CN105534968A - Application of diterpenoid compound to preparation of drug for treating prostate cancer - Google Patents
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Abstract
The invention relates to application of a diterpenoid compound obtained by separation from dry girald daphne bark to the preparation of a drug for treating prostate cancer. The diterpenoid compound is reported for the first time and can be obtained by extraction, separation and purification from the dry girald daphne bark. In-vitro tests prove that the compound has an inhibiting effect on prostate cancer cell lines PC3 and DU145, and the inhibiting effect has a concentration-time dependence relation. The compound (I) may become a potential choice for the treatment of the advanced prostate cancer and can be developed into the drug for treating the prostate cancer.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to be separated the diterpene-kind compound that the obtains medical usage before the treatment in row adenocarcinoma from dry Daphne giraldii Nitsche.
Background technology
Daphne giraldii Nitsche is thymelaeceae (Thymelaeaceae) plant Daphne Giraldii Nitsche (DaphnegiraldiiNitsche), retuseleaf daphne bark (D.retusaHemsl), the root bark of the sweet winter daphne in Shan (having another name called Tang Gute winter daphne) (D.tanguticaMaxim) and peel of stem, it is warm in nature, acrid in the mouth is bitter, there is wind-damp dispelling, effect of promoting blood circulation and stopping pain.Autumn gathers, removing radicula, strips root bark and peel of stem and then dries and form.Another name: dog skin willow, ancestral's silk fiber crops etc.Mainly be distributed in the ground such as Shanxi, Shaanxi, Sichuan, Qinghai, Gansu.Daphne giraldii Nitsche is mainly used in treatment eliminating stasis to stop pain, dispelling wind and removing obstruction in the collateral, stomachache, headache, numb limbs and tense tendons, rheumatic arthralgia, traumatic injury, arthritis, rheumatoid arthritis etc.This medicine is rather good in disease effects such as Turkey's folk therapy rheumatoid arthritis and lumbago and skelalgia.
The chemical constitution study of Daphne giraldii Nitsche starts from nineteen seventies, is separated first during Wang Ming etc. and obtains coumarin kind compound, just carry out gradually subsequently to the chemical constitution study of Daphne giraldii Nitsche from Daphne giraldii Nitsche.Now there are some researches show except containing except main active coumarin kind compound in Daphne giraldii Nitsche, also containing multiple compounds such as flavonoid, lignanoids, Diterpeneses.
Daphne giraldii Nitsche is mainly used in the aspects such as antiinflammatory, analgesia and malaria clinically.Find that Daphne giraldii Nitsche also has significant effect in antitumor and treatment glomerulonephritis etc. by further studying in recent years.Existing commercially available patent medicine mainly comprises Giraldi daphne tablet, Daphne giraldii Nitsche rheumatism plaster, Daphne giraldii Nitsche pain-relieving plaster for arthritis, Daphne Capsules, Daphne giraldii Nitsche slow-release micro-pill, Daphne giraldii Nitsche injection liquid etc.
Summary of the invention
The object of this invention is to provide a kind of a kind of diterpene-kind compound obtained that is separated from dry Daphne giraldii Nitsche and prepare the medical usage in the medicine for the treatment of carcinoma of prostate.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
The application of diterpene compound formula I in the medicine of preparation treatment carcinoma of prostate, it is separated from Daphne giraldii Nitsche and obtains, and has the compound (I) of following structural formula:
As preferably, compound (I) is separated from Daphne giraldii Nitsche and obtains, and separating step comprises:
S01: Daphne giraldii Nitsche is dry, pulverizing, extract with 75 ~ 85% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste, use petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
S02: acetic acid ethyl ester extract 10% ethanol elution, 8 column volumes in step S01, then use 75 ~ 85% ethanol elution, 10 column volumes, collect 75 ~ 85% ethanol elution, concentrating under reduced pressure obtains ethanol elution thing extractum;
S03: in step S02, ethanol elution extractum purification on normal-phase silica gel is separated, obtains 5 components with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1 successively;
S04: in step S03, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1 successively;
S05: in step S04, component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10 ~ 12 column volume eluents, eluent concentrating under reduced pressure obtains pure compound (I);
The concentration of above-mentioned ethanol is concentration expressed in percentage by volume.
