CN102603856B

CN102603856B - Anti-tumor saponin in anemone plants and preparation method thereof as well as application

Info

Publication number: CN102603856B
Application number: CN201110462335.1A
Authority: CN
Inventors: 路金才; 贾凌云; 王晶; 吕重宁; 林杨; 薛娇
Original assignee: Shenyang Pharmaceutical University Benxi Medical Technology Co ltd; Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University Benxi Medical Technology Co ltd; Shenyang Pharmaceutical University
Priority date: 2011-12-31
Filing date: 2011-12-31
Publication date: 2015-03-25
Anticipated expiration: 2031-12-31
Also published as: CN102603856A

Abstract

The invention relates to a preparation method of a triterpenoid saponin compound in anemone plants and a mechanical application, in particular relates to an anti-tumor application. The molecular formula of the compound is C47H76O16, and a chemical name of the compound is oleanolic acid-3-0-alpha-L-rhamanopyranosyl-(1->2)-[beta-D-glucopyranosyl-(1->4)]-alpha-L-arabinopyranosid. The extraction preparation process of the compound disclosed by the invention is applicable to the industrial production and can be used for converting other saponin compounds in medicinal materials into the saponin through an alkaline hydrolysis production, so that the content of the target saponin compound is greatly improved. Shown by anti-tumor experiments in vivo and vitro, the compound plays a role in suppressing a plurality of kinds of tumor cells to generate, and approximately has no suppression and toxic actions on generation of normal cells. Therefore, the triterpenoid saponin compound can be used for treating tumor diseases.

Description

A kind of antitumor saponin(e and its production and use in thimbleweed

Technical field

The present invention relates to medical art, relate to extraction and isolation from thimbleweed and obtain a kind of compound with anti-tumor activity, and in the application of anti-tumor aspect.

Background technology

Cancer is the disease that a kind of common and M & M is all rising year by year.But there is no effective means at present and medicine can be cured, the cancer therapy drug therefore finding a kind of safe toxic side effect little by exploitation natural product becomes current focus.

Anemone (Anemone) is one maximum in Ranunculaceae (Ranunculaceae) genus, and total plant more than 150 is planted, and all has distribution all over the world.Thimbleweed has more than 50 in China and plants, and except Hainan Island, all there is distribution other each province.Thimbleweed has long medicinal history in China, and more at the medical species of this platymiscium among the people, effect is extensive.Conventional kind is as follows: anemone altaica (Anemonealtaica), Anemone amurensis (Korsh) Kom. (A.amurensis), southwest Anemone cathayensis Kitag. (A.davidii), thin stem Anemone cathayensis Kitag. (A.rossii), comospore Anemone cathayensis Kitag. (A.bacalensis), Patenthairy Anemone (A.demissa), two discrimination Anemone cathayensis Kitag.s (A.dichotoma), the shady Anemone cathayensis Kitag. (A.flaccida) of woods, Hupeh Anemone Herb (A.hupehensis), Japanese anemone (A.hupehensis var.japonica), anemone raddeana Regel (A.raddeana), Anemone reflex (A.reflexa), Herba Veronicastri Sibirici (A.rivularis), little Hua Herba Veronicastri Sibirici (A.rivularis var.flore-minore), large fire weed (A.tomentosa), bar leaf Anemone cathayensis Kitag. (A.trullifolia var.linearis), Herba anemones vitifoliae (A.vitifolia) etc.Chemical composition contained by thimbleweed mainly contains: triterpene saponin, coumarins, flavonoid, steroid, phenols and organic acid etc.Wherein triterpene and glycoside thereof are extensively a large amount of in thimbleweed exists, and is study a class the most deep in thimbleweed.Its aglycon is mainly Oleanolic Acid, hederagenin, Folium seu Cortex Nerii acid etc.This platymiscium primary efficacy has: wind-damp dispelling, subduing inflammation, and anti-inflammatory is antipyretic, analgesia, antitumor, anticonvulsion etc.Existing research data shows these plants mostly containing triterpenoid saponins.Be separated from thimbleweed at present and obtained more than 100 triterpene saponin componds, further pharmacological experiment study result shows, wherein great majority all have good antitumous effect.

Summary of the invention

The object of the invention is to provide a kind of antitumor saponin(e and its production and use in a kind of thimbleweed, extracts the method preparing AR17 and the application in treatment tumor disease thereof from Anemone various plants.

