CN108125993A - A kind of preparation of the tuckahoe extracts of energy reverse multiple drug resistance of tumor - Google Patents
A kind of preparation of the tuckahoe extracts of energy reverse multiple drug resistance of tumor Download PDFInfo
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Abstract
The invention discloses a kind of tuckahoe extracts, fuling triterpene content is 40% 90% in the tuckahoe extracts, is prepared via a method which to obtain：Poria cocos is crushed, ethanol in proper amount solution is added in, is extracted under heating condition, filter, obtain filtrate, merging filtrate, concentration add in a small amount of water, and ethyl acetate extraction is multiple, after being dried under reduced pressure, methanol dissolves, filtering, and filtrate decompression concentration, freeze-drying obtains solid-state tuckahoe extracts.Tuckahoe extracts prepared by the present invention are derived from traditional Chinese medicine, and experiment in vitro shows that the extract has antitumor activity, and can reverse the multidrug resistance of MCF/ADR.
Description
Technical field
The present invention relates to a kind of tuckahoe extracts with reverse multiple drug resistance of tumor, preparation method and application.
Background technology
Malignant tumour is that malignant tumour is to endanger one of principal disease of human health, and incidence and case fatality rate are still
The trend constantly risen is presented.Operation, chemotherapy and radiotherapy are current most common treatment means, and single chemotherapeutics is not
Needs can be met, drug combination has become the inexorable trend for the treatment of tumour.Many active materials are equal in discovered in recent years Chinese medicine
With antineoplastic action, clinically also there is certain effect.At present, the death of malignant tumour has 94% more with tumour
The generation of medicine drug resistance is related, and the method for seeking the reverse multiple drug resistance of tumor of high-efficiency low-toxicity has become research hotspot.
While multi-drug resistance of the tumor (MDR) refers to that tumour cell generates drug resistance to a kind of antitumor drug, to knot
The phenomenon that structure and other completely different antitumor drugs of mechanism of action generate cross resistance.The mechanism that MDR is generated is main
Have：1. the MDR of memebrane protein mediation, the classical MDR including the mediation of P-g glycoprotein is theoretical, multidrug resistance associated protein mediation
MDR, the MDR of breast drug-resistance protein mediation, the MDR of lung resistance-related protein mediation etc.；2. the MDR of enzyme mediation, including：Paddy Guang
MDR transferase mediated sweet peptide S, the MDR of topoisomerase II mediation, the MDR of protein kinase C mediation etc.；3. apoptosis regulation base
Because of the MDR of mediation, include the MDR of 2 gene mediateds of bcl, p53 genes etc.；MDR caused by 4. DNA is repaired extremely.Tumour it is more
Medicine drug resistance Producing reason is more complicated, and natural drug has the characteristics that less toxic, resourceful, action target spot is more.In recent years,
Many scholars are had found in Chinese medicine containing there are many drug resistant compositions of reverse multidrug.
Poria cocos is the dry sclerotia of porous section fungi Poria cocos Poria cocos (schw) Wolf, sweet in flavor, light, flat.Have
Clearing damp and promoting diuresis, invigorating the spleen, calming heart function, cure mainly oedema oliguria, spleen eating less, loose stool diarrhea has no peace of mind and waits diseases.It is main in Poria cocos
Ingredient is wanted as pachymaran and fuling triterpene, wherein pachymaran research is more, and fuling triterpene mainly has radicals scavenging work(
Can, antibacterial, it is anti-inflammatory and antitumor the effects that.It has been investigated that Poria cocos ethanol extract can be by activating mitochondria caspase
Access inhibits the proliferation of A549 cells, mainly by reducing mitochondrial membrane potential, and discharges cromoci to enter cytoplasm molten
Caspase precursors are cracked into caspase-9, so as to reach inhibiting effect by glue.And it has studied one in Poria cocos ethanol extract
Kind is soil not sour composition, and it is the main ingredient for generating cytotoxicity to find it.And point out that tuckahoe extracts can inhibit non-small
Cell lung cancer.And the composition pachymic acid in Poria cocos can trigger PANC-1 the and MIA PaCa-2 pancreases for having drug resistance to gemcitabine
The apoptosis of adenocarcinoma cell.Find that pachymic acid can activate heat shock response to react dependency basis with expansion albumen by gene comparative analysis
Cause is so as to cause endoplasmic reticulum stress response, i.e., by increasing XBP-1s, ATF4, Hsp70, CHOP and phospho-eIF2 α
Expression.Meanwhile experiment in vivo shows that the PA of 25mg/kg can be by causing the Apoptosis at tumor tissues position should with endoplasmic reticulum
The expression of GAP-associated protein GAP is swashed so as to significantly inhibit the growth of MIA PaCa-2 tumours and not have toxicity.