Further, step S02 before acetic acid ethyl ester extract is with 10% ethanol elution, 8 column volumes, to acetic acid ethyl ester extract macroporous resin remove impurity.
As preferably, in described step S02, macroporous resin is AB-8 type macroporous adsorbent resin.
As preferably, in described step a, national expenditures alcohol heat reflux extracts the concentration of alcohol adopted is 80%.
The present invention, when preparing the application in the medicine for the treatment of carcinoma of prostate, directly can use, or use with the form of pharmaceutical composition.This pharmaceutical composition contains the compounds of this invention (I) for the treatment of effective dose, and all the other are acceptable on materia medica, nontoxic to humans and animals and pharmaceutically suitable carrier of inertia and/or excipient.By CCK-8 method and colony-forming test, inventor has verified that above-claimed cpd (I) is to the effect of Prostatic cancer cell lines PC3 and DU145, and inhibitory action has concentration---time-dependent sexual relationship.Therefore compound (I) may become a potential selection of tool in therapy approaches of advanced prostate cancer.Therefore, compound of the present invention (I) has the medical usage in the medicine of preparation treatment 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 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.
Embodiment 1: compound (I) is separated 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: dry Daphne giraldii Nitsche (10kg) is pulverized by (a), (25L × 3 time) are extracted with 80% alcohol heat reflux, merge extractive liquid, be concentrated into without alcohol taste (3L), use petroleum ether (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated n-butyl alcohol (3L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 75% ethanol elution, 10 column volumes again, collect 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing extractum (157g); C in () step (b), 75% ethanol elution extractum purification on normal-phase silica gel is separated, successively with volume ratio be 75:1 (8 column volumes), the methylene chloride-methanol gradient elution of 45:1 (8 column volumes), 20:1 (8 column volumes), 12:1 (8 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (49g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (8 column volumes), the methylene chloride-methanol gradient elution of 12:1 (10 column volumes) and 5:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (25g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10-12 column volume eluent, eluent concentrating under reduced pressure obtains pure compound (I) (35mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z511.2308, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
27h
36o
8, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 500MHz): H-1 (2.47, t, J=13.5), H-1 (1.57, dd, J=13.5, 3.5), H-2 (2.54, m), H-3 (5.69, t, J=5.0), H-4 (3.01, d, J=5.0), H-7 (1.75, ddd, J=15.5, 12.5, 2.0), H-7 (1.26, m), H-8 (1.92, m), H-8 (1.54, t, J=12.5), H-9 (3.37, dd, J=9.5, 4.5), H-11 (3.01, d, J=2.5), H-12 (3.36, d, J=2.5), H-16 (0.91, d, J=7.5), H-17 (1.07, s), H-18 (0.93, s), H-19 (0.79, s), H-20 (1.11, s), H-2 ' (8.05, d, J=7.0), H-3 ' (7.44, t, J=7.0), H-4 ' (7.52, t, J=7.0), H-5 ' (7.44, t, J=7.0), H-6 ' (8.05, d, J=7.0), 9-OH (4.55, d, J=4.5), 11-OH (5.36, s), 12-OH (5.34, s), 15-OH (6.45, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 125MHz): 42.3 (CH
2, 1-C), 35.7 (CH, 2-C), 78.4 (CH, 3-C), 54.1 (CH, 4-C), 212.3 (C, 5-C), 49.0 (C, 6-C), 31.8 (CH
2, 7-C), 29.2 (CH
2, 8-C), 79.5 (CH, 9-C), 38.8 (C, 10-C), 79.3 (CH, 11-C), 70.1 (CH, 12-C), 56.0 (C, 13-C), 208.1 (C, 14-C), 85.1 (C, 15-C), 16.2 (CH
3, 16-C), 18.9 (CH
3, 17-C), 25.1 (CH
3, 18-C), 14.7 (CH
3, 19-C), 10.5 (CH
3, 20-C), 131.1 (C, 1 '-C), 130.2 (CH, 2 '-C), 129.3 (CH, 3 '-C), 133.5 (CH, 4 '-C), 129.3 (CH, 5 '-C), 130.2 (CH, 6 '-C), 167.2 (C, 7 '-C), carbon atom labelling is see Fig. 1.Infrared spectrum shows that this compound contains hydroxyl (3583,3502 and 3378cm
-1), carbonyl (1708 and 1689cm
-1) and aromatic ring (1605 and 1496cm
-1) group.