The invention provides a kind of antitumor saponin(e, its molecular formula is C ₄₇h ₇₆o ₁₆, chemical name is 3-O-α-L-rhamanopyranosyl-(1 → 2)-[β-D-Glucose base-(1 → 4)]-α-L-arabinose base-Oleanolic Acid (oleanolicacid-3-O-α-L-rhamanopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 4)]-α-L-arabinopyranoside).Be called for short AR17.

The invention still further relates to a kind of mode by basic hydrolysis from Ranunculaceae Anemone various plants, extract the method that preparation has a kind of compd A R17 of anti-tumor activity.

In the present invention, the extracting and preparing technique step of AR17 is as follows:

(1) pulverized at the medicinal material positions such as the stem of the root and rhizome of multiple for Anemone medicinal plant, ground, leaf, flower or fruit, grinding particle size is 5-30 order.Described thimbleweed is selected from: anemone raddeana Regel (Anemoneraddeana), Anemone amurensis (Korsh) Kom. (A.amurensis), thin stem Anemone cathayensis Kitag. (A.rossii), comospore Anemone cathayensis Kitag. (A.bacalensis), southwestern Anemone cathayensis Kitag. (A.davidii) or From Anemone Rupestris Ssp. Gelida (A.rupestris) etc.

(2) Extraction solvent ethanol used before extraction by medicinal material, water or ethanol alkaline solution, alkali aqueous solution is according to medicinal material: solvent=1kg: 4-12L carries out immersion 1-12h, and wherein alcohol concn is 20%-90%, alkali concn is: 0.05-2.0M, and alkali used is NaOH or KOH or Na ₂cO ₃or K ₂cO ₃or Na ₂s or K ₂s or NH ₄the aqueous solution of OH.

(3) medicinal material is through normal pressure condensation heating refluxing extraction 2-4 time, and each 1-3h, merges each extracting solution.

(4) ethanol extract of medicinal material reclaims ethanol or aqueous extract and is concentrated into certain volume, add above-mentioned alkaline solution, heating also constantly stirs hydrolysis 1-6 hour, adding 2%-3% hydrochloric acid regulates pH value to neutral, or this walks uncomfortable neutrality, then by medicinal material: weight resin ratio is 1: 2-3 upper macroporous resin column HPD300, HPD400, HPD450 or D101 type, Static Adsorption 4-10h, use distilled water again, with 1kg medicinal material: 20-25L distilled water, elution speed is that 0.8-1L/h carries out wash-out removal of impurities, and then with 50%-90% ethanol elution, amount of alcohol used is with 1kg medicinal material: 20-25L ethanol, elution speed is that 0.5-0.8L/h carries out wash-out removal of impurities collection ethanol eluate, reclaim ethanol.

(5) medicinal material also can adopt direct alkali ethanol or potass extraction, alkali ethanol extract reclaims ethanol, or potass extraction liquid is concentrated into certain volume, adds 2%-3% hydrochloric acid and regulates pH value to neutral, or this walks uncomfortable neutrality, then by 4) item same operation.

(6) by 4) or 5) products therefrom is by the further separation and purification of silica gel column chromatography, with ethyl acetate: methyl alcohol: water ratio: 6-11: 0.5-2: 0.05-O.2 is moving phase wash-out, target product AR17 is detected by TLC, TLC condition used is developping agent is chloroform: methyl alcohol: water=13: when 7: 1, target product Rf is 0.35-0.5, collect elutriant, recycling design.

(7) also can by 3) directly go up silica gel column chromatography, by 6 after the extracting solution recycling design of gained) operation.

(8) by product ethyl acetate that above-mentioned various different extraction and separation method obtains: methyl alcohol: water=6-11: 0.5-2: 0.05-0.2, or ethanol: methyl alcohol=8-12: 3-1 or methyl alcohol: water=8-10: 2-0.1 or ethanol: water=1-9: 9-1 recrystallization, obtain purity higher than 90% AR17.

It should be noted that, crucial part of the present invention is basic hydrolysis, shows through this research herein, and basic hydrolysis goes for the arbitrary step in sample extraction, enrichment or purifying, and alkali used is NaOH or KOH or Na ₂cO ₃or K ₂cO ₃or Na ₂s or K ₂the aqueous solution of S or NH4OH, the buck of hydrolysis or the alkali concn of alkali ethanolic soln are: O.05-2.0M.