The triterpene composition contained in Poria cocos is more, there is certain report to its extractive technique at present.Most of triterpene composition
For lipid soluble ingredients, therefore organic solvent extracting method is in the majority.The present invention is extracted with ethanol solution, is adopted and is extracted with ethyl acetate, first
A kind of triterpene extracts with certain antitumor activity and reverse multiple drug resistance of tumor are obtained after alcohol filtering.
Invention content
The object of the present invention is to provide a kind of fuling triterpene extracts, especially methanolic extract；
It is a further object of the present invention to provide a kind of preparation methods of fuling triterpene extract；
It is a still further object of the present invention to provide use of the above-mentioned fuling triterpene extract in terms of reverse multiple drug resistance of tumor
On the way.
The realization process of the present invention is as follows：
A kind of fuling triterpene extract, is obtained by following steps：Poria cocos is crushed, the extraction for adding in 4-15 times of volume is molten
Liquid, extraction solution concentration are 50%-90%, are extracted 1-4 times under 50-110 DEG C of heating condition, and a length of 1-5h during extraction, takes out every time
Filter, merging filtrate add in after suitable quantity of water dispersion and carry out extraction 2-5 times with ethyl acetate, be concentrated and dried, methanol dissolution filter, into
One step concentrates, water-dispersible and be dried to obtain the fuling triterpene extract containing pachymic acid and dehydrotumulosic acid.
The present invention is using ethanol solution as Extraction solvent, and the ethanol solution of 4-15 times of volume, preferred alcohol volume is 8
Times；The ethanol solution concentration used is 50%-90%, preferred concentration 75%；The heating condition used is 50-110 DEG C, preferably
It is 75 DEG C；Extraction time is 1-4 times, preferably 3 times；A length of 1-5h, preferably 3h during extraction, ethyl acetate extraction times are 2-5
It is secondary, preferably 4 times.
It is low-temperature vacuum drying or freeze-drying that final step of the present invention, which is concentrated and dried the method used,.
The fuling triterpene extract that present invention extraction obtains, is measured wherein using vanillic aldehyde-glacial acetic acid-perchloric acid development process
Triterpene content is more than 80%, and extracting method of the present invention is stablized, reliably.
In the fuling triterpene extract that present invention extraction obtains, main active contains pachymic acid and dehydrotumulosic acid,
The two total content is more than 50%, is 53%.
Extracting method of the present invention is reliable and stable, and the pachymic acid contained and dehydrotumulosic acid content are higher, by cell in vitro
Experiment finds that it has tumour cell certain lethal effect, and has reverse multidrug drug-resistant effect to MCF/ADR cells,
With preferable application prospect.
Description of the drawings
Fig. 1 is the chemical structural formula of pachymic acid.
Fig. 2 is the chemical structural formula of dehydrotumulosic acid.
Fig. 3 is the chromatogram of 1 fuling triterpene extract of embodiment.
Fig. 4 is the chromatogram of pachymic acid.
Fig. 5 is the chromatogram of dehydrotumulosic acid.
Fig. 6 is toxic effect of the fuling triterpene extract to cell.
Fig. 7 DOX are to the toxic effect of MCF, MCF/ADR cell.
