CN105712878A - Diterpene compound and preparation method thereof - Google Patents
Diterpene compound and preparation method thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
- C07C69/33—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with hydroxy compounds having more than three hydroxy groups
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
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- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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Abstract
The invention discloses a novel diterpene compound and a preparation method thereof and provides a compound structure. This compound is reported for the first time and is a novel-structure diterpene compound that may be extracted, separated and purified from dried tuber of Ranunculus ternatus. In-vitro experiments prove that the compound has inhibitory action on prostate cancer cell lines PC 3 and DU145, the inhibitory action has a concentration-time dependent relation, and the compound may be developed into drugs for treating prostate cancer.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to separate a kind of diterpene compound with treatment effect on prostate carcinoma of obtaining and preparation method thereof from the dried root of Radix Ranunculi Ternati.
Background technology
Radix Ranunculi Ternati RanunculiTernatiRadix is the dried root of ranunculaceae plant Seynuns Kugelann RanunculusternatusThunb..This product is spindle, and many 5～6 cluster, and is similar to ram's horn, and long 3～10mm, diameter 2～3mm, there are the residual stem of yellowish-brown or stem trace in top.Surface yellowish-brown or lark, deposit color and luster for a long time and deepen, and micro-have vertical wrinkle, and has point-like mark of fibrous root and residual fibrous root.Matter is solid, section off-white color or yellow-white, hollow or solid, mealiness.Feeble QI, taste is micro-sweet.Radix Ranunculi Ternati another name three dissipates grass (Zhejiang), ram's horn grass (Henan), ram's horn (Henan), flipper (Anhui), Herba Stenolomatis Chusani (Guangxi).Radix Ranunculi Ternati is warm in nature, sweet in the mouth, pungent, enters liver, lung meridian, has the effects such as heat-clearing and toxic substances removing, dispersing swelling and dissipating binds, eliminating phlegm and stopping cough, is clinically used for the diseases such as treatment pulmonary tuberculosis, tuberculous lymphadenitis, pharyngolaryngitis, malaria.Among the people treat lymphoid tuberculosis with Radix Ranunculi Ternati, no matter tuberculosis size or whether suppurate and all have good curative effect.In recent years because it has good antitumous effect to become study hotspot.
Research for Radix Ranunculi Ternati chemical composition is less in recent years, and it mainly contains the compound such as flavonoid and glycoside, alkaloid, volatile oil, organic acid.
Summary of the invention
It is an object of the invention to provide a kind of a kind of diterpene compound with treatment effect on prostate carcinoma separating from the dried root of Radix Ranunculi Ternati and obtaining and preparation method thereof.
The above-mentioned purpose of the present invention is achieved by the techniques below scheme:
A kind of diterpene compound, has the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operating procedure: the dried root of Radix Ranunculi Ternati is pulverized by (a), extract with 75～85% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste, successively with petroleum ether, ethyl acetate and water saturated n-butanol extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract；B acetic acid ethyl ester extract macroporous resin remove impurity in () step (a), first with 8 column volumes of 15% ethanol elution, then with 10 column volumes of 75% ethanol elution, collects 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing extractum；In (c) step (b) 75% ethanol elution extractum with purification on normal-phase silica gel separate, successively with volume ratio be 85:1,45:1,20:1,10:1 and 1:1 methylene chloride-methanol gradient elution obtain 5 components；D in () step (c), component 3 separates further by purification on normal-phase silica gel, successively with volume ratio be 25:1,20:1 and 10:1 methylene chloride-methanol gradient elution obtain 3 components；E reverse phase silica gel that in () step (d), component 2 is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collecting 8～10 column volume eluents, eluent concentrating under reduced pressure obtains pure compound (I).
Further, in step (a), extract with 80% alcohol heat reflux, united extraction liquid.
