CN104693261B - To cast off a skin steroidal compounds, containing its pharmaceutical composition and its preparation method and application - Google Patents
To cast off a skin steroidal compounds, containing its pharmaceutical composition and its preparation method and application Download PDFInfo
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- CN104693261B CN104693261B CN201510168115.6A CN201510168115A CN104693261B CN 104693261 B CN104693261 B CN 104693261B CN 201510168115 A CN201510168115 A CN 201510168115A CN 104693261 B CN104693261 B CN 104693261B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
- C07J7/0005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21
- C07J7/001—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group
- C07J7/0015—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa
- C07J7/002—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa not substituted in position 16
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Abstract
The invention discloses steroidal compounds of casting off a skin, containing its pharmaceutical composition and its preparation method and application, belong to technical field of pharmaceuticals, be specifically related to be separated from Root or herb of Clethra Loosestrife the one obtained and cast off a skin steroidal compounds, containing its pharmaceutical composition and its preparation method and application.This compound is reported first, and can obtain by extracting and developing purifying from Root or herb of Clethra Loosestrife, purity is high.In vitro tests proves that this compound can suppress the propagation of ovarian cancer cell, and is less than the toxic action of cis-platinum to it to the toxic action of normal Epithelium Cells, can be used for being developed to low toxicity, efficient medicine for the treatment of ovarian cancer.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to be separated from Root or herb of Clethra Loosestrife the one obtained and cast off a skin steroidal compounds, containing its pharmaceutical composition and its preparation method and application.
Background technology
From natural product, find the biologically active substance of novel structure or pharmacological action uniqueness, and find that new drug has been one of important means of generally acknowledged new drug development as lead compound by methods such as structure of modification.Ratify in the 1073 kinds of small-molecule drugs gone on the market between 1981-2010, have 50% to derive from natural product or relevant with natural guide structure.From last century, the medicine of many widespread uses clinically is all excavated out, as Artemisinin, taxol etc. from natural product.
China uses natural drug to have a long history, and defines oneself unique theory of medicine, and much traditional Chinese medicine preparation also shows good effect so far in clinical application.Meanwhile, China region is wide, various places Meteorological difference great disparity, natural product aboundresources, and of a great variety, natural resources of Chinese medicinal materials reaches kind more than 12000 especially.Therefore, along with natural drug more and more comes into one's own, active chemical components is extracted from herbal medicine, its curative effect is determined through pharmacology, clinical trial, then various preparation is made for Clinical practice, become after China joined WTO, found independent intellectual property right new drug, grown the important means of China's medicinal industry.
Root or herb of Clethra Loosestrife
lysimachiaclelhroidesduby is Primulaceae Lysimachia plant, has another name called Herba Lysimachiae Clethroids, Arisaema balansae Engl. grass etc.All herbal medicine, is distributed widely in each provinces and regions on the south North China and the Changjiang river, has the effects such as clearing heat and detoxicating, promoting blood flow to regulate menstruation, inducing diuresis to remove edema, among the people be used for the treatment of oedema, heat pourings, yellow subcutaneous ulcer, band under, through closing, injury and bone fracture, the treatment of breast pain drag, snakebite etc.Modern science grinds to make internal disorder or usurp proves that Lysimachia main chemical compositions is flavones and triterpenoid saponin, also has benzene a pair of horses going side by side lactone, quinones, phenolic acid, alkaloid, steroidal saponin and lignanoid etc., mainly has the multiple biological activitys such as cell toxicant, antisepsis and anti-inflammation, immunomodulatory.
Summary of the invention
The object of this invention is to provide a kind of one obtained that is separated from Root or herb of Clethra Loosestrife to cast off a skin steroidal compounds, containing its pharmaceutical composition and its preparation method and application.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula:
Described pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The preparation method of described compound (I), it is characterized in that, comprise following operation steps: pulverize after (a) Root or herb of Clethra Loosestrife dries in the shade, extract with 70% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B acetic acid ethyl ester extract in step (a) D101 macroporous resin is removed sugar by (), use 20% ethanol and 50% ethanol elution successively, collect 50% elutriant, concentrating under reduced pressure; C 50% ethanol elution enriched material purification on normal-phase silica gel in step (b) is separated by (), obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 45:1,25:1 and 10:1; D component 3 in step (c) is separated by purification on normal-phase silica gel by () further, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,10:1 and 5:1; E the reverse phase silica gel of component 2 in step (d) with octadecylsilane bonding is separated by (), be the methanol aqueous solution isocratic elution of 65% by concentration expressed in percentage by volume, collect cut according to thin layer plate, concentrating under reduced pressure obtains pure compound (I).
