CN104961791B - A kind of new triterpenoid, preparation method and purposes in Radix potentillae anserinae - Google Patents
A kind of new triterpenoid, preparation method and purposes in Radix potentillae anserinae Download PDFInfo
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Abstract
The invention discloses a kind of new triterpenoid, preparation method and purposes in Radix potentillae anserinae, belong to technical field of pharmaceuticals.This compound is reported first, is a kind of novel Cycloartane type triterpene compound of structure, extracting and developing purification can obtain from Radix potentillae anserinae rhizome.In vitro tests proves that this compound can suppress the propagation of melanoma cell, can be used to develop into the medicine for the treatment of melanoma.
Description
Technical field
The invention belongs to technical field of pharmaceuticals is and in particular to separate a kind of cycloartane triterpenoids obtaining from Radix potentillae anserinae
Compound, containing its pharmaceutical composition and its preparation method and application.
Background technology
Medicine Radix potentillae anserinae is Rosaceae potentilla plants potentilla anseriana(Potentilla anserineL.)Tuber,
There is promoting the production of body fluid to quench thirst, invigorating spleen and reinforcing stomach, vigorate qi and replenish the blood, astringing to arrest bleeding, antidiarrheal, cough-relieving, sharp expectorant, cure mainly haematemesis, hematochezia, collapse
In, the disease such as malaria ulcerative carbuncle, insufficiency of the spleen diarrhoea, dysentery.18 kinds of aminoacid of Radix potentillae anserinae rich in starch, fatty acid and needed by human body and many
Plant vitamin, there is higher medical treatment and nutritive value.《Dictionary of medicinal plant》、《Chinese herbal medicine is commonly used in Tibet》Deng book, this medicine is all had
Record.This product in Qinghai Province's wild resource very abundant, for a long time, particularly apply more in Tibetan medicine by Medicinal.
Triterpenoid compound is to remove, by several isoprene, the material constituting that joins end to end after hydroxyl.Major part is 30
Carbon atom, small part contains the terpenoid of 27 carbon atoms.Triterpenes components are very wide in distributed in nature, shark oil, Radix Glycyrrhizae,
There is triterpene substance in the effective ingredient of Fructus Schisandrae Chinensis.Triterpenoid compound functional group and different side chains according to contained by it, also
It is divided into different basic frameworks.
Obtain triterpenoid in three pentacyclic triterpenes except Hong Xia etc. separates from Radix potentillae anserinae rhizome(Hong Xia etc., Chinese herbal medicine, the
Volume 37 the 2nd phase, page 165 ~ page 168), there is not yet in Radix potentillae anserinae other triterpenoids report.
Content of the invention
It is an object of the invention to provide a kind of separate, from Radix potentillae anserinae, a kind of Cycloartane type triterpene compound obtaining, containing it
Pharmaceutical composition and its preparation method and application.
The above-mentioned purpose of the present invention is achieved by techniques below scheme:
There is the triterpenoid I of following structural formula:
The preparation method of described compounds I, comprises following operating procedure:(a)Radix potentillae anserinae rhizome is pulverized, with 75% ethanol heat
Reflux, extract, united extraction liquid, it is concentrated into no alcohol taste, use petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, point
Do not obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;(b)Step(a)Middle acetic acid ethyl ester extract is used
Macroporous resin remove impurity, successively with 15% ethanol and 70% ethanol elution, collects 70% eluent, it is dense that concentrating under reduced pressure obtains 70% ethanol elution
Contracting thing;(c)Step(b)In 70% ethanol elution concentrate with purification on normal-phase silica gel separate, successively use volume ratio be 40:1、20:1、10:1
With 5:1 methylene chloride-methanol gradient elution obtains 4 components;(d)Step(c)Middle component 4 is divided further with purification on normal-phase silica gel
From, successively use volume ratio be 8:1、5:1 and 2:1 methylene chloride-methanol gradient elution obtains 3 components;(e)Step(d)In
The reverse phase silica gel that component 2 octadecylsilane is bonded separates, and the methanol aqueous solution being 65% with concentration expressed in percentage by volume is isocratic to be washed
De-, collect 7-9 column volume eluent, eluent is concentrated under reduced pressure to give pure compounds I.
Further, described macroporous resin is D101 type macroporous adsorbent resin.