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
The compounds of this invention is at field of medicaments, it is possible to as antiprostate cancer, it is possible to directly use, or uses with the form of pharmaceutical composition.This pharmaceutical composition contains the compounds of this invention (I) of therapeutically effective amount, and all the other are acceptable on materia medica, humans and animals is nontoxic and pharmaceutically suitable carrier of inertia and/or excipient.In vitro tests proves that this compound on prostate JEG-3 PC3 and DU145 has inhibitory action, and inhibitory action has concentration-time dependency relationships, and therefore, the above-claimed cpd (I) of the present invention can be used to develop into the medicine for the treatment of carcinoma of prostate.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula；
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value；
Fig. 3 is PC3 and DU145 survival rate after variable concentrations compound (I) effect 72h；
Fig. 4 is PC3 and DU145 survival rate after 10.0mg/L compound (I) effect different time.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, it is possible to technical scheme is modified or equivalent replacement, without deviating from the spirit and scope of technical solution of the present invention.
Embodiment 1: compound (I) separates preparation and structural identification
Reagent source: ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dried root (10kg) of (a) Radix Ranunculi Ternati is pulverized, (25L × 3 time) are extracted with 80% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste (3L), extract with petroleum ether (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated n-butyl alcohol (3L × 3 time) successively, respectively obtain petroleum ether extract, acetic acid ethyl ester extract (398g) and n-butyl alcohol extract；Acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in (b) step (a), first with 8 column volumes of 15% ethanol elution, again with 10 column volumes of 75% ethanol elution, collecting 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing extractum (163g)；C in () step (b), 75% ethanol elution extractum 200-300 order purification on normal-phase silica gel separates, obtain 5 components with the methylene chloride-methanol gradient elution that volume ratio is 85:1 (10 column volumes), 45:1 (9 column volumes), 20:1 (8 column volumes), 10:1 (8 column volumes) and 1:1 (5 column volumes) successively；D in () step (c), component 3 (49g) 200-300 order purification on normal-phase silica gel separates further, obtain 3 components with the methylene chloride-methanol gradient elution that volume ratio is 25:1 (8 column volumes), 20:1 (10 column volumes) and 10:1 (5 column volumes) successively；E reverse phase silica gel ODS-C18 that in () step (d), component 2 (21g) is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collecting 8～10 column volume eluents, eluent concentrating under reduced pressure obtains pure compound (I) (43mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z489.2808, can obtain molecular formula in conjunction with nuclear-magnetism feature is C26H42O7, degree of unsaturation is 6.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6, 600MHz): H-1 (1.16, m), H-1 (2.03, ddd, J=13.7, 8.7, 1.8), H-2 (4.21, m), H-3 (1.91, m), H-3 (1.28, m), H-4 (2.20, ddd, J=14.9, 7.2, 3.6), H-6 (1.48, m), H-6 (1.64, dt, J=14.0, 2.8), H-7 (1.31, m), H-7 (1.12, m), H-8 (1.45, m), H-10 (1.71, d, J=13.7), H-11 (1.11, m), H-11 (1.34, m), H-12 (1.94, m), H-14 (6.33, dd, J=17.6, 10.9), H-15 (5.13, d, J=17.6), H-15 (4.93, d, J=10.8), H-16 (4.93, s), H-16 (4.82, s), H-17 (0.76, d, J=6.8), H-18 (6.38, d, J=7.2), H-19 (6.03, s), H-20 (0.84, s), H-2 ' (2.15, td, J=7.3, 1.4), H-3 ' (1.52, J=7.3), H-4 ' (0.84, t, J=7.3), H-2 " (1.80, s)；Carbon-13 nmr spectra data δC(ppm, DMSO-d6, 150Hz): 29.1 (CH2, 1-C), 66.4 (CH, 2-C), 29.4 (CH2, 3-C), 45.3 (CH, 4-C), 49.2 (C, 5-C), 22.9 (CH2, 6-C), 27.0 (CH2, 7-C), 36.9 (CH, 8-C), 36.1 (C, 9-C), 34.7 (CH, 10-C), 32.3 (CH2, 11-C), 26.3 (CH2, 12-C), 144.4 (C, 13-C), 139.4 (CH, 14-C), 111.3 (CH2, 15-C), 114.4 (CH2, 16-C), 14.6 (CH3, 17-C), 97.2 (CH, 18-C), 99.2 (CH, 19-C), 25.7 (CH3, 20-C), 171.2 (C, 1 '-C), 35.4 (CH2, 2 '-C), 17.2 (CH2, 3 '-C), 12.7 (CH3, 4 '-C), 168.8 (C, 1 "-C), 20.5 (CH3, 2 " and-C)；Carbon atom labelling is referring to Fig. 1.13CNMR spectrum shows 26 signals, comprises two methyl (δ C14.6,17-C；25.7,20-C), two conjugated double bonds (δ C144.4,13-C；139.4,14-C；111.3,15-C；114.4,16-C), and acetoxyl group (δ C168.8,1 "-C；20.5,2 " and-C), butyryl acyloxy (δ C171.2,1 '-C；35.4,2 '-C；17.2,3 '-C；12.7,4 '-C).Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as it is shown in figure 1, absolute configuration is followed successively by 2R, 4S, 5R, 8S, 9S, 10R, 18S, 19R；Spatial configuration is determined by ECD test further, theoretical value and experiment value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Prostatic cancer cell lines PC3 (ArCC-CRL-1435), Prostatic cancer cell lines DU145 (ATCC-HTB-81).Compound (I) is made by oneself, and HPLC normalization purity, more than 98%, is configured to, with dimethyl sulfoxide (DMSO), the storage liquid that concentration is 1.0g/L standby.CCK-8 test kit is Japan's colleague's chemistry institute product.RPMI-1640 culture medium is purchased from Gibco company.Hyclone (FBS) is purchased from Hyelone company.Green grass or young crops/streptomycin is Shanghai pioneer's Pharmaceutical product.Trypsin is purchased from Huamei Bio-Engrg Co.,.Dimethyl sulfoxide (DMSO) is purchased from Shanghai Hua Shun bio-engineering corporation.Agar (Agarose), dithiothreitol, DTT (DTT), benzyl sulfonephthalein fluorine (PMSF), tetramethylethylenediamine (TEMED) are Sigma Products.Dodecyl sodium sulfate (SDs), trichloromethyl alkyl methane (Tris) Tris-Hcl, Tritonx-100 are Promega Products.Propylene phthalein amine, persulfuric acid money (AP) are purchased from Suzhou chemical reagent factory product.
CO2Cell culture incubator (ShellLab), inverted phase contrast microscope (Nikon), superclean bench (Suzhou Decontamination Equipment Plant), flow cytometer (BD), F039300A type microplate reader (Sunrise), autoclave sterilizer (HirayamaHA-300MD), low temperature supercentrifuge (Kubota3740), Universal table (Jiangsu kylin medical apparatus factory TS-92), electrophresis apparatus (Gibco company), LXJ-II type centrifuge (Shanghai medical analytical instrument factory), DK600 type electric heating constant-temperature water-bath tank (Shanghai microtest equipment company), electronic balance (METTLERTOLEDO), desk-top drying baker (Shanghai gloomy reliable test Instrument Ltd.), UV detector (Beckman).
Two, test method
1, cell is cultivated and maintenance
1.1 cells are cultivated
Cell strain is incubated at central laboratory of tumour hospital.Be incubated in the RPMI-1640 culture medium containing 10% hyclone, another add paddy ammonia phthalein amine (2mmol/L) and antibiotic (l00U/ penicillin and 100mg/L streptomycin), put 37 DEG C, saturated humidity, containing 5%CO2The incubator of gas is cultivated.
1.2 passages
1. under inverted phase contrast microscope, observation of cell covers with adherent, can go down to posterity.With 75% alcohol wipe superclean bench through ultraviolet radiation and both hands before going down to posterity.2. the old culture fluid in culture bottle is sucked with suction pipe, with PBS 3 times.3. add 0.02%EDTA-0.25% trypsin solution 2mL to digest, stand about 5 minutes, and frequently observe under inverted phase contrast microscope, when kytoplasm bounces back, when no longer connecting in blocks between cell, add the appropriate fresh culture containing serum and terminate tryptic effect.4. with dropper, the cell digested is blown and beaten into cell suspension, suck equilibrium centrifugation (1000 revs/min) 5 minutes in 10mL centrifuge tube.5. abandoning supernatant, adds 2mL culture fluid, blows and beats cell with dropper gently and make cell suspension, and 1:2～3 are distributed into new culture bottle, adds culture fluid and continues in right amount to cultivate in incubator.