1this compound of HNMR spectrum display contains a benzoyl moiety δ H8.05 (2H, d, J=7.0Hz, H-2 ' and H-6 '), 7.52 (1H, t, J=7.0Hz, H-4 ') and 7.44 (2H, t, J=7.0Hz, H-3 ' and H-5 '); Four containing oxygen methine δ H5.69 (t, J=5.0Hz, H-3), 3.37 (dd, J=9.5,4.5Hz, H-9), 3.01 (d, J=2.5Hz, H-11) and 3.36 (d, J=2.5Hz, H-12); Five methyl δ H0.91 (d, J=7.5Hz, H
3-16), 1.07 (H
3-17), 0.93 (H
3-18), 0.79 (H
3-19) and 1.11 (H
3-20).Except benzoyl moiety,
13cNMR and DEPT composes display 20 carbon signals, also comprise except the functional unit that correspondence is above-mentioned six quaternary carbons (two carbonyl carbon δ C212.3,208.1 and one containing oxygen carbon δ C85.1).Show that this compound is a diterpene alcohol benzoate compounds according to nuclear magnetic data analysis.In HMBC spectrum, H
2-1 and C-2, C-3, C-4, C-15 and C-16; H-3 and C-1, C-15 and C-7 '; H
3-16 and C-1, C-2 and C-3; The dependency of OH-15 and C-4 and C-14, and in conjunction with the chemical shift of these protons with resonance carbon, show that this compound contains the five-membered ring that is connected with methyl and benzoyl fragment, C-15 position is connected with an oh group in addition.H-4 and C-5 and C-6 in being composed by HMBC; H
3-17 and C-5, C-6 and C-13; H
3-20 and C-6, C-13 and C-14; The dependency of OH-15 and C-14, known Isosorbide-5-Nitrae-cyclohexanedione is connected with five-membered ring by C-4 with C-15, and C-6 and C-13 position is connected with two methyl.In addition, H-9 and H-11 and C-18 and C-19; H-11 and C-12 and C-13; H-12 and C-13 and C-20; H
3-17 and C-7; H
3-18 and H
3-19 and C-9, C-10 and C-11; H
3-20 and C-12; With the dependency of C-10, OH-9 and C-9 shows that octatomic ring is connected with hexatomic ring by C-6 with C-13, C-9, C-11 and C-12 position is respectively connected with a hydroxyl, and C-10 position is connected with two methyl.In NOESY spectrum, H-12 and H
3the dependency of-19 shows that they are beta configuration.In addition, H-3 and H-2 and H-4; H-4 and H-2, H-3 and H
3-17; H-9 and H-11 and H
3the dependency of-18 shows that these protons are α-configuration.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 shown in Figure 1, spatial configuration is determined further by ECD test, theoretical value and experiment value basically identical (Fig. 2).
Embodiment 2: the pharmacological action test in the simple application process of compound (I)
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 is greater than 98%, and being mixed with concentration with dimethyl sulfoxide (DMSO) is that the storage liquid of 1.0g/L is for subsequent use.CCK-8 test kit is Japanese 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.
CO
2cell 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 culture and maintenance
1.1 cell culture
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%CO
2cultivate in the incubator of gas.