Feature of the present invention is, under adopted AR17 extracting and preparing technique parameter, AR17 yield is high, and the content of AR17 significantly increases after critical base hydrolysis step.

When treating tumor disease, the AR17 that the present invention prepares can be used alone, and is mixed with different dosage form and re-uses after also can mixing with other medicines auxiliary material.

Pharmacological experiment study result of the present invention shows, AR17 to human liver cancer cell Hep G2, human esophagus cancer cell Eca-109,

Human oral epidermoid carcinoma cell KB, human liver cancer cell Hep 3B, people lung cancer A549, the kinds of tumor cells such as human bladder cancer cell 5637 all have restraining effect, and prompting may be used for oncotherapy.

The method is simple, reliable and stable, and yield is high, be applicable to suitability for industrialized production, and adopt present method medicinal material through direct basic hydrolysis extraction or traditional extraction again after basic hydrolysis, other saponin(es in medicinal material can be made to be converted into AR17, thus make target saponin A R17 content significantly increase nearly 5 times.Anti-tumor experiment result display AR17 is inhibited to kinds of tumor cells growth in inside and outside.Can as the raw material of anti-tumor medicinal preparation.

Accompanying drawing explanation

Fig. 1 is AR17's ¹h-NMR spectrogram.

Fig. 2 is AR17's ¹³c-NMR spectrogram.

Fig. 3 is the DEPT spectrogram of AR17.

Fig. 4 is the H-Hcosy spectrogram of AR17.

Fig. 5 is the HMBC spectrogram of AR17.

Fig. 6 is the HMQC spectrogram of AR17.

Embodiment

One, AR17 preparation

Saponin A R17 prepares example one

Pulverize 500g anemone raddeana Regel rhizome, cross 10 mesh sieves, add the 65% ethanolic sodium hydroxide alkaline solution of 8 times amount 0.2M, after soaking 6h, refluxing extraction three (each alcohol adding amounts 8 times, 8 times, 8 times), each 1.5h, united extraction solution, be recycled to without alcohol taste, liquid distilled water suitably dilutes, with HPD-400 macroporous resin adsorption 5h, amount of resin used is 1kg, first use 12L distilled water, with the removal of impurities of 1L/h speed wash-out, use 12L 80% ethanol with 0.8L/h speed wash-out again, collect 80% ethanol elution part, be spin-dried for, upper silicagel column, with ethyl acetate: methyl alcohol: water=65: 30: 8 wash-outs, this step elution process is monitored by TLC, collect AR17 part, be spin-dried for, with ethanol: methyl alcohol=6: 1 recrystallization, products therefrom AR17 4.7g, purity is 95.6%.

Saponin A R17 prepares example two

Pulverize 500g anemone raddeana Regel medicinal material, cross 10 mesh sieves, add the 50% ethanolic sodium hydroxide alkaline solution of 6 times amount 0.3M, after soaking 10h, refluxing extraction three times (6 times, 6 times, 6 times), each 2h, united extraction solution, adjust neutral with 2% hydrochloric acid, be recycled to without alcohol taste, liquid distilled water suitably dilutes, with D101 macroporous resin adsorption absorption 3h, amount of resin used is 2kg, first use 10L distilled water, with the removal of impurities of 0.8L/h speed wash-out, be washed till colourless, use 12L 80% ethanol with 0.5L/h speed wash-out again, collect 80% ethanol elution part, be spin-dried for, upper silicagel column, with ethyl acetate: methyl alcohol: water=70: 30: 7 wash-outs, this step elution process is monitored by TLC, collect AR17 part, be spin-dried for, with ethanol: methyl alcohol=3: 1 recrystallization, products therefrom AR17 6.0g, purity is 92.3%.