2, morphological observation
Inverted microscope is observed: exponential phase PC3 and DU145 cell are inoculated in 6cm culture dish, adds 10.0mg L after cultivating 24h-1, compound (I) processes after 24,48,72h observation of cell morphologic change record under inverted phase contrast microscope respectively.
3, cytotoxicity test (CCK-8 method)
1. the cell dissociation in culture bottle becomes single cell suspension, according to 3 × 10 after cell counting4Individual cells/well is inoculated in 96 orifice plates, and every hole adds 0.lmL culture medium, and cellar culture is in 37 DEG C, containing 5%CO2In the incubator of gas.2. packet is tested: setting negative control group (having cell but not dosing, 0.1%DMSO), blank group (acellular only culture fluid), compound (I) is 2.5mg/L, 5.0mg/L, 10.0mg/L and 20.0mg/L totally 6 groups respectively.3. after 24 hours, observation of cell adherent growth is good, is grouped to dosing in 96 orifice plates by above-mentioned test respectively, and often group sets 6-8 repeating hole.4. after dosing, 96 orifice plates are moved into 37 DEG C, containing 5%CO2The incubator of gas continues cultivate 24,48 and 72 hours respectively.When 5. often organizing off-test, every hole adds CCK-810 μ L, continues absorbance (OD) value in microplate reader detection 450 every holes after cultivating 4 hours in 37 DEG C of incubators, and mensuration wavelength is 450nm, and reference wavelength is 600nm.6. calculating cell survival rate (cellviability) according to below equation, be then plotted into chart, the value that survival rate is when 50% is IC50.Cell survival rate (%)=[(As-Ab)/(Ac-Ab)] × 100%.Wherein As is test hole, and Ac is control wells, and Ab is blank well.
4, colony-forming test
1. take the logarithm trophophase cell, with 3 × l0 after counting2Individual 6 orifice plates that are inoculated in, every hole adds 2mL culture medium, and cellar culture is in 37 DEG C, containing 5%CO2In the incubator of gas.2. after 24h, observation of cell adherent growth is good, is separately added into variable concentrations (0,2.5,5.0,10.0,20.0mg/L) compound (I) and processes, and often group sets 3 repeating holes.3. after dosing, 6 orifice plates are moved into 37 DEG C, containing 5%CO2The incubator of gas continues 14 days.4. 10% methanol is fixed, and Giemsa dyes, and calculates every hole colony number (being calculated as a colony of >=50 cells).5. colony-forming efficiency is calculated: colony sum/inoculating cell number × 100%.
Three, result and conclusion
1, compound (I) gas impact on PC3 and DU145 cellular morphology
Seeing cellular control unit adherent growth under mirror, flanking cell merges in flakes, and cell is similar round or fusiformis, and volume is relatively big, and closely, the smooth of the edge, kytoplasm is full, and the structure outline such as nuclear membrane, kernel is obvious, and Growth of Cells is rapid in arrangement；After 10.0mg/L compound (I) processes, cell density is gradually lowered, and the speed of growth substantially slows to and almost stagnates, and cell comes off gradually and floats in culture fluid.Cell volume reduces, after birth shrinkage, becomes small circular or irregular form, common fine granularity material in born of the same parents.Drug treating time is more long, and morphological changes of cell is more obvious.
2, cytotoxicity test
The growth of Prostatic cancer cell lines PC3 and DU145 is all had inhibitory action by variable concentrations compound (I).Survival rate (table l and table 2) as shown in the table after 2.5,5.0,10.0,20.0mg/L compounds (I), two kinds of cells 24,48 of effect and 72h.Wherein, 10.0mg/L compound (I) acts on the cell survival rate after PC3 and DU145 cell 24,45,72h respectively 56.2%, 42.5%, 24.3% and 50.8%, and 32.6%, 20.7%；2.5,5.0,10.0,20.0mg/L, the survival rate respectively 54.3%, 37.7%, 24.3%, 13.2% and 52.4%, 32.8%, 20.7% after compound (I) two kinds of cell 72h of effect, 11.2% (see Fig. 3 and Fig. 4).Two analysis of variance show that between variable concentrations and different time process group, difference has statistical significance (P < 0.05), and the growth inhibited of PC3 and DU145 is that time-concentration relies on by prompting compound (I).