1.2 passage
1. under inverted phase contrast microscope, observation of cell covers with adherent, can go down to posterity.Before going down to posterity by 75% alcohol wipe through the superclean bench of ultraviolet radiation and both hands.2. suck the old culture fluid in culture bottle with suction pipe, clean 3 times with PBS.3. add 0.02%EDTA-0.25% trypsin solution 2mL to digest, leave standstill about 5 minutes, and frequently observe under inverted phase contrast microscope, when kytoplasm retraction, when no longer connecting in blocks between cell, add the appropriate fresh culture containing serum and stop tryptic effect.4. with dropper, the cell digested is blown and beaten into cell suspension, equilibrium centrifugation (1000 revs/min) 5 minutes in suction 10mL centrifuge tube.5. abandoning supernatant, adds 2mL culture fluid, and blow and beat cell gently with dropper and make cell suspension, 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 10.0mgL after cultivating 24h
-1, observation of cell morphologic change record under inverted phase contrast microscope respectively after compound (I) process 24,48,72h.
3, cytotoxicity test (CCK-8 method)
1. the cell dissociation in culture bottle becomes single cell suspension, according to 3 × 10 after cell counting
4individual cells/well is inoculated in 96 orifice plates, and every hole adds 0.lmL culture medium, cellar culture in 37 DEG C, containing 5%CO
2in the incubator of gas.2. grouping is tested: establish negative control group (to have cell but not dosing, 0.1%DMSO), blank group (acellular only have culture fluid), compound (I) is 2.5mg/L respectively, 5.0mg/L, 10.0mg/L and 20.0mg/L totally 6 groups.3. after 24 hours, observation of cell adherent growth is good, and divide into groups to dosing in 96 orifice plates by above-mentioned test respectively, often group establishes 6-8 repeating hole.4. after dosing, 96 orifice plates are moved into 37 DEG C, containing 5%CO
2continue in the incubator of gas to cultivate 24,48 and 72 hours respectively.When 5. often organizing off-test, every hole adds CCK-810 μ L, continue in 37 DEG C of incubators cultivation after 4 hours microplate reader detect absorbance (OD) value in 450 every holes, mensuration wavelength is 450nm, and reference wavelength is 600nm.6. go out cell survival rate (cellviability) according to following formulae discovery, be then depicted as chart, value when survival rate is 50% is IC
50.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. to take the logarithm trophophase cell, with 3 × l0 after counting
2individually be inoculated in 6 orifice plates, every hole adds 2mL culture medium, cellar culture in 37 DEG C, containing 5%CO
2in the incubator of gas.2. after 24h, observation of cell adherent growth is good, and add variable concentrations (0,2.5,5.0,10.0,20.0mg/L) compound (I) process respectively, often group establishes 3 repeating holes.3. after dosing, 6 orifice plates are moved into 37 DEG C, containing 5%CO
2continue 14 days in the incubator of gas.4. 10% methanol is fixed, and Giemsa dyes, and calculates every hole colony number (the counting 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 is on the impact of PC3 and DU145 cellular morphology
See cellular control unit adherent growth under mirror, flanking cell merges in flakes, and cell is similar round or fusiformis, and volume is comparatively large, and arrangement is tight, the smooth of the edge, and kytoplasm is full, and the structure outline such as nuclear membrane, kernel is obvious, and Growth of Cells is rapid; After 10.0mg/L compound (I) process, cell density reduces gradually, and the speed of growth obviously 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 longer, and morphological changes of cell is more obvious.
2, cytotoxicity test
Variable concentrations compound (I) growth to Prostatic cancer cell lines PC3 and DU145 all has inhibitory action.2.5,5.0,10.0,20.0mg/L compound (I) acts on the survival rate after two kinds of cells 24,48 and 72h (table l and table 2) as shown in the table.Wherein, the cell survival rate after 10.0mg/L compound (I) acts on PC3 and DU145 cell 24,45,72h is respectively 56.2%, 42.5%, 24.3% and 50.8%, 32.6%, 20.7%; 2.5,5.0,10.0,20.0mg/L, the survival rate after compound (I) effect two kinds of cell 72h is respectively 54.3%, 37.7%, 24.3%, 13.2% and 52.4%, 32.8%, 20.7%, 11.2% (see Fig. 3 and Fig. 4).Two analysis of variance show that between variable concentrations and different time processed group, difference has statistical significance (P<0.05), and prompting compound (I) the growth inhibited effect to PC3 and DU145 is that time-concentration relies on.