Pharmaceutical composition, wherein containing the compounds I described in therapeutically effective amount and pharmaceutically acceptable carrier.
Application in the medicine of preparation treatment melanoma for the described compounds I.
Application in the medicine of preparation treatment melanoma for the described pharmaceutical composition.
When the compounds of this invention is used as medicine, can directly use, or be used in the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention I of therapeutically effective amount, and remaining is pharmaceutically acceptable, to people
Nontoxic with animal and inert pharmaceutical acceptable carrier and/or excipient.
Described pharmaceutical acceptable carrier or excipient are to be selected from solid, semi-solid and liquid diluent, filler for one or more
And pharmaceutical preparation adjuvant.The pharmaceutical composition of the present invention is used in the form of per weight dose.Medicine of the present invention can
Patient in need for the treatment of is applied to by form that is oral or injecting.During for being administered orally, tablet, slow releasing tablet, control can be made into
Release piece, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc.;During for injecting, can be made into sterilizing
Aqueouss or oily solution, aseptic powder injection, liposome or Emulsion etc..
Figure of description
Fig. 1 is compounds I structural formula;
Fig. 2 is compounds I two dimension hsqc spectrum;
Fig. 3 is compounds I two dimension1H-1H COSY composes;
Fig. 4 is compounds I two dimension HMBC spectrum;
Fig. 5 is compounds I two dimension NOESY spectrum;
Fig. 6 calculates ECD and experiment ECD figure for compounds I.
Specific embodiment
Further illustrate the essentiality content of the present invention with reference to embodiment, but present invention protection model is not limited with this
Enclose.Although being explained in detail to the present invention with reference to preferred embodiment, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
Main material, reagent source and instrument type:
Ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are that analysis is pure, limited purchased from Shanghai Ling Feng chemical reagent
Company, methanol, analysis is pure, purchased from Jiangsu Han Bang chemical reagent company limited.
People's A375 melanoma cell strain is given by Third Military Medical University.Compounds I, purity 99%, for self-control.DMEM trains
Foster base, 0.25% pancreatin are purchased from Gibco company of the U.S.;MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromine
Salt;Methyl thiazoly tetrazolium assay, DMSO(Dimethyl sulfoxide), BrdU be purchased from Sigma Co., USA;Mus anti-BrdU polyclonal antibody is purchased
From American AB cam company;Mountain sheep anti mouse two resists purchased from Cell signaling company of the U.S.;Annexin V-FITC/PI cell
Apoptosis detection kit is purchased from Promega company of the U.S..
Constant temperature CO2Incubator, general refrigerator, -80 DEG C of refrigerators are Forma company of the U.S.;Superclean bench is purchased from China
Suzhou cleaning project company;Inverted microscope, inverted fluorescence microscope is purchased from olympus company;Electro-heating standing-temperature cultivator is purchased from
Chinese Shanghai leap medical apparatus and instruments factory;Automatically microplate reader is purchased from Japanese Wako company;UV detector is purchased from the U.S.
Beckman company;J6-HC high speed centrifuge is purchased from Beckman company of the U.S.;Low-temperature trace centrifuge is purchased from Germany
Eppendorf company;Vibration shaking table is purchased from Forma company of the U.S..
Embodiment 1:Compounds I separates preparation and structural identification
(a)Radix potentillae anserinae rhizome(10kg)Pulverize, extracted with 75% alcohol heat reflux(30L × 3 time), united extraction liquid, it is concentrated into
No alcohol taste(6L), use petroleum ether successively(6L × 3 time), ethyl acetate(6L × 3 time)With water saturated n-butyl alcohol(6L × 3 time)Extraction
Take, respectively obtain petroleum ether extract, acetic acid ethyl ester extract(375g)And n-butyl alcohol extract;(b)Step(a)Middle acetic acid second
The D101 macroporous resin remove impurity of ester extract, uses 15% ethanol successively(8L)With 70% ethanol(12L)Eluting, collects 70% eluent,
Concentrating under reduced pressure obtains 70% ethanol elution concentrate(125g);(c)Step(b)In 70% ethanol elution concentrate purification on normal-phase silica gel divide
From, successively use volume ratio be 40:1、20:1、10:1 and 5:1 methylene chloride-methanol gradient elution obtains 4 components;(d)Step
Suddenly(c)Middle component 4(25g)Separated further with purification on normal-phase silica gel, use volume ratio to be 8 successively:1、5:1 and 2:1 dichloromethane-first
Alcohol gradient elution obtains 3 components;(e)Step(d)Middle component 2(12g)Separated with the reverse phase silica gel of octadecylsilane bonding,
The methanol aqueous solution isocratic elution being 65% with concentration expressed in percentage by volume, collects 7-9 column volume eluent, eluent concentrating under reduced pressure
Obtain pure compounds I(35mg).