To MCF/ADR cytotoxic effects after Fig. 8 fuling triterpenes, CsA and DOX drug combinations.
Specific embodiment
The present invention will be further elaborated below.
Total triterpene contents assay method (ultraviolet spectrophotometry)：
The preparation of reference substance solution：Ursolic acid 2mg is weighed in 2ml volumetric flasks, ethyl acetate, which dissolves and is diluted to graduation mark, to be taken
The 1ml solution is put in 5ml volumetric flasks, is diluted to graduation mark, obtains the ursolic acid reference substance solution of 200 μ g/ml.
5% vanillic aldehyde-glacial acetic acid solution is prepared：0.5g vanillic aldehydes are weighed in 10ml volumetric flasks, acetic acid is diluted to scale
Line obtains 5% vanillic aldehyde-glacial acetic acid solution.
Precision pipettes 0.0,0.1,0.2,0.3,0.4,0.5ml standard solution in test tube, is heated under 85 DEG C of water-baths and volatilizes solvent.
0.4ml vanillic aldehydes-glacial acetic acid solution is separately added into each test tube after volatilizing solvent, 1ml perchloric acid is uniformly mixed, is placed in 60
It is moved into cold bath after DEG C heating water bath 15min and cools down 10min.It is cooled to room temperature in backward each test tube and adds in 5ml acetic acid, shake up
15 minutes are stood, absorbance is measured under 545nm wavelength.
Embodiment 1
Fuling triterpene extracting method is as follows：
100g Indian Breads are taken, add in 75% ethanol solution refluxing extraction 3 times, each 2h, ethanol consumption 800ml, are extracted
Temperature is 75 DEG C, filters, filtrate is merged, and concentration is evaporated, and adds in moisture and dissipates, and carries out extraction 4 times using ethyl acetate, collects second
Ethyl acetate layer is concentrated to dryness, and adds in methanol dissolution filter, removes insoluble matter, and solution concentration is evaporated, and moisture is added to dissipate, in 45 DEG C of items
It is dried in vacuo under part.Fuling triterpene extract 60.5mg is obtained, it is 81.32% to measure total triterpene contents.
The content of dehydrotumulosic acid in extract and pachymic acid is measured, method is as follows：
1st, instrument and reagent Shimadzu efficient liquid phase, LC-20AT pumps, SPD-20A UV detector, SIL-20A sampling systems,
LCsolution work stations, Kromasil 100-5-C18 chromatographic columns (4.6 × 250mm), methanol (chromatographic grade, TED), glacial acetic acid
(Chinese medicines group Co., Ltd), pachymic acid reference substance (Shanghai source leaf biology Co., Ltd, content ＞ 98.0%), dehydrotumulosic acid
Reference substance (Chengdu De Site biologies Co., Ltd, content ＞ 98.0%) (structural formula such as attached drawing 1-2)
2nd, testing conditions mobile phase：- 0.2% acetic acid water (80: 20) of methanol, flow velocity 1.0ml/min, Detection wavelength 242nm,
Sample size is 20 μ L.
3rd, the preparation of reference substance solution：Precision weighs pachymic acid in right amount in 10ml volumetric flasks, adds methanol dissolved dilution to scale
Line obtains the pachymic acid reference substance solution of 197 μ g/ml；Precision weighs dehydrotumulosic acid in right amount in 10ml volumetric flasks, adds methanol
It dissolves and is diluted to graduation mark, obtain the dehydrotumulosic acid reference substance solution of 202 μ g/ml.Reference substance solution is diluted to respectively
A series of concentration, and centrifuge, Aspirate supernatant sample introduction.
4th, the preparation of test solution：Precision weighs fuling triterpene extract 2mg in 10ml volumetric flasks, adds in methanol dissolving simultaneously
Graduation mark is diluted to, solution 6000rpm × 5min is centrifuged, takes supernatant, sample introduction.
5th, assay method：Precision draws above-mentioned reference substance solution and each 20 μ L injections liquid chromatograph of test solution, obtains Fu
Siberian cocklebur triterpene extract and the attached 3-5 of reference substance chromatogram.