3, colony-forming test
Test display, the colony-forming efficiency of matched group PC3 and DU145 is respectively 67.7 and 64%, and 2.5,5.0,10.0,20.0mg/L compound (I) processed group is 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 compound (I) has inhibited proliferation to PC3 and DU145 cell.
Conclusion, verify that compound (I) is to the effect of Prostatic cancer cell lines PC3 and DU145, and inhibitory action has concentration in this test by CCK-8 method and colony-forming test---time-dependent sexual relationship.Compound (I) may become a potential selection of tool in therapy approaches of advanced prostate cancer.
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 DU145 of table 3 variable concentrations compound (I) effect
Embodiment 3
Make the application of tablet medicine: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, excipient is added, pelletizing press sheet than the ratio for 1:9 in itself and excipient weight.
Embodiment 4
Make the medicinal application of drink form: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
Make the medicinal application of capsule or granular form: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, add excipient in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
Make the medicinal application of injection form: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, inject with water routinely, fine straining, injection is made in embedding sterilizing.
Embodiment 7
Make the medicinal application of sterile powder form: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, be 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 is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.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 protection domain of technical solution of the present invention.
Claims (5)
1. the application of diterpene compound formula I in the medicine of preparation treatment carcinoma of prostate, is characterized in that: it is separated from Daphne giraldii Nitsche and obtains, and has the compound (I) of following structural formula;
2. the application of diterpene compound formula I according to claim 1 in the medicine of preparation treatment carcinoma of prostate, is characterized in that:
Compound (I) is separated from Daphne giraldii Nitsche and obtains, and separating step comprises:
S01: Daphne giraldii Nitsche is dry, pulverizing, extract with 75 ~ 85% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste, use petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
S02: acetic acid ethyl ester extract 10% ethanol elution, 8 column volumes in step S01, then use 75 ~ 85% ethanol elution, 10 column volumes, collect 75 ~ 85% ethanol elution, concentrating under reduced pressure obtains ethanol elution thing extractum;
S03: in step S02, ethanol elution extractum purification on normal-phase silica gel is separated, obtains 5 components with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1 successively;
S04: in step S03, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1 successively;
S05: in step S04, component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10 ~ 12 column volume eluents, eluent concentrating under reduced pressure obtains pure compound (I);
The concentration of above-mentioned ethanol is concentration expressed in percentage by volume.
3. the application of diterpene compound formula I according to claim 2 in the medicine of preparation treatment carcinoma of prostate, is characterized in that:
Step S02 before acetic acid ethyl ester extract is with 10% ethanol elution, 8 column volumes, to acetic acid ethyl ester extract macroporous resin remove impurity.
4. the application of diterpene compound formula I according to claim 3 in the medicine of preparation treatment carcinoma of prostate, is characterized in that:
In described step S02, macroporous resin is AB-8 type macroporous adsorbent resin.
5. the application of diterpene compound formula I according to claim 2 in the medicine of preparation treatment carcinoma of prostate, is characterized in that:
In described step a, national expenditures alcohol heat reflux extracts the concentration of alcohol adopted is 80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610040371.1A CN105534968A (en) | 2016-01-20 | 2016-01-20 | Application of diterpenoid compound to preparation of drug for treating prostate cancer |
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| CN105330714A (en) * | 2015-11-25 | 2016-02-17 | 杨飞杰 | Novel limonin compound as well as preparation method and medical application thereof in treatment of prostatic cancer |
| 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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| CN105330714A (en) * | 2015-11-25 | 2016-02-17 | 杨飞杰 | Novel limonin compound as well as preparation method and medical application thereof in treatment of prostatic cancer |
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