CN105523937A - Diterpene compound with medical application and preparation method thereof - Google Patents
Diterpene compound with medical application and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a diterpene compound for treating prostate cancer and a preparation method of the diterpene compound. The compound is first reported, is a diterpene compound with a novel structure, and can be obtained by means of extracting, separating and purifying dried bark of girald daphne. Proved by in vitro tests, the compound has an inhibiting effect on prostate cancer cell strains PC3 and DU145, and the inhibiting effect has a concentration-time dependency relationship. A compound (I) probably becomes a potential choice for treating advanced prostate cancer, and can be used for being developed into a drug for treating prostate cancer.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from dry Daphne giraldii Nitsche, be separated obtain a kind of and there is diterpene-kind compound for the treatment of effect on prostate carcinoma and preparation method thereof.
Background technology
Daphne giraldii Nitsche is thymelaeceae (Thymelaeaceae) plant Daphne Giraldii Nitsche (DaphnegiraldiiNitsche), retuseleaf daphne bark (D.retusaHemsl), the root skin of the sweet winter daphne in Shan (having another name called Tang Gute winter daphne) (D.tanguticaMaxim) and stem skin, it is warm in nature, taste is arduous, there is wind-damp dispelling, effect of promoting blood circulation and stopping pain.Autumn gathers, removing radicula, strips Gen Pi and stem skin and then dries and form.Another name: dogskin 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 blood stasis removing analgesic, dispelling wind and removing obstruction in the collateral, stomachache, headache, numbness of the limbs, rheumatic arthralgia, wound, sacroiliitis, 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 ingredient 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 anti-inflammatory, analgesia and anti-malarial clinically.Find that Daphne giraldii Nitsche also has significant effect in antitumor and treatment glomerulonephritis etc. by further research in recent years.Existing commercially available patent medicine mainly comprises Giraldi daphne tablet, Daphne giraldii Nitsche's rheumatism plaster, Daphne giraldii Nitsche's pain-relieving plaster for arthritis, Daphne Capsules, Daphne giraldii Nitsche's sustained release pellet, Daphne giraldii Nitsche injection liquid etc.
Summary of the invention
The object of this invention is to provide and a kind ofly from dry Daphne giraldii Nitsche, be separated the diterpene-kind compound that obtain a kind of has treatment effect on prostate carcinoma, another goal of the invention of the present invention is to provide above-mentioned diterpene-kind compound preparation method.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
Have a diterpene compound for medicinal use, it 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, united extraction liquid, is 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;
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 eluates, concentrating under reduced pressure obtains ethanol elution thing medicinal extract;
S03: in step S02, ethanol elution medicinal extract 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 elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
An above-mentioned preparation method with the diterpene compound of medicinal use, comprises following operation steps:
A, dry Daphne giraldii Nitsche to be pulverized, extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, is 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;
Acetic acid ethyl ester extract macroporous resin removal of impurities in b, step a, first uses 10% ethanol elution, 8 column volumes, then uses 75% ethanol elution, 10 column volumes, collects 75% ethanol eluate, and concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract;
In c, step b, 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, and obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1;
In d, step c, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1;
In e, steps d, component 2 reverse phase silica gel of octadecylsilane bonding is separated, and be the methanol aqueous solution isocratic elution of 75% by concentration expressed in percentage by volume, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I);
The concentration of above-mentioned ethanol is concentration expressed in percentage by volume.
Further, in described step b, macroporous resin is AB-8 type macroporous adsorbent resin.
Further, in described step a, national expenditures alcohol heat reflux extracts the alcohol concn adopted is 80%.
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.By CCK-8 method and colony-forming test, contriver has verified that above-claimed cpd (I) is to the effect of Prostatic cancer cell lines PC3 and DU145, and restraining effect has concentration---time-dependent sexual intercourse.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 medicine effect.
Above-mentioned 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.
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 different concns compound (I) effect 72h;
Fig. 4 is PC3 and DU145 survival rate after 10.0mg/L compound (I) effect different time.
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.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: 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.