6th, result calculates：It is computed, Poria cocos acid content is 53.34%, and dehydrotumulosic acid content is 2.68%, and the two total content is
56.02%.
Embodiment 2
Fuling triterpene extracting method is as follows：
500g Indian Breads are taken, add in 80% ethanol solution refluxing extraction three times, each 2h, Extracting temperature is 115 DEG C, and ethyl alcohol is used
It measures as 2000ml, filters, filtrate merges concentration, adds in suitable quantity of water and is disperseed, ethyl acetate extracts 4 times, by ethyl acetate layer
Merging is evaporated, and with methanol dissolution filter, obtained solution, after revolving is dry plus moisture dissipates, and is freeze-dried.Obtain fuling triterpene
Extract 280.5mg, wherein total triterpene contents are 80.52% after measured.Both pachymic acid and dehydrotumulosic acid total content account for
55.20%.
Embodiment 3
Fuling triterpene class antitumor activity of compound is studied：This experiment is extracted by the use of A549, HepG-2 cell as fuling triterpene class
Object antitumor activity Effect study object.
1st, laboratory apparatus：Sterilization tank (Nanjing Chi Yuan bio tech ltd), CO2Incubator (Mode1311 Thermo
Scientific), Biohazard Safety Equipment (Su Jing is safe and sound), liquid-transfering gun (ACURA 825), centrifuge (Hunan instrument), electric heating constant temperature water-bath
Pot (Guo Hua Electrical Appliances Co., Ltd), microplate reader (BioTek).Reagent：Sodium chloride (Xi Long science limited company), di(2-ethylhexyl)phosphate
Hydrogen potassium (Xilong Chemical Co., Ltd), disodium hydrogen phosphate dodecahydrate (Nanjing Chemistry Reagent Co., Ltd.), potassium chloride is (forever
Magnificent scientific and technological (Jiangsu) Co., Ltd of chemistry), MTT (Solarbio), RPMI-1640 complete culture solutions (the triumphant base biotechnology in Jiangsu
Limited company), dimethyl sulfoxide (Solution on Chemical Reagents in Shanghai Co., Ltd), 96 orifice plates (BIOFIL), 15,50ml centrifuge tubes
(BIOFIL)。
2nd, the preparation of PBS solution：0.2g potassium chloride, 0.2g potassium dihydrogen phosphates, 12 water phosphorus of 8g sodium chloride and 3.15g are weighed respectively
Sour disodium hydrogen adds 1000ml water.4 DEG C of preservations after sterilizing.Prepare MTT solution：20mgMTT is weighed, pays attention to encasing shading with tinfoil,
4mlPBS solution is dissolved in, a concentration of 5mg/ml solution is made into, is preserved under 4 DEG C of shadings.1640th, the preparation of DMEM complete mediums：
Complete medium adds 10% serum, 1% Pen .- Strep solution.0.0487mg fuling triterpene extracts are weighed, are added in
1.218mIDMSO is configured to the fuling triterpene solution of 40mg/ml, and after 0.22 μm of filtering with microporous membrane, 4 DEG C of refrigerators preserve.Take 100
μ l fuling triterpene solution, is diluted to 10ml solution with DMEM culture mediums, RPMI culture mediums respectively, obtains the Poria cocos three of 400 μ g/ml
Terpene solution A is diluted to the solution of 20,40,60,80,100,150,200,400 μ g/ml respectively, is diluted using corresponding culture medium
Into the solution of 3ml, a series of fuling triterpene solution of concentration is obtained.