3, colony-forming test
Test display, the colony-forming efficiency of matched group PC3 and DU145 respectively 67.7 and 64%, and 2.5,5.0,10.0,20.0mg/L compound (I) process group respectively 60.7%, 51.3,39.3%, 27.0% and 49.7%, 34.0%, 27.7%, 15.7% (see table 3).This further demonstrates that PC3 and DU145 cell is had inhibited proliferation by compound (I).
Conclusion, verifies the compound (I) effect to Prostatic cancer cell lines PC3 and DU145 by CCK-8 method and colony-forming test in this test, and inhibitory action has concentration one time dependency relationships.Compound (I) is likely to become in therapy approaches of advanced prostate cancer and has potential selection.
Table l variable concentrations compound (I) acts on the cell survival rate after PC3
| Time/concentration | 2.5mg/L | 5.0mg/L | 10.0mg/L | 20.0mg/L |
| 24h | 0.825 | 0.731 | 0.562 | 0.422 |
| 48h | 0.69 | 0.53 | 0.425 | 0.226 |
| 72h | 0.543 | 0.377 | 0.243 | 0.132 |
Table 2 variable concentrations compound (I) acts on the cell survival rate after DU145
| Time/concentration | 2.5mg/L | 5.0mg/L | 10.0mg/L | 20.0mg/L |
| 24h | 0.809 | 0.604 | 0.508 | 0.375 |
| 48h | 0.646 | 0.481 | 0.326 | 0.173 |
| 72h | 0.542 | 0.328 | 0.207 | 0.112 |
The colony-forming efficiency of lower PC3 and the DU145 of table 3 variable concentrations compound (I) effect
Embodiment 3
The preparation of tablet: first prepare compound (I) by embodiment 1 method, and utilize the salt that organic acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid makes, excipient, pelletizing press sheet is added with excipient weight than the ratio for 1:9 in it.
Embodiment 4
The preparation of oral liquid: first prepare compound (I) by embodiment 1 method, and utilizing the salt that organic acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid makes, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: first prepare compound (I) by embodiment 1 method, and utilize the salt that organic acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid makes, add excipient with excipient weight than the ratio for 1:9 in it, make capsule or granule.
Embodiment 6
The preparation of injection: first prepare compound (I) by embodiment 1 method, and utilize the salt that organic acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid makes, injecting routinely and use water, fine straining, injection is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: first prepare compound (I) by embodiment 1 method, and utilize the salt that organic acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid makes, it is dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic fine straining again, is sub-packed in ampoule, and after frozen drying, aseptic sealing by fusing obtains injectable powder.
The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit protection scope of the present invention with this.It will be understood by those within the art that, it is possible to technical scheme is modified or equivalent replacement, without deviating from essence and the protection domain of technical solution of the present invention.
Claims (4)
1. a diterpene compound, it is characterised in that: it is the compound (I) with following structural formula,
2. the preparation method of the compound (I) described in claim 1, it is characterized in that: comprise following operating procedure: the dried root of Radix Ranunculi Ternati is pulverized by (a), extract with 75～85% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste, successively with petroleum ether, ethyl acetate and water saturated n-butanol extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract；B acetic acid ethyl ester extract macroporous resin remove impurity in () step (a), first with 8 column volumes of 15% ethanol elution, then with 10 column volumes of 75% ethanol elution, collects 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing extractum；In (c) step (b) 75% ethanol elution extractum with purification on normal-phase silica gel separate, successively with volume ratio be 85:1,45:1,20:1,10:1 and 1:1 methylene chloride-methanol gradient elution obtain 5 components；D in () step (c), component 3 separates further by purification on normal-phase silica gel, successively with volume ratio be 25:1,20:1 and 10:1 methylene chloride-methanol gradient elution obtain 3 components；E reverse phase silica gel that in () step (d), component 2 is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collecting 8～10 column volume eluents, eluent concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), it is characterised in that: in step (a), extract with 80% alcohol heat reflux, united extraction liquid.
4. the preparation method of compound according to claim 2 (I), it is characterised in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
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| CN105503800A (en) * | 2016-02-20 | 2016-04-20 | 杭州富阳伟文环保科技有限公司 | Novel eremophilanolides type compound and preparation method and medical application thereof |
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Application publication date: 20160629 |