dries, obtain the object of the invention active component.
Obtained component LTJ-AC-7 is further isolated and purified and the hydrocarbon spectrum analysis of NMR by preparative liquid chromatography, is determined Mainly contain saponins and other substances.
Embodiment 2:
Method of the present embodiment from middle extraction active component pointed at both ends, comprising the following steps:
(1) room temperature pointed at both ends is dried in the shade after processing, in 40 DEG C of drying and dewaterings, then in -10 DEG C of ultramicro grinding 5min to get two The sharp Ultramicro-powder of head.
(2) above-mentioned Ultramicro-powder 1Kg pointed at both ends is taken, 8.0L analysis ethyl acetate is added in 50 DEG C of extraction 4h, every 1h stirring one It is secondary, filtered after the static 3h of room temperature, collect filtered solution, take filter residue to be repeated 3 times, merge gained filtered solution be concentrated under reduced pressure into paste and in 65 DEG C of baking oven drying process, that is, obtain ethyl acetate extract pointed at both ends.
(3) chromatographic grade n-hexane-chromatographic grade ethyl alcohol binary mixed solvent that 8 times of volumes are added is resulting small by step (2) The ratio of molecule extractive progress ultrasonic dissolution, n-hexane and ethyl alcohol is 4:1.0.22 μm of organic filter membrane is crossed after dissolution to get arriving The sample solution of ethyl acetate extract pointed at both ends.
(4) by the sample solution of the ethyl acetate extract pointed at both ends of acquisition using filler as positive separation material Silica It is isolated and purified in the preparation chromatography of (8 μm of partial size), the separation chromatography column diameter used is adopted for 150mm, column length 250mm It is n-hexane with A, B is that ethyl alcohol binary-mobile phase system is isolated and purified;The condition isolated and purified are as follows: 0~7min, 100% A, 0%B;7~27min, 90%A, 10%B;27~40min, 5%A, 95%B elute sample according to gradient;Detection Wavelength: 210nm;Sample volume: 5g;Flow velocity: 600mL/min.It is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (b), receives Collect at 26-28min eluent (peak LTJ-AC-7), is concentrated under reduced pressure and dries, obtain the object of the invention active component.
Obtained component LTJ-AC-7 is further isolated and purified and the hydrocarbon spectrum analysis of NMR by preparative liquid chromatography, is determined Mainly contain saponins and other substances.
Embodiment 3:
Method of the present embodiment from middle extraction active component pointed at both ends, comprising the following steps:
(1) room temperature pointed at both ends is dried in the shade after processing, in 50 DEG C of drying and dewaterings, then -15 DEG C of ultramicro grinding 10min to get Rhizome Ultramicro-powder pointed at both ends.
(2) above-mentioned Ultramicro-powder 1Kg pointed at both ends is taken, 5L analysis ethyl alcohol is added in 40 DEG C of extraction 2h, every 0.5h stirring is primary, room It is filtered after the static 4h of temperature, collects filtered solution, filter residue is taken to be repeated 3 times, merged gained filtered solution and be concentrated under reduced pressure into paste and in baking oven 65 DEG C of drying process, that is, obtain ethyl acetate extract pointed at both ends.
(3) chromatographic grade n-hexane-chromatographic grade ethyl alcohol binary mixed solvent that 5 times of volumes are added is resulting small by step (2) The ratio of molecule extractive progress ultrasonic dissolution, n-hexane and ethyl alcohol is 4:1.0.22 μm of organic filter membrane is crossed after dissolution to get arriving The sample solution of ethyl acetate extract pointed at both ends.
(4) by the sample solution of the ethyl acetate extract pointed at both ends of acquisition using filler as positive separation material Silica It is isolated and purified in the preparation chromatography of (8 μm of partial size), the separation chromatography column diameter used is adopted for 150mm, column length 250mm It is n-hexane with A, B is that ethyl alcohol binary-mobile phase system is isolated and purified;The condition isolated and purified are as follows: 0~7min, 100% A, 0%B;7~27min, 90%A, 10%B;27~40min, 5%A, 95%B elute sample according to gradient;Detection Wavelength: 210nm;Sample volume: 5g;Flow velocity: 600mL/min.It is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (c), receives Collect at 26-28min eluent (peak LTJ-AC-7), is concentrated under reduced pressure and dries, obtain the object of the invention active component.
Obtained component LTJ-AC-7 is further isolated and purified and the hydrocarbon spectrum analysis of NMR by preparative liquid chromatography, is determined Mainly contain saponins and other substances.
(a), (b), (c) in comparison diagram 1, it can be deduced that, the present invention is repeated from the middle method for extracting active component pointed at both ends Property it is good, batch-to-batch consistency is high, can operational degree it is strong, be suitble to industrialization promotion production.
Embodiment 4: anti-lung cancer activity experiment
(1) in 5%CO2, 37 DEG C, under saturated humidity, with F-12K (10%FBS+1%PS) culture medium culture human lung cancer A549 cell chooses the cell of logarithmic phase growth as experimental cell.It is diluted to after cell count with culture medium certain density Cell suspension.
(2) cell real-time monitor is put into 5%CO2, in 37 DEG C of saturated humidity incubators.8 orifice plates are taken, every hole is added 150 μ LF-12K culture mediums, are put into cell real-time monitor and walk baseline, take out octal plate after covering baseline, dilution is added in every hole Whether equal good 345 μ L of A549 cell suspension observes cell until every hole cell number about 20,000, stands 3min under inverted microscope It is even.The sample that 5 μ L have diluted is added in every hole, and final concentration is set as 50 μ g/ml.Use the culture medium containing 0.1%DMSO as blank Control group is put into cell real-time monitor and detects, and obtains being allocated as under using in each test group of ethyl acetate extract pointed at both ends The real-time vegetative map of lung cancer A549 cell, test result such as Fig. 2.Experimental result illustrates the group in ethyl acetate extract pointed at both ends Divide LTJ-AC-7 that there is good inhibiting effect to the proliferation of lung cancer A549 cell when concentration is 50 μ g/ml.Therefore, of the invention Purpose active component LTJ-AC-7 has exploitation at the potentiality of medicines resistant to liver cancer.
Ethyl acetate extract pointed at both ends respectively tests component: LTJ-AC-1, LTJ-AC-2, LTJ-AC-3, LTJ-AC-4, LTJ-AC-5, LTJ-AC-6, LTJ-AC-7, LTJ-AC-8 are the 5- collected in embodiment 1 according to Fig. 1 (a) chromatographic isolation respectively 6min, 6-8min, 8-9min, 14-15min, 16-18min, 20-22min, 26-28min, 34-36min eluent.
The present invention carries out the monitoring of resisting liver cancer activity experiment using the real-time n cell analytical technology of RTCA.RTCA is real-time N cell analytical technology be it is a kind of established based on novel cytoanalyze have real-time monitoring, high information quantity, nothing Need the detection technique of the particular advantages such as label, full-automatic, highly sensitive and high accuracy.The cytoanalyze is by being embedded in E- On plate plate the microelectronics inductor impedance variations of bottom hole go the presence or absence of permissive cell and it is adherent, stick and extent of growth Change.It, can the cell biologicals such as real-time, intuitive reacting cells proliferation, survival, apoptosis, metamorphosis in cytotoxicity detection Learn variation.The technology makes cell experiment become more time-saving and efficiency.
The present invention carries out external anti-lung cancer activity detection using the real-time n cell analytical technology of RTCA, has prison in real time Survey, without label, full-automatic, highly sensitive and high accuracy the advantages of, can in real time, it is so intuitive that show lung carcinoma cell increasing It grows, survive, the cell biologies variation such as apoptosis, so that the cell experiment becomes more time-saving and efficiency.
The present embodiments relate to the material arrived, reagent and experimental facilities, are to meet traditional Chinese medicine extraction unless otherwise instructed Commercial product.
The above is merely a preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise of not departing from core of the invention technology, improvements and modifications can also be made, these improvements and modifications are also answered Belong to scope of patent protection of the invention.With any change in the comparable meaning and scope of claims of the present invention, all It is considered as being included within the scope of the claims.