3rd, cell culture：A549, MCF-7, HepG-2 are thawed respectively and are placed in complete medium 1640, cell can paste culture bottle
Wall is bred, and culture external condition is 5%CO2, temperature be 37 DEG C, humidity 95%.Liquid is changed, outwells liquid in original culture bottle, PBS
5 times of amounts of buffer solution are washed, then add 10~15ml complete mediums.Waste liquid in original culture bottle, 5 times of amounts of PBS buffer solution are outwelled in passage
It washes, adds pancreatin cell dissociation buffer vitellophag 1min, 3 times of amount complete mediums is added to terminate digestion.1000rpm × 1min is centrifuged,
Cell is made to be deposited in bottom of the tube.Incline supernatant, and complete medium is added to be transferred to new culture bottle in piping and druming convolution in centrifuge tube.
4th, the tumour cell long-term to inteilectual is taken, is digested with pancreatin, the culture medium containing serum accordingly is added in and terminates digestion, it will
It is blown and beaten into single cell suspension, and by cell suspension and is diluted to 3 × 104/ ml is inoculated in 96 orifice plates, and 200 μ l are added in per hole,
It is placed in 37 DEG C, saturated humidity is cultivated for 24 hours in 5%CO2 incubators.Culture medium in 96 orifice plates is sucked out, adds in the Fu of respective concentration
Siberian cocklebur triterpene drug solution adds in 200 μ l, 6 multiple holes of every group of setting per hole.After 24/48h, MTT solution (basal mediums are added in
Prepare, 5mg/ml), per 20 μ l of hole, after being incubated 4h in incubator, MTT solution is sucked out, the DMSO of 150 μ l is added in, with oscillator
On shake 10min, after purple dissolving to be crystallized, absorbance value is measured in 490nm.
The experimental results showed that fuling triterpene extract has certain lethal effect, and right to A549 and HepG-2 cells
HepG-2 cells are more sensitive.Upper chart is bright, and fuling triterpene extract increases over time work to the lethal effect of A549 cells
With becoming apparent from, and for HepG-2 cells, cytotoxicity enhances with the increase of concentration, but cell is killed with 48h for 24 hours
Wound acts on no significant difference.
Fuling triterpene extract acts on MCF/ADR cell reversals multidrug resistance
1st, laboratory apparatus and reagent：Electric-heated thermostatic water bath (Shanghai-perseverance scientific instrument Co., Ltd), CO2 incubators (Thermo
Scientific), Biohazard Safety Equipment (SuZhou Antai Air Tech Co., Ltd. of Su Jing groups), microplate reader (BioTek
Instruments Inc.), MTT (triumphant base biology Co., Ltd), DMSO (Sinopharm Chemical Reagent Co., Ltd.), pancreatin
(triumphant base biology Co., Ltd), DMEM culture mediums (triumphant base biology Co., Ltd), DOX (lot numbers：150802), CsA (Nanjing all Lays
Biological Co., Ltd), disodium hydrogen phosphate dodecahydrate (Nanjing chemistry tries Co., Ltd), potassium dihydrogen phosphate (western Gansu Province chemical industry share
Co., Ltd), sodium chloride (Xilong Chemical Co., Ltd), potassium chloride (Shanghai Ling Feng chemical reagent Co., Ltd).
2nd, solution prepares fuling triterpene solution：40mg fuling triterpene extracts are weighed, add in 1ml DMSO dissolvings, 4 DEG C of refrigerators are put
It puts, it is spare.The preparation of DOX solution：40mg DOX are weighed, add in 1ml DMSO dissolvings, 4 DEG C of refrigerators are placed, spare.CsA solution
Preparation：40mg CsA are weighed, add in 1ml DMSO dissolvings, 4 DEG C of refrigerators are placed, spare.
3rd, cell culture：MCF, MCF/ADR cell adherent growth in RP1640 culture mediums (fetal calf serum for including 10%), put
In 37 DEG C, 95% saturated humidity, 5%CO2Incubator in cultivate, change a not good liquor within every two days, when cell 80% merges, use
0.05% pancreatin cell dissociation buffer digestion, 1: 2-1: 3 passages are primary.