Saponin A R17 prepares example three

Pulverize 500g Anemone amurensis (Korsh) Kom. rhizome, cross 16 mesh sieves, add 5 times amount 80% ethanolic solns, soak after 12h, and refluxing extraction three times (5 times, 5 times, 5 times), each 1h, united extraction solution, is recycled to without alcohol taste, add NaOH, make 1.5MNaOH extracting solution, 80 degree of heating 3h, and constantly stir.Liquid distilled water suitably dilutes, and with HPD-300 macroporous resin adsorption 8h, amount of resin used is 1.5kg, first use 11L distilled water, with the removal of impurities of 0.9L/h speed wash-out, then use 12.5L 80% ethanol with 0.6L/h speed wash-out, collect 80% ethanol elution part, be spin-dried for, upper silicagel column, with ethyl acetate: methyl alcohol: water=6: 0.5: 1 wash-out, collects elution fraction, products therefrom AR17 5.8g, purity is 91.6%.

Saponin A R17 prepares example four

Pulverize 500g anemone raddeana Regel medicinal material, cross 10 mesh sieves, add the KOH aqueous solution of 10 times amount 0.5M, after soaking 10h, heating extraction three times (10 times, 10 times, 10 times), each 2h, united extraction solution, adjust neutral with 2% hydrochloric acid, in decompression removing liquid after part water, with D101 macroporous resin adsorption absorption 3h, amount of resin used is 2.5kg, first use 12L distilled water, with the removal of impurities of 0.8L/h speed wash-out, be washed till colourless, use 12L 80% ethanol with 0.5L/h speed wash-out again, collect 80% ethanol elution part, be spin-dried for, upper silicagel column, with ethyl acetate: methyl alcohol: water=80: 20: 7 wash-outs, this step elution process is monitored by TLC, collect AR17 part, be spin-dried for, with ethanol: water=6: 1 recrystallization, products therefrom AR17 5.5g, purity is 90.3%.

Saponin A R17 prepares example five

Pulverize 500g thin stem Anemone cathayensis Kitag. rhizome, cross 10 mesh sieves, add the 12 times amount aqueous solution, after soaking 10h, refluxing extraction three times (12 times, 12 times, 12 times), each 2h, united extraction solution, in decompression removing liquid after part water, add KOH, make 1MKOH liquid, 80 degree of heating 3h, with HPD400 macroporous resin adsorption absorption 5h, amount of resin used is 2kg, first use 12L distilled water, with the removal of impurities of 1L/h speed wash-out, be washed till colourless, use 10L 80% ethanol with 0.6L/h speed wash-out again, collect 80% ethanol elution part, be spin-dried for, upper silicagel column, with ethyl acetate: methyl alcohol: water=90: 10: 7 wash-outs, this step elution process is monitored by TLC, collect AR17 part, be spin-dried for, with ethanol: water=6: 1 recrystallization, products therefrom AR17 5.2g.Purity is 93.8%.

Saponin A R17 prepares example five

Pulverize 500g anemone raddeana Regel rhizome, cross 20 mesh sieves, add the 70% ethanolic sodium hydroxide alkaline solution of 8 times amount 0.2M, after soaking 6h, refluxing extraction three (each alcohol adding amounts 8 times, 8 times, 8 times), each 1.5h, united extraction solution, be recycled to without alcohol taste, liquid is adjusted neutral with 2% hydrochloric acid, evaporate to dryness, upper silicagel column, with ethyl acetate: methyl alcohol: water=65: 30: 8 wash-outs, this step elution process is monitored by TLC, collects AR17 part, be spin-dried for, with ethanol: methyl alcohol=6: 1 recrystallization, obtain product 5.1g AR17, and purity is 92.6%.The spectroscopic data of the nuclear magnetic resonance of saponin A R17 is as follows:

Table 1 saponin A R17's ¹³c-NMR spectral data (δ in C ₅d ₅n)

Two, with the pharmaceutical preparation being used for the treatment of tumor disease that saponin A R17 makes for raw material

Example one

AR17 capsule

Get AR17 20.0g, add medical starch 56.0g, Icing Sugar 18.0g, Xylo-Mucine 6.0g, mixes, and adds starch slurry, mixing, crosses 14 mesh sieve mixing granulations, dry, crosses the whole grain of 12 mesh sieve, incapsulates, make 1000, and every containing AR17 20mg.

Example two

AR17 freeze-dried powder

Get AR17 10g, add in appropriate distilled water for injection, regulate PH to 8.0 with sodium hydroxide solution, get appropriate N.F,USP MANNITOL simultaneously and add in water for injection and make it dissolve, then liquid and mannitol solution is merged, add water for injection to 1000ml, every 1ml is containing AR17 10mg, N.F,USP MANNITOL 8mg, filter (0.22um), sterile filling, lyophilize, often props up containing AR17 10mg.