Structural identification:Faint yellow acicular crystal, is soluble in chloroform, ethyl acetate, acetone and methanol;HR-ESIMS shows [M
+Na]+For m/z 489.6542, can obtain molecular formula in conjunction with nuclear-magnetism feature is C30H42O4, degree of unsaturation is 10.Infrared IR is shown in
3442,1732 and 1715cm-1There is absorption, point out there is hydroxyl and carbonyl.Hydrogen nuclear magnetic resonance modal data δH(ppm, Pyridine-d 5 , 400MHz):H-1(5.55, br, s), H-2(2.53, m), H-2(2.26, m), H-3(3.78, dd,J=6.2,10.1),
H-5(2.36, m), H-6(2.64, m), H-6(2.38, m), H-7(5.46, t,J=6.4), H-11(5.38, br, d,J=5.2),
H-12(2.31, br, d,J=17.4), H-12(2.16, dd,J=17.4,6.0), H-15(2.41, d,J=17.8), H-15
(2.10, d,J=17.8), H-17(2.39, d,J=9.0), H-18(0.76, s), H-19(3.22, d,J=13.5), H-19
(3.13, d,J=13.5), H-20(2.73, m), H-21(1.10, d,J=6.6), H-22(4.09, br, d,J=17.1), H-24
(4.41, s), H-26(1.62, s), H-27(1.58, s), H-28(1.03, s), H-29(1.30, s), H-30(0.95, s);Core
Magnetic resonance carbon modal data δC(ppm, Pyridine-d 5 , 100MHz):120.7(CH, 1-C), 33.2(CH2, 2-C), 73.6
(CH, 3-C), 39.0(C, 4-C), 51.1(CH, 5-C), 25.3(CH2, 6-C), 125.6(CH, 7-C), 142.5(C, 8-C),
137.9(C, 9-C), 139.0(C, 10-C), 121.1(CH, 11-C), 36.6(CH2, 12-C), 43.4(C, 13-C), 45.8(C,
14-C), 47.9(CH2, 15-C), 218.3(C, 16-C), 60.0(CH, 17-C), 16.6(CH3, 18-C), 43.8(CH2, 19-
C), 27.4(CH, 20-C), 20.4(CH3, 21-C), 47.4(CH2, 22-C), 214.3(C, 23-C), 84.4(CH, 24-C),
72.4(C, 25-C), 27.5(CH3, 26-C), 26.3(CH3, 27-C), 25.1(CH3, 28-C), 25.1(CH3, 29-C), 14.6
(CH3, 30-C);Carbon atom labelling is referring to Fig. 1.Composed by DEPT, have in 30 carbon 7 methyl, 6 methylene, 8 secondary
Methyl and 9 quaternary carbons.In hydrogen spectrum, typical δ H 0.76,1.62,1.58,1.03,1.30,0.95 6 is connected on quaternary carbon
Methyl signals and δ H 1.10(D, J=6.6)The methyl signals being connected on tertiary carbon to point out this compound may be 9,19- ring A Er
Court of a feudal ruler type triterpenoid(cyclolanostane triterpenoid).Alkene hydrogen δ H 5.38 in hydrogen spectrum (1H, d, J=5.2
Hz, H-11), 5.46 (1H, t, J=6.4 Hz, H-7) and 5.55 (1H, s, H-1) correspond to three pairs of alkene in carbon spectrum
Carbon δ C, i.e. 120.7 (C-1) and 139.0 (C-10), 125.6 (C-7) and 142.5 (C- 8), 121.1 (C-11) and
137.9 (C-9).Two carbonyl carbon δ C 218.3 (C-16), 214.3 (C-23) and three company oxygen carbon δ C 73.6 in carbon spectrum
(C-3), 84.4 (C- 24), 72.4 (C-25) point out this compound is highly oxidized Fourth Ring 9,10-seco-9,19-
Cyclolanostane triterpene.The planar structure of this compound can pass through HSQC(Fig. 2)、1H-1H COSY(Fig. 3)And HMBC
(Fig. 4)Two-dimensional spectrum determines.In HMBC spectrum, H3- 29/30 related to C-3, C-4, C-5 it may be determined that hydroxyl be located at C-3 on;H-1 with
C-2, C-3, C-5 are related, and H-7 is related to C-5, C-6, C-9, C-14, and H-11 is related to C-8, C-13, H2- 19 with C-1, C-8,
C-9, C-10, C-11 are related, comprehensive1H-1H-1/H in H COSY spectrum2-2/H-3、H2-6/H-7、H-11/H2- 12 correlations can determine that
Three double bonds(C-1 and C-10, C-7 and C-8, C-9 and C-11)Position.H in being composed by HMBC2- 15 is related to C-16, H2-