Claims (8)

1. a kind of from the middle method for extracting active component pointed at both ends, it is characterised in that the following steps are included:
(1) with pointed at both ends for raw material, room temperature is dried in the shade after processing, then drying and dewatering at a temperature of 40~60 DEG C carries out Ultramicro-powder Broken processing obtains Ultramicro-powder;
(2) ethyl acetate is added by the solid-liquid ratio of 1g ︰ (4~12) mL in step (1) resulting Ultramicro-powder, extracts 1~6h, extracts Temperature is 40~65 DEG C, and every 0.5~1h stirring is primary, filters after the static 1~5h of room temperature, collects filtered solution, take filter residue repeat 1~ 3 times, merges gained supernatant and be concentrated under reduced pressure into paste, obtain small molecular extract pointed at both ends;
(3) 5~10 times of n-hexane-ethyl alcohol binary mixed solvent is added by mass volume ratio, carries out ultrasonic dissolution, mistake after dissolution Organic filter membrane obtains the sample solution of small molecular extract pointed at both ends;
(4) sample solution that step (3) obtain is separated in the preparation chromatography that filler is positive separation material Silica Purifying, using A is the binary-mobile phase system of ethyl alcohol, the condition isolated and purified are as follows: 0~7min100%A is isocratic for n-hexane, B Elution, 7~27min90%A-10%B isocratic elution, 27~40min5%A-95%B isocratic elution, according to ultra-violet absorption spectrum 26-28min eluent is collected, is concentrated under reduced pressure and dries, obtain the object of the invention active component.
2. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (1) Middle ultramicro grinding carries out at a temperature of -20~4 DEG C, and the time is 5~20min.
3. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (3) The volume ratio of middle n-hexane-ethyl alcohol binary mixed solvent n-hexane and ethyl alcohol is 4 ︰ 1.
4. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (3) In organic filter membrane aperture be 0.22~0.45 μm.
5. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (4) The diameter of middle reverse phase separation chromatographic column is 150mm, column length 250mm, and the partial size of positive separation material Silica is 8~10 μm.
6. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (4) In Detection wavelength when isolating and purifying be 210nm, sample volume 5g, flow velocity 600mL/min.
7. according to claim 1 from the middle method for extracting active component pointed at both ends, it is characterised in that: the step (2), (4) it is concentrated under reduced pressure in and is carried out using Rotary Evaporators, process conditions are 65 DEG C of temperature, vacuum degree 0.08Mpa.
8. the method described in claim 1 from middle extraction active component pointed at both ends obtains active component pointed at both ends and is preparing anti-lung Application in cancer drug or anti-lung cancer health food.
CN201410707851.XA 2014-11-28 2014-11-28 From the middle method and its application for extracting active component pointed at both ends Active CN105641078B (en)

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