Example three

The preparation of AR17 dripping pill

Get AR17 10.0g, add matrix PEG6000 or PEG 4000 990g of melting, be placed in stirred in water bath and make it mix, pour dripping pill device into, in instillation dimethyl silicone oil, collect dripping pill, removing condensing agent, every dripping pill is containing AR17 2mg.

Three, the pharmacodynamic experiment of AR17

(1) anti tumor activity in vitro test-results

1. experiment material

1.1 cell

HeLa Cells, human melanoma A375-S2 cell, MCF-7 Human Breast Cancer Cells and typeⅡ pneumocyte are purchased from American Type Culture Collection (ATCC, Rockville, MD, USA), SGC-7901gastriccarcinomacellline is obtained by total institute of the military region, Shenyang ground force.Human bladder cancer cell 5637, people's promyelocytic leukemia cell HL-60, human gastric adenocarcinoma SGC-7901, human lung adenocarcinoma cell SPC-A-1, human glioma cells U251, human esophagus cancer cell Eca-109, human oral epidermoid carcinoma cell KB, human colon cancer cell HT-29, human liver cancer cell Hep 3B, human pancreatic cancer cell CFPAC-1, human liver cancer cell Hep G2 is purchased from national cancer institute.Cell is seeded in the RPMI-1640 nutrient solution containing 10% foetal calf serum, 2% glutamine, at 37 DEG C, and 5%CO ₂cultivate in incubator.

1.2 medicines and reagent

AR17 prepares by method described in this patent embodiment one, and under aseptic condition, after dissolving by dimethyl sulfoxide (DMSO) (DMSO), be diluted to desired concn with RPMI RPMI-1640, DMSO final concentration is less than 0.1%.

Foetal calf serum, Beijing unit Heng Shengma biotechnology research institute

5 FU 5 fluorouracil (5-FU), trypsinase, glutamine, penicillin, Streptomycin sulphate, Hepes, dimethyl sulfoxide (DMSO) (DMSO), tetramethyl-azo azoles (MTT) are purchased from Sigma Co., USA.

1.3 instrument

CO2gas incubator (NuAir, USA), enzyme immunoassay instrument (Tecan, Austria), 96 well culture plates (Corning, USA), inverted microscope (Motic, China).

2. experimental technique

MTT reduction method

Operation steps:

2.1 attached tumor cells selecting logarithmic phase, after trysinization, are made into 5 × 10 with RPMI 1640 substratum containing 10% calf serum ⁴the cell suspension of/ml, is seeded in 96 well culture plates, every hole 100l, 37 DEG C, 5%CO ₂cultivate 24h.

The nutrient solution containing different concns sample that 2.2 experimental group more renew, control group then changes the nutrient solution containing equal-volume solvent, and often group establishes 3 parallel holes, 37 DEG C, 5%CO ₂cultivate 48h.

2.3 abandoning supernatant, carefully wash 2 times with PBS, and every hole adds the freshly prepared substratum containing 0.5mg/ml MTT of 100l, and 37 DEG C are continued to cultivate 4h.Careful supernatant discarded, and add 150lDMSO, after microoscillator mixing 10min, measure optical density value by microplate reader at 492nm place.

Result evaluation:

Be calculated as follows the inhibiting rate of drug on tumor Growth of Cells:

Growth of tumour cell inhibiting rate (%)

=[A ₄₉₂(negative control)-A ₄₉₂(dosing group)]/A ₄₉₂(negative control) × 100%

Therefrom obtain the half-inhibition concentration (IC of sample ₅₀).

Table 2 each tumour cell IC50 value (μm ol/L)

Experiment in vitro shows, AR17 all has restraining effect to above-mentioned 15 tumour cells.

(2) AR17 tests Normocellular restraining effect

1. experiment material

Test medicine: AR17 prepares by method described in this patent embodiment one, and experimental cell is mesangial cell strain HBZY-1

2. experimental technique

The 2.1 HBZY-1 cells selecting logarithmic phase, after trysinization, be made into the cell suspension of 5000-10000/ml with the DMEM substratum containing 10% foetal calf serum, be seeded in 96 well culture plates, 100 μ l are inoculated in every hole, 37 DEG C, and 5%CO2 cultivates 24h.