22/H-24 is related to C-23 to can determine that two carbonyls(C-16 and C-23)Position.Relative configuration can by coupling constant and
NOESY spectrum determines(Fig. 5).From H-3(δ H 3.78, dd,J= 10.1, 6.2Hz)Coupling constant understands that C-3 is beta comfiguration.NOESY
In spectrum, H-3/H3- 29 related and H-3/H-5 correlations are it can be inferred that H-3 is α configuration;Finally, by ECD(Fig. 6)Determine this chemical combination
The absolute configuration of thing is 3S, 5R, 13R, 14R, 17R, 20R, 24S, and experiment value is basically identical with theoretical value.Therefore, may be used
Determine that this compound structure is as shown in Figure 1.
Embodiment 2:Compounds I pharmacological action is tested
First, test method
1st, cell culture
1.1 cell recovery
Frozen A375 melanoma cell in liquid nitrogen container is taken out, puts into rapidly in 37 DEG C of warm water, be shaken gently for
It is made to melt as early as possible(About lmin).Then suction out cell suspension, be added in the centrifuge tube added with 2mL culture medium, gently
Piping and druming mixes, 800r/min, is centrifuged 5min, discards upper strata culture medium.With the penicillium sp G/ streptomycin containing 10%FBS and 1%
After DMEM culture medium 8mL makes cell suspension, it is seeded in 10cm culture plate.It is subsequently placed in 5% CO2, 37 DEG C of constant incubators
In cultivated.
1.2 passage
Basis of microscopic observation cell fusion degree reaches and can be passed on during 80%-90%.First use 2mLPBS washed cell 2 times,
It is subsequently adding 0.25% pancreatin lmL.Gently horizontal wave and culture disk, makes digestion liquid energy cover the surface of cell, is subsequently placed in 37
About lmin is digested in DEG C incubator.It is placed under microscope and sees that cell rounding bounces back, be then rapidly added the culture medium that 1mL contains FBS
Terminate digestion.Gently blow and beat and be uniformly dispersed to cell, then according to cell density according to 1:2 or 1:3 pass on, and are re-seeded into new
Culture plate, in 5%CO2, cultivate in 37 DEG C of constant incubators.
1.3 cell cryopreservation
1 disk is taken to be in the cell of exponential phase, pancreatin digests and collects in centrifuge tube, 1000r/min is centrifuged 5min,
Abandon supernatant.It is subsequently adding lmL frozen stock solution, gently dispels cell and make cell suspension, cell suspension is added to the frozen of 1.5mL
Guan Zhong.In the title of cryopreservation tube subscript clear-cellss, holding time, the name of preserver.First cryopreservation tube is put into 4 DEG C of refrigerators, put
Put about 30min.Then it is placed in -20 DEG C of refrigerators, place about 2h.Then it is put in -80 DEG C of ultra cold storage freezers and stand overnight.Second day
Frozen cell is placed in liquid nitrogen container and preserves for a long time.
2nd, cell counting draws cell growth curve
(1)Take the logarithm trophophase A375 melanoma cell, count, adjustment cell density is 2 × 104/mL.
(2)By cell suspension inoculation in 12 orifice plates, 1.5mL/ hole.