The application of described compound (I) in the medicine of preparation treatment ovarian cancer.
The application of described composition in the medicine of preparation treatment ovarian cancer.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.This pharmaceutical composition contains the compounds of this invention I for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
figure of description
Fig. 1 is compound (I) structural formula;
Fig. 2 is that compound (I) is to the restraining effect of HEY cell proliferation;
Fig. 3 is that compound (I) is to the restraining effect of SKVO3 cell proliferation;
Fig. 4 is the restraining effect of cis-platinum to HEY cell proliferation;
Fig. 5 is the restraining effect of cis-platinum to SKVO3 cell proliferation;
Fig. 6 is that compound (I) is on the impact of FTE-187 cell proliferation;
Fig. 7 is the impact of cis-platinum on FTE-187 cell proliferation.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
1. main agents: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
2. material and instrument
People's Epithelium Cells system FTE-187, (M199 substratum+10%FBS; Abortion syndrome HEK, DMEM substratum+10%FBS; Abortion syndrome SKOV-3, McCoy's5A substratum+10%FBS; M199 substratum, DMEM substratum, McCoy's5A substratum, cis-platinum, foetal calf serum are all purchased from Gibco company of the U.S.; Chemical compounds I, purity >99%, MTT are purchased from Sigma company; Tissue Culture Dish and 96 orifice plates are purchased from CorningCostar company; Trypsin Trypsin) be purchased from Shanghai biotech company; Dimethyl sulfoxide (DMSO) (DMSO) chromatographically pure, is purchased from Shanghai bio-engineering corporation.
Electronic balance, Sartorius company of the U.S.; Ultralow Temperature Freezer, NewBrunswickScientific company of the U.S.; Normal temperature table model high speed centrifuge and freezing table model high speed centrifuge, Thermo company of the U.S.; Pipettor, German Eppendorf company; Inverted phase contrast microscope, Japanese Olymlpus company; CO
2cell culture incubator, German Heraeus company; The multi-functional microplate reader of Wallac1420, PerkinElmer company of the U.S.; DK-S24 type electric-heated thermostatic water bath, the limited public affairs of Shanghai gloomy letter laboratory apparatus.
Embodiment 1
(9kg) is pulverized a () Root or herb of Clethra Loosestrife dries in the shade after, extract with 70% alcohol heat reflux, 5L × 3 time, each 2 hours, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil (2L × 3 time), ethyl acetate (2L × 3 time) and water saturated propyl carbinol (2L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (205g) and n-butyl alcohol extract respectively; B the acetic acid ethyl ester extract in () step (a) except sugar with D101 macroporous resin, is used 20% ethanol and 50% ethanol elution successively, is collected 50% elutriant, concentrating under reduced pressure (165g); C 50% ethanol elution enriched material purification on normal-phase silica gel in () step (b) is separated, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 45:1,25:1 and 10:1; Component 3(34g in (d) step (c)) be separated further by purification on normal-phase silica gel, obtain 3 components with the methylene chloride-methanol gradient elution that volume ratio is 15:1,10:1 and 5:1 successively; Component 2(22g in (e) step (d)) be separated with the reverse phase silica gel of octadecylsilane bonding, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 65%, collect cut according to thin layer plate, concentrating under reduced pressure obtains pure compound (I) (18mg).