2.2 settings, 5 dosage groups, 1 control group and 1 blank group, 5 dosage group drug levels are respectively: 103,102,10,1,0.1ug/ml. medicine DMSO is dissolved in corresponding substratum after dissolving, control group then changes the substratum containing equal-volume solvent, and DMSO is no more than 0.1%, and often group establishes 5 parallel holes, 37 DEG C, 5%CO2 cultivates 48 hours.

2.3 every holes add the freshly prepared substratum .37 DEG C of continuation containing 5mg/ml MTT of 20 μ l and cultivate 4h.Carefully abandon supernatant, and add 100 μ l DMSO, vibration, in microplate reader, wavelength is 492nm place colorimetric, records OD value.

2.4 result evaluations: the inhibiting rate being calculated as follows medicine cell growth:

Inhibitory rate of cell growth %=(1-OD experiment/OD contrasts) × 100%

3. experimental result, in table 3

The cell inhibitory rate of table 3 HBZY-1

Control group OD average is 0.88.

4. conclusion:

When medicine maxima solubility, AR17 does not have restraining effect to normal cell.

(3) Study on antitumor effect in AR17 Mice Body

1. experiment material:

Tested material AR17, prepares by method described in this patent embodiment three.Positive drug: Cyclophosphamide for injection, specification: 0.2g/ props up, lot number: 20100307, production unit: SHANXI POWERDONE PHARMACEUTICAL.,LTD.Sodium chloride injection, specification: 500ml, lot number: 08012103 production unit: Zhiying Pharmaceutical Factory, Shenyang.Experimental cell strain title: rat liver cancer H22 provide unit: institute of oncology of Chinese Medical Sciences University.Kunming mouse, sex: male and female half and half, body weight: 18-22g, provides unit: Shenyang Pharmaceutical University's Experimental Animal Center, conformity certification number: SCXK (the Liao Dynasty) 2003-008

2. experimental technique

The foundation of 2.1 bearing mouse model

External recovery rat liver cancer H22 knurl strain cell, dilute by stroke-physiological saline solution, choose healthy mice, every only with above-mentioned cell suspension 0.2ml abdominal injection, the obvious swell of Mice Inoculated belly after about one week, extract ascites, in sterile test tube, dilute oncocyte Particle density by stroke-physiological saline solution is 2 × 107/ml.Inoculate buying Kunming mouse with above-mentioned ascites dilutions, every subcutaneous vaccination 0.2ml after armpit place sterilization skin, sets up rat liver cancer H22.

2.2 experiment grouping and administrations

By tumor-bearing mice random packet, often organize 10, be respectively model control group, high, medium and low group of tested material, positive drug endoxan group.

After modeling second day start administration, its cyclophosphamide group is intraperitoneal injection, the next day once, i.e. administration in the 2nd, 4,6,8 day after lotus knurl, dosage is 20mg/kg, and the continuous gastric infusion of test medicine 3 days, once a day, administration volume is 20ml/kg.

2.3 claim knurl weigh and calculate tumour inhibiting rate

After modeling the 9th day, cervical dislocation put to death animal, and dissect and peel off knurl block, electronic balance claims knurl weight.According to formulae discovery tumour inhibiting rate below:

Tumour inhibiting rate=(the average knurl weight of 1-administration group average knurl weight/control group) × 100%

Experimental data is all with mean+SD represent.

3 experimental results:

The restraining effect of rat liver cancer H22 is tested

Compared with model control group, AR17 has significant restraining effect to the growth of rat liver cancer H22 in range of doses, and in obvious dose-effect relationship.Experiment repetition three batches, the results are shown in Table 4.

Table 4 AR17 is to the restraining effect of rat liver cancer H22

*P＜0.05.**P＜0.01。

Claims

1. antitumor saponin(e and the application of a kind of medicinal compositions in preparation treatment antitumor drug in thimbleweed, described tumour is human bladder cancer, human oral epidermoid carcinoma or human esophagus cancer; In described thimbleweed, antitumor saponin(e is 3-O-α-L-rhamanopyranosyl-(1 → 2)-[β-D-Glucose base -(1 → 4)]-α-L-arabinose base-Oleanolic Acid, its structure as shown in the formula (I):

（I）；

Described medicinal compositions, the excipient comprising described antitumor saponin(e and pharmaceutically accept, and prepare the tablet, injection, powder, pill, capsule, micro-capsule, the paste that accept clinically.