4th, it takes the logarithm MCF, MCF/ADR cell in growth period, is digested with 0.05% pancreatin, with 8 × 10 after cell count3It is a thin
Born of the same parents/hole is inoculated on 96 orifice plates, after cultivating 24 hours, adds in DOX, CsA, the fuling triterpene extract of various concentration, concentration point
It Wei not 0.01,0.1,1,5,10,20,50 μ g/ml.Dosing group and control group are set up in experiment, and control group is not added with drug.Every group sets 6
A secondary orifices.After cultivating 48h, MTT20 μ L are added in per hole and continue culture 4 hours.After being incubated 4 hours, carefully discard in 96 orifice plates
Liquid, then the DMSO of 150 μ l is added in into each hole, it is densitometric value at 490nm in wavelength after the grain dissolution in hole.
Calculate each group cell IC50, drug resistance multiple=IC50 (MCF/ADR)/IC50 (MCF).
IC50 values, respectively MCF are calculated using spss19.0：0.377 μ g/ml, MCF/ADR：18.583 μ g/ml, drug resistance multiple
For：55.14 times.
5th, it takes the logarithm MCF, MCF/ADR cell in growth period, is digested with 0.05% pancreatin, it is thin with 8 × 103 after cell count
Born of the same parents/hole is inoculated on 96 orifice plates, after cultivating 24 hours, adds in relative medicine.Experimental group be DOX+5 μ g/ml triterpenes, DOX+25 μ
G/ml triterpenes, DOX+50 μ g/ml triterpenes, positive controls are DOX+5 μ g/mlCsA, negative control group DOX.Experiment, which is set up, to be added
Medicine group and control group, control group are not added with drug.Every group sets 6 secondary orifices.After cultivating 48h, MTT20 μ L are added in per hole and continue culture 4
Hour.After being incubated 4 hours, the liquid in 96 orifice plates is carefully discarded, then the DMSO of 150 μ l is added in into each hole, treat in hole
It is densitometric value at 490nm in wavelength after grain dissolving.
IC50 is calculated, and calculate reversal index using spss19.0, each group reversal index result is as follows,
Tab1. fuling triterpene extract and CsA are to the reversal index of MCF/ADR multidrug resistances
It is seen from the above data that the reverse effect of fuling triterpene extract increases with the increase of concentration, and 48h, triterpene
During a concentration of 5 μ g/ml, reversal index is 1.67 times, and sigmaplot12.0 examines display compared with negative control group without conspicuousness
Difference, there are significant differences with DOX+5 μ g/mlCsA groups for DOX groups.Table Tab1. illustrates fuling triterpene extract to MCF/ADR
Cell multidrug resistance has certain reverse effect, and effect enhances with the increase of concentration.Have in Chinese medical extract compared with
More compositions have the function of reverse multiple drug resistance of tumor, as investigated damp wind grass extract pair in patent CN104606272A
The reverse effect of a variety of mdr cells, damp wind grass extract concentrations are respectively 50 μ g/ml, 20 μ g/ml, after acting on 48h, to various
Between the reversal index of cell is 1-20 times, and fuling triterpene extract is under the same conditions, has preferable reverse effect.Text
It offers《Ganoderma lucidum, Rabdosia rubescens extract reverse the research of human breast carcinoma MCF/ADM cell multidrug resistances》In, ganoderma lucidum, Rabdosia rubescens carry
Object is taken to have certain reverse effect to MCF/ADR cells, and through Verapamil, ganoderma lucidum ethyl acetate (8 μ g/ml, 40 μ g/
Ml), ganoderma lucidum n-butanol (8 μ g/ml, 40 μ g/ml), Rabdosia rubescens ethyl acetate (4 μ g/ml, 20 μ g/ml), Rabdosia rubescens chloroform (4 μ g/
Ml, 20 μ g/ml) after position processing MCF-7/ADR cells, reversal index is respectively 3.76,1.78,3.56,2.11,3.47,
1.87th, 4.02,2.16,4.60, compared with tuckahoe extracts, the reverse multiple drug resistance of tumor effect of tuckahoe extracts is brighter
It is aobvious, illustrate that tuckahoe extracts have preferable prospect in terms of reverse multiple drug resistance of tumor.Document《Multidrug Resistance of Gastric Cancer
Reverse and Mechanism Study》In screened plurality of Chinese ethanol extract, and ability is reversed to investigate it, wherein Poria cocos pass through
After ethyl alcohol extraction, cell experiment is directly used in, it is found that it does not have reverse multidrug drug-resistant effect to SGC7901/VCR cells；Mesh
Before, in terms of tuckahoe extracts are used as multi-drug resistance of the tumor reverse there are no patent.In conclusion the Poria cocos described in this patent
Extract is worth people further to study with opening as having stronger researching value in terms of tumor multi-drug resistance reversal agents
Hair.