Structural identification: white amorphous powder, is soluble in chloroform, acetone and methyl alcohol, is insoluble in water; HR-ESIMS shows [M+Na]
+for m/z383.1844, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
21h
28o
5.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, CDCl
3, 600MHz): 1.60(1H, dd, 1-Ha), 1.35(1H, dd, 1-Hb), 3.54(1H, m, 2-H), 3.54(1H, m, 3-H), 1.78(1H, m, 4-Ha), 1.53(1H, m, 4-Hb), 2.10(1H, dd, 5-H), 6.01(1H, s, 7-H), 2.56(1H, d, 9-H), 5.43(1H, dd, 11-H), 5.37(1H, d, 12-H), 1.78(1H, m, 15-Ha), 1.53(1H, m, 15-Hb), 1.79(1H, m, 16-Ha), 1.54(1H, m, 16-Hb), 2.30(1H, dd, 17-H), 1.01(3H, s, 18-H), 0.94(3H, s, 19-H), 1.98(3H, s, 21-H), 5.40(1H, s, 2-OH), 5.40(1H, s, 3-OH), 4.77(1H, s, 14-OH), carbon-13 nmr spectra data δ
c(ppm, CDCl
3, 150MHz): 42.0(CH
2, 1-C), 68.2(CH, 2-C), 68.0(CH, 3-C) and, 32.4(CH
2, 4-C), 51.8(CH, 5-C), 203.5(C, 6-C) and, 124.0(CH, 7-C), 162.6(C, 8-C), 36.1(CH, 9-C), 21.5(C, 10-C) and, 131.7(CH, 11-C), 136.0(CH, 12-C), 41.0(C, 13-C), 91.5(C, 14-C) and, 33.4(CH
2, 15-C), 17.4(CH
2, 16-C), 52.7(CH, 17-C), 16.3(CH
3, 18-C), 14.1(CH
3, 19-C), 209.5(C, 20-C), 31.3(CH
3, 21-C), carbon atom mark is see accompanying drawing 1.
Embodiment 2
One, test method
1, cell cultures
1.1 cell recovery
From-80 DEG C of refrigerators or liquid nitrogen, take out fast the cell that will recover, cryopreservation tube dress being deposited cell moves to rapidly oneself the water-bath Cao through in advance temperature being adjusted to 40 DEG C, rocks fast, makes it dissolve as early as possible.Within about 1 minute, find to determine CL, the liquid rotating containing cell is moved to ready cultivation in advance and get over Cao, the centrifugal 4min of whizzer 800rpm.After removing Cheongju, by the resuspended precipitation of 1mL substratum, be transferred in Tissue Culture Dish after mixing and cultivate.Change fresh culture after 24h to remove the cell that the death in base is supported in old mill, continue to cultivate and observation of cell growing state.
1.2 passage
In advance substratum, PBS, pancreatin are taken out from refrigerator and make its temperature rising room temperature.From ` constant temperature temperature incubator, take out the cell needing Secondary Culture, general cell density reaches about 80% and goes down to posterity.Former substratum is removed, uses PBS to rinse to add appropriate pancreatin after twice and digest, when finding that cell comes off from the bottom of ware, add appropriate cultivate carry out Cao and, move in glass centrifuge tube.After the centrifugal 4min of whizzer 800rpm, remove and disappear and use fresh culture re-suspended cell to precipitate, go down to posterity according to research purpose and in corresponding Tissue Culture Dish, continue constant incubator Cao cultivate.
1.3 cell counting
Cell dissociation step is the same, centrifugal abandon supernatant after to add suitable volumes nutrient solution resuspended, get l0 μ L cell suspension with pipettor, slowly squeeze into from tally edge, make suspension be full of space between tally and cover plate completely, avoid producing bubble.(10 ×) counting cells under just putting microscope, cell density calculation formula is as follows: cell suspension cell count/mL=(4 large lattice cell count summation/4) × 10000.