(3)After 12h, treat cell attachment, change 1mL serum-free DMEM culture medium, and respectively with 5 μm of ol/L compounds Is and
DMSO (for DMSO dilution compounds I control DMSO concentration below 1/1000 it is ensured that harmless to cell) process, every group of cell
If 3 parallel holes, it is placed in 5%CO2, cultivated in 37 DEG C of constant incubators.
(4)Terminate culture respectively at 0h, 24h, 48h, 72h, 96h, collect each group cell, add 0.25% trypsin to disappear
Change, centrifugation, resuspended.
(5)Cell counting:Draw cell suspension 10 μ l, add isopyknic phenol blue area to divide living cells, draw 10 μ L
Mixed liquor gently adds in cell counting count board groove it is ensured that tight and bubble.Under inverted microscope, count four, periphery generous
Cell number in lattice, substitutes into formula:Archeocyte suspension concentration (cell number/mL)=(cell number/4 of 4 block plaid) × 104
× extension rate.
(6)Calculate number of viable cells, draw cell growth curve.
3rd, MTT method detection cell proliferation
(1)Take the logarithm the A375 cell of trophophase, digestion, centrifugation, resuspended, count, cell in adjustment cell suspension
Density is 2 × 104/mL.
(2)By each group cell suspension inoculation in 96 well culture plates, the every hole of 200 μ L, 3 parallel holes of every group of cell, with
When set blank control wells(Only add culture medium).
(3)12h, after cell attachment, changes 200 μ L serum-free DMEM culture medium, and respectively with 5 μm of ol/L chemical combination
Thing I and DMSO process.
(4)Terminate culture respectively at 0,1,2,3,4 days.
(5)Every hole adds the Methyl thiazoly tetrazolium assay 20 μ L that concentration is 5mg/mL to continue cultured cells 4h.
(6)Inhale the culture medium abandoning each little in the hole, add 200 μ LDMSO, microoscillator vibrates 10min, makes crystallization
Thing fully dissolves.
(7)With blank control wells zeroing, the absorption photometric value at each hole 570 nm is measured using automatic microplate reader(OD
Value), ability of cell proliferation is represented with corresponding OD value, each group takes the meansigma methodss of 3 parallel holes, with the time as transverse axis, with each
Absorbance draws cell growth curve for the longitudinal axis.
4th, BrdU Immunofluorescence test cell proliferation
(1)Prepare creep plate:Creep plate is put into immersion 24h in 75% ethanol disinfect.
(2)Aseptic creep plate is placed in 24 well culture plates, the A375 melanoma cell of trophophase of taking the logarithm, digestion, from
The heart, makes cell suspension, 2 × 104cell/ hole, is inoculated in the hole being placed with creep plate.
(3)12h, after cell attachment, changes 1mL plasma-free DMEM medium, and respectively with 5 μm of ol/L compounds Is and DMSO
Process.
(4)After 72h, add 10 μ lBrdU (1mg/mL), 37 DEG C of culture 1h in the medium.
(5)Take out 24 orifice plates, cold PBS washes 5min × 2 time, 4% paraformaldehyde fixes cell, room temperature 30min.
(6)PBS washes 5min × 3 time.
(7)2mol/L HCl process, after room temperature 10min, 37 DEG C of 20min.
(8)The penetrating process in cell 1%Triton × -100, washes 5min × 3 time.
(9)10% lowlenthal serum closing, room temperature, lh.
(10)Add BrdU monoclonal antibody (1:300 dilutions), 37 DEG C of 1.5h.
(11)L × PBST shakes and washes 5min × 3 time.
(12)Plus BrdU bis- resists (1:300), after room temperature lucifuge 1h, l × PBST washes 3 times.
(13)DAPI dyeing liquor contaminates nucleus 15min.
(14)PBS washes 5min × 3 time.
(15)Plus 1 anti-fluorescent quenching mounting liquid, neutral gum mounting, fluorescence microscope, photograph.
5th, statistical analysiss
Application SPSS 17.0 statistical analysis software, is carried out using two independent sample t inspections and one factor analysis of variance
Data analysiss, data is represented with X ± S, P<0.05 is statistically significant.
2nd, data analysiss
Cellular morphology observe and count display, compared with DMSO matched group, compounds I process A375 cell 24h, 48h,
After 72h, viable count significantly reduces and reaches 44.5%.The results are shown in Table 1(*P <0.05, ** P <0.01 versus DMSO
group).