Claims (10)
1. a kind of tuckahoe extracts of energy reverse multiple drug resistance of tumor, which is characterized in that triterpene content is in tuckahoe extracts
40%-90% is obtained by the following method：Poria cocos is crushed, suitable extraction solution is added in, is extracted under heating condition, is filtered
Liquid merges, and after concentration plus suitable quantity of water, ethyl acetate are extracted, dissolved after being dried under reduced pressure with methanol, are filtered, and filtrate concentration is dry
It is dry, tuckahoe extracts are obtained, main ingredient contains pachymic acid and dehydrotumulosic acid in extract.
2. fuling triterpene extract according to claim 1, which is characterized in that the extraction solution is methanol, ethyl alcohol, third
One or more kinds of mixed solvents in alcohol, isopropanol, butanol, the mass volume ratio of the Poria cocos and extraction solution
It is 1: 4-1: 15, extracts a concentration of 50%-90% of solution, the heating condition is 50-110 DEG C, and the extraction time is 1-4
Secondary, each extract time is 1-5h, and the extraction times are 2-5 times；Main ingredient has pachymic acid and dehydrogenation soil not in product
Acid, total content 30%-80%.
3. a kind of preparation method of tuckahoe extracts described in claim 1, which is characterized in that include the following steps：
(1) Poria cocos is crushed, be sieved；
(2) in proportion with extracting solution mixing；
(3) under heating condition, extraction is multiple；
(4) it filters, merging filtrate, concentration, ethyl acetate extraction is multiple, is dried under reduced pressure；
(5) methanol is added in, removes insoluble matter, it is dry.
4. the preparation method of tuckahoe extracts according to claim 3, which is characterized in that in step (1), smash it through
200 mesh screens.
5. the preparation method of tuckahoe extracts according to claim 3, which is characterized in that in step (2), Poria cocos and extraction
The ratio between quality volume of solution is 1: 4-1: 15, and extraction solution concentration is 50%-90%, and extracting solution is methanol or ethyl alcohol.
6. the preparation method of tuckahoe extracts according to claim 3, which is characterized in that in step (3), heating condition is
50-110 DEG C, extraction time is 1-4 times.
7. the preparation method of tuckahoe extracts according to claim 3, which is characterized in that in step (4), ethyl acetate extraction
It is 2-5 times to take number.
8. the preparation method of tuckahoe extracts according to claim 3, which is characterized in that in step (5), the drying side
Formula is low-temperature vacuum drying or freeze-drying.
9. according to the preparation method of the tuckahoe extracts described in claim 2, which is characterized in that main in obtained extract
Composition is triterpene, and containing pachymic acid and dehydrotumulosic acid, Poria cocos acid content is 30%-60%, and dehydrotumulosic acid content is 1%-
20%, the two total content is 30%-80%.
10. according to described in claim 1 it is a kind of can reverse multiple drug resistance of tumor tuckahoe extracts prepare it is antitumor
Application in drug.
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| CN111494393A (en) * | 2019-01-30 | 2020-08-07 | 中国科学院微生物研究所 | Preparation method and application of poria peel triterpene composition |
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| US20230041859A1 (en) * | 2021-07-01 | 2023-02-09 | Arjil Biotech Holding Company Limited | Use of terpenoids in the treatment or prevention of fibrotic diseases |
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