2, cell agent-feeding treatment
(1) get the cell being in logarithmic phase and carry out cell counting, by proper density paving bottle; After (2) second days cell attachments, outwelled by original substratum, add the fresh culture of fixed volume, dosing simultaneously processes, and establishes solvent control simultaneously; (3) chemical compounds I adding consistency gradient: during MTT experiment, the normal Epithelium Cells of people and ovarian cancer cell drug treating concentration are 1 μM, 10 μMs, 20 μMs, 50 μMs and 100 μMs; Solvent control is DMSO process.During the flow cytomery cell cycle, Proliferation of Human Ovarian Cell drug treating concentration is solvent control is 10 μMs.During detection apoptosis, Proliferation of Human Ovarian Cell drug treating concentration is 10 μMs, and solvent control group is DMSO process.During WesternBlot, abortion syndrome drug treating concentration is 10 μMs, and solvent control group is DMSO process; (4) cis-platinum working concentration: during MTT experiment, the normal Epithelium Cells of people and ovarian cancer cell drug treating concentration are 0.1 μM, 0.5 μM, 2 μMs, 5 μMs and 10 μMs; Solvent control is PBS process.During the flow cytomery cell cycle, Proliferation of Human Ovarian Cell drug treating concentration is l μM, and solvent control group is DMSO process; Detecting Proliferation of Human Ovarian Cell drug treating concentration when tune is died is l μM, and solvent control group is PBS process.During WesternBlot, Proliferation of Human Ovarian Cell drug treating concentration is 1 μM, and solvent control group is PBS process.
3, tetrazolium salts (MTT) colorimetry
MTT colorimetry is a kind of method of conventional detection cell survival and growth.Principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-fast bluish voilet knot product first a ceremonial jade-ladle, used in libation and be deposited in cell, and dead cell can not be like this.First a ceremonial jade-ladle, used in libation in DMSO energy dissolved cell, measures its light absorption value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Within the scope of certain cell count, the amount that MTT ties product formation is directly proportional to viable count.Detailed process: (1) observation of cell, when cell is in logarithmic phase, obtains inferior cell suspension by the method for trysinization, cell counting count board counting under microscope.Cell concn is adjusted to 2-5 × 10
4individual/mL, the 96 every hole of orifice plate about 2000-5000 cell.(2) set loading hole in advance, each hole adds 100 μ L cell suspensions, and 96 orifice plates are placed in CO
2constant temperature 37 DEG C cultivation in incubator; (3), after overnight incubation, carry out changing liquid.Carefully removed by former substratum in every hole, add the fresh culture containing different pharmaceutical concentration prepared in advance, each drug level group arranges 4 multiple holes.96 orifice plates are positioned over CO
2constant temperature 37 DEG C cultivation is continued in incubator.(4) after 24h, 48h, take out Tissue Culture Plate respectively, each hole adds MTT(5mg/ml) 20 μ L, continue to be positioned in incubator and cultivate 4 hours; (5) tilted by 96 orifice plates and carefully remove Shang Cheongju, each hole adds the DMSO of 150 μ L, and room temperature places 10min or low speed shaking table placement 5min makes precipitation fully dissolve.Operation microplate reader is measured light absorption value in 490nm place and is calculated cell survival rate.
4, statistical analysis
Data statistic analysis uses SPSS13.0 software to carry out two tail independent t test, and all data are expressed as mean+/-standard error; P<0.05 thinks that significant difference, P<0.01 think that difference is extremely remarkable.
Two, interpretation of result
1, chemical compounds I has obvious restraining effect to Proliferation of Human Ovarian Cell HEY, SKOV3
Tetrazolium salt colorimetric assay (MTT) analysis shows, the growth of chemical compounds I to human ovarian cancer HEY cell has obvious restraining effect, and this effect is time-dependent manner and dose-dependently (Fig. 2).The chemical compounds I of same concentration, along with the prolongation gradually (24h, 48h) of administration time, increases gradually to the restraining effect of HEY cell proliferation.When 48h, the restraining effect of chemical compounds I peaks, and the difference between each group has statistical significance (P<0.05).The chemical compounds I of different concns, at one time, along with the increase gradually of drug level, it also increases gradually to the restraining effect of the propagation of HEY cell.Difference between each group of different concns has statistical significance (P<0.05).After with 1 μM, 10 μMs, 20 μMs, 50 μMs, 100 μMs chemical compounds I process HEY cell 24h, the survival rate of HEY cell is about 87%, 61%.57%, 42%, 34% respectively.But the concentration and time effect of chemical compounds I to SKOV3 there is no evident regularity (Fig. 3).After with 1 μM, 10 μMs, 20 μMs, 50 μMs, 100 μMs chemical compounds I treatment S KOV3 cell 24h, the survival rate of SKOV3 cell is about 65%, 55%, 54%, 51%, 48% respectively.Same concentration chemical compounds I process different time, its inhibition also not presentative time dependency.