MTT result shows, compounds I can significantly inhibit A375 cell proliferation, and OD value is in concentration(5μmol/L、10μ
mol/L、20μmol/L)Dependency decline reaches 27.3%, and difference is statistically significant.The results are shown in Table 2(*P <0.05, ** P <
0.01 versus DMSO group).
Being further characterized by compounds I by BrdU immunofluorescence dyeing can suppress melanoma cell to breed, 5 μm of ol/L
Compounds I processes A375 cell after 3 days, and compares(26.07±2.59%)Compare, experimental group BrdU positive cell percentage
(7.55±2.76%)Significantly reduce(P=0.000).
This experiment have detected the impact that compounds I is bred to melanoma cell, and cell counting and MTT analysis result are equal
Prove that compounds I can significantly inhibit melanoma cell growth, and cell number is in that dependency declines with compounds I concentration.BrdU
After immunofluorescence dyeing displays that compounds I process, BrdU positive cell percentage is substantially less than matched group, illustrates that compounds I can
Suppression A375 melanoma cell propagation.Compounds I is probably the efficient targeting medicine of melanoma.
The suppression to A375 melanoma cell for the table 1 determination of cell count compounds I(×l04/ml)
Table 2 mtt assay measures the suppression to A375 melanoma cell vigor for the compounds I
Embodiment 3
The preparation of tablet:First compounds I is obtained by embodiment 1 method, and utilizes organic acid such as tartaric acid or citric acid
Or the salt that formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by its with excipient weight than for 1:7 ratio
Example adds excipient, pelletizing press sheet.
Embodiment 4
The preparation of oral liquid:First compounds I is obtained by embodiment 1 method, and utilizes organic acid such as tartaric acid or Fructus Citri Limoniae
The salt that acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, routinely oral liquid preparation method make oral
Liquid.
Embodiment 5
Capsule or the preparation of granule:First compounds I is obtained by embodiment 1 method, and utilizes organic acid such as winestone
The salt that acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by itself and excipient weight
Than for 1:9 ratio adds excipient, makes capsule or granule.
Embodiment 6
The preparation of injection:First compounds I is obtained by embodiment 1 method, and utilizes organic acid such as tartaric acid or Fructus Citri Limoniae
The salt that acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, routinely plus water for injection, fine straining, fills
Injection is made in envelope sterilizing.
Embodiment 7
The preparation of aseptic powder injection:First compounds I is obtained by embodiment 1 method, and utilizes organic acid such as tartaric acid or lemon
The salt that lemon acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, is dissolved in sterile water for injection,
Stirring makes molten, is filtered with aseptic suction funnel, more aseptic fine straining, is sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains
Injectable powder.
Claims (2)
1. there is the triterpenoid of following structural formula
Preparation method it is characterised in that comprising following operating procedure:A () Radix potentillae anserinae rhizome is pulverized, carried with 75% alcohol heat reflux
Take, united extraction liquid, be concentrated into no alcohol taste, use petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, respectively obtain
Petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;Acetic acid ethyl ester extract macropore tree in (b) step (a)
Fat remove impurity, successively with 15% ethanol and 70% ethanol elution, collects 70% eluent, concentrating under reduced pressure obtains 70% ethanol elution and concentrates
Thing;C in () step (b), 70% ethanol elution concentrate is separated with purification on normal-phase silica gel, use volume ratio to be 40 successively:1、20:1、10:1
With 5:1 methylene chloride-methanol gradient elution obtains 4 components;D in () step (c), component 4 is divided further with purification on normal-phase silica gel
From, successively use volume ratio be 8:1、5:1 and 2:1 methylene chloride-methanol gradient elution obtains 3 components;In (e) step (d)
The reverse phase silica gel that component 2 octadecylsilane is bonded separates, and the methanol aqueous solution being 65% with concentration expressed in percentage by volume is isocratic to be washed
De-, collect 7-9 column volume eluent, eluent is concentrated under reduced pressure to give pure compound.
2. triterpenoid preparation method according to claim 1 it is characterised in that:Described macroporous resin is that D101 type is big
Macroporous adsorbent resin.
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