2. apply as claimed in claim 1, it is characterized in that, described antitumor saponin(e is prepared by the following method:

1) stem of the root and rhizome of Anemone medicinal plant, ground, leaf, flower or fruit are pulverized;

2) medicinal material carries out immersion 1-12h with Extraction solvent before extraction, and described Extraction solvent is ethanol alkaline solution, alkali aqueous solution, medicinal material: the ratio of solvent is 1kg:4-12L, and wherein alcohol concn is 20%-90%, and alkali concn is: 0.05-2.0M;

3) medicinal material after soaking is through normal pressure condensation heating refluxing extraction 2-4 time, and each 1-3h, merges each extracting solution;

4) the alkali ethanol extract of medicinal material reclaims ethanol or potass extraction liquid and is concentrated into certain volume, then medicinal material is pressed: weight resin is than being macroporous resin column on 1:2-3, Static Adsorption 4-10h, use distilled water again, with 1kg medicinal material: 20-25L distilled water, elution speed is that 0.8-1L/h carries out wash-out removal of impurities, and then with 50%-90% ethanol elution, amount of alcohol used is with 1kg medicinal material: 20-25L ethanol, and elution speed is that 0.5-0.8L/h carries out wash-out collection ethanol eluate, reclaims ethanol;

5) by 4) products therefrom by the further separation and purification of silica gel column chromatography, with ethyl acetate: methyl alcohol: water ratio: 6-11:0.5-2:0.05-0.2 is moving phase wash-out, detect target product AR17 by TLC, collect elutriant, recycling design;

6) by product recrystallization, obtaining antitumor saponin(e is 3-O-α-L-rhamanopyranosyl-(1 → 2)-[β-D-Glucose base -(1 → 4)]-α-L-arabinose base-Oleanolic Acid, its structure as shown in the formula (I):

（I）。

3. apply as claimed in claim 2, it is characterized in that, described Anemone medicinal plant is selected from anemone raddeana Regel anemone raddeana, Anemone amurensis (Korsh) Kom. a. amurensis, thin stem Anemone cathayensis Kitag. a. rossii, comospore Anemone cathayensis Kitag. a. bacalensis, southwestern Anemone cathayensis Kitag. a. davidiior From Anemone Rupestris Ssp. Gelida a. rupestris.

4. apply as claimed in claim 2 or claim 3, it is characterized in that, the macroporous resin column described in step 4) is HPD300, HPD400, HPD450 or D101 type; Described TLC condition is developping agent is chloroform: methyl alcohol: water=13:7:1.

5. apply as claimed in claim 2 or claim 3, it is characterized in that, solvent selected by described recrystallization is ethyl acetate: methyl alcohol: water=6-11:0.5-2:0.05-0.2, or ethanol: methyl alcohol=8-12:3-1 or methyl alcohol: water=8-10:2-0.1 or ethanol: water=1-9:9-1.

6. apply as claimed in claim 4, it is characterized in that, solvent selected by described recrystallization is ethyl acetate: methyl alcohol: water=6-11:0.5-2:0.05-0.2, or ethanol: methyl alcohol=8-12:3-1 or methyl alcohol: water=8-10:2-0.1 or ethanol: water=1-9:9-1.

7. apply as claimed in claim 2 or claim 3, it is characterized in that, described alkali is selected from NaOH, KOH, Na ₂cO ₃, K ₂cO ₃, Na ₂s, K ₂s or NH ₄the aqueous solution of OH.

8. apply as claimed in claim 4, it is characterized in that, described alkali is selected from NaOH, KOH, Na ₂cO ₃, K ₂cO ₃, Na ₂s, K ₂s or NH ₄the aqueous solution of OH.

9. apply as claimed in claim 5, it is characterized in that, described alkali is selected from NaOH, KOH, Na ₂cO ₃, K ₂cO ₃, Na ₂s, K ₂s or NH ₄the aqueous solution of OH.

10. apply as claimed in claim 6, it is characterized in that, described alkali is selected from NaOH, KOH, Na ₂cO ₃, K ₂cO ₃, Na ₂s, K ₂s or NH ₄the aqueous solution of OH.