2, the growth of cisplatin on human ovarian cancer HEY, SKOV3 cell has the restraining effect weak compared with chemical compounds I
After conventional clinically chemotherapeutic drugs Cisplatin handler ovarian cancer cell HEY, SKOV3, through MTT detect find, in HEY cell be 24h also sufficient 48h cis-platinum to the restraining effect of cell be not especially obviously and do not demonstrate time and concentration dependent (Fig. 4).Contrast with the situation after chemical compounds I process in Fig. 2 and find, in HEY cell, the chemical compounds I of low dosage can produce obvious restraining effect.Even and if the cis-platinum of high dosage does not reach that effect of chemical compounds I yet as shown in Figure 4, we show that chemical compounds I suppresses the effect of HEY Growth of Cells stronger than cis-platinum thus.
In SKOV3 cell, by Fig. 5, we find out to have concentration dependent when 48h to cis-platinum, and along with the rising of concentration, restraining effect is also stronger.Contrast with Fig. 4 and find that the cis-platinum of same concentrations is stronger to the restraining effect of SKOV3 cell, this illustrates for these two kinds of cells of HEY, SKOV3, and SKOV3 is for cis-platinum rdativery sensitive, and HEY relative tolerance.Chemical compounds I, cis-platinum be treatment S KOV3(Fig. 3, Fig. 5 respectively) after, we can find out that the restraining effect of chemical compounds I is stronger, and just can reach the effect of cis-platinum high dosage when low dosage, effectively reduce the toxic side effect of medicine.
3, chemical compounds I to the toxic action of Epithelium Cells lower than the toxic action of cis-platinum to it
In Epithelium Cells FTE-187,20 μMs of chemical compounds I process are after 48 hours, still have an appointment 50% cell survival (Fig. 6), and 5 μMs of cisplatin treated are after 48 hours, only have the cell survival (Fig. 7) of about 20%.We select normal Epithelium Cells FTE-187 to carry out testing is exactly toxic side effect in order to understand the medicine normal tissue when medication, found through experiments chemical compounds I less than the toxic action of cis-platinum, and with tumour cell to its reacting phase than chemical compounds I to Normocellular toxic side effect very little (Fig. 2, Fig. 3, Fig. 6).After 20 μMs of chemical compounds I process FTE-18748h, the survival rate of cell is about 60%, and after 20 μMs of chemical compounds I process HEY, SKOV348h, survival rate is only about 40%.
Chemical compounds I has obvious restraining effect to ovarian cancer cell HEY.Although it also has certain restraining effect to normal Epithelium Cells, this restraining effect is lower than the restraining effect to tumour cell.The toxic action of chemical compounds I to normal Epithelium Cells is less than the toxic action of cis-platinum to it.Low toxicity, efficiently antitumor drug, have unusual meaning to the treatment of tumour.
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:9 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, the salt made, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filters with aseptic suction funnel, more aseptic essence filter, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
Claims (1)
1. cast off a skin steroidal compounds (I's) preparation method, the structural formula of compound (I) is as follows,
It is characterized in that, comprise the steps: that (a) Root or herb of Clethra Loosestrife is pulverized after drying in the shade, extract with 70% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B acetic acid ethyl ester extract in step (a) D101 macroporous resin is removed sugar by (), use 20% ethanol and 50% ethanol elution successively, collect 50% elutriant, concentrating under reduced pressure; C 50% ethanol elution enriched material purification on normal-phase silica gel in step (b) is separated by (), obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 45:1,25:1 and 10:1; D component 3 in step (c) is separated by purification on normal-phase silica gel by () further, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,10:1 and 5:1; E the reverse phase silica gel of component 2 in step (d) with octadecylsilane bonding is separated by (), with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 65%, collect cut according to thin layer plate, concentrating under reduced pressure obtains pure compound (I).
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