Preparation method: dry Daphne giraldii Nitsche (10kg) pulverizes by (a), (25L × 3 time) are extracted with 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (3L), use sherwood oil (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated propyl carbinol (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 removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 75% ethanol elution, 10 column volumes again, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract (157g); C in () step (b), 75% ethanol elution medicinal extract 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 elutriant, elutriant 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 mark is see Fig. 1.Infrared spectra 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 methyne 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-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-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 six-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, steric configuration is determined further by ECD test, theoretical value and experimental 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 method purity is greater than 98%, and being mixed with concentration with dimethyl alum (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 substratum is purchased from Gibco company.Foetal calf serum (FBS) is purchased from Hyelone company.Green grass or young crops/Streptomycin sulphate is Shanghai pioneer's medicine company product.Trypsinase is purchased from Huamei Bio-Engrg Co..Dimethyl sulfoxide (DMSO) (DMSO) is purchased from Shanghai Hua Shun bio-engineering corporation.Agar (Agarose), dithiothreitol (DTT) (DTT), phenmethyl sulfonephthalein fluorine (PMSF), Tetramethyl Ethylene Diamine (TEMED) are Sigma Products.Sodium laurylsulfonate (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), Bechtop (Suzhou Decontamination Equipment Plant), flow cytometer (BD), F039300A type microplate reader (Sunrise), autoclave sterilizer (HirayamaHA-300MD), low temperature ultracentrifuge (Kubota3740), Universal table (Jiangsu kylin medical apparatus factory TS-92), electrophoresis apparatus (Gibco company), LXJ-II type whizzer (Shanghai medical analytical instrument factory), DK600 type electric heating constant-temperature water-bath tank (Shanghai microtest equipment company), electronic balance (METTLERTOLEDO), desk-top loft drier (Shanghai gloomy reliable test Instrument Ltd.), UV detector (Beckman).
Two, test method
1, cell cultures and maintenance
1.1 cell cultures
Cell strain is incubated at centralab of tumour hospital.Be incubated in the RPMI-1640 substratum containing 10% foetal calf serum, another add paddy ammonia phthalein amine (2mmol/L) and microbiotic (l00U/ penicillin and 100mg/L Streptomycin sulphate), 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 Bechtop of uviolizing and both hands.2. suck the old nutrient solution in culturing 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 nutrient solution, and blow and beat cell gently with dropper and make cell suspension, 1:2 ~ 3 are distributed into new culturing bottle, adds nutrient solution and continues in right amount to cultivate in incubator.
2, morphological observation
Inverted microscope is observed: logarithmic 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 culturing 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 substratum, 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 nutrient solution), 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 absorbancy (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 holes, and Ac is control wells, and Ab is blank well.
4, colony-forming test
1. to take the logarithm cell in vegetative period, with 3 × l0 after counting
2individually be inoculated in 6 orifice plates, every hole adds 2mL substratum, 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 different concns (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% methyl alcohol 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 form
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 nutrient solution.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
Different concns compound (I) growth to Prostatic cancer cell lines PC3 and DU145 all has restraining effect.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 different concns and different time treatment group, difference has statistical significance (P<0.05), and prompting compound (I) the growth-inhibiting effect to PC3 and DU145 is that time-concentration relies on.
3, colony-forming test
Test display, the colony forming efficiency of control group PC3 and DU145 is respectively 67.7 and 64%, and 2.5,5.0,10.0,20.0mg/L compound (I) treatment 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 restraining effect has concentration in this test by CCK-8 method and colony-forming test---time-dependent sexual intercourse.Compound (I) may become a potential selection of tool in therapy approaches of advanced prostate cancer.
Table l different concns 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 different concns 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 different concns compound (I) effect
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
Prepared by 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, 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, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
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. there is a diterpene compound for medicinal use, it is characterized in that: it has the compound (I) of following structural formula,
2. the diterpene compound with medicinal use according to claim 1, 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, united extraction liquid, is 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;
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 eluates, concentrating under reduced pressure obtains ethanol elution thing medicinal extract;
S03: in step S02, ethanol elution medicinal extract 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 elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. a preparation method with the diterpene compound of medicinal use according to claim 1, is characterized in that: comprise following operation steps:
A, dry Daphne giraldii Nitsche to be pulverized, extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, is 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;
Acetic acid ethyl ester extract macroporous resin removal of impurities in b, step a, first uses 10% ethanol elution, 8 column volumes, then uses 75% ethanol elution, 10 column volumes, collects 75% ethanol eluate, and concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract;
In c, step b, 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, and obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1;
In d, step c, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1;
In e, steps d, component 2 reverse phase silica gel of octadecylsilane bonding is separated, and be the methanol aqueous solution isocratic elution of 75% by concentration expressed in percentage by volume, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I);
The concentration of above-mentioned ethanol is concentration expressed in percentage by volume.
4. the preparation method with the diterpene compound of medicinal use according to claim 3, is characterized in that:
In described step b, macroporous resin is AB-8 type macroporous adsorbent resin.
5. the preparation method of compound according to claim 3 (I), is characterized in that:
In described step a, national expenditures alcohol heat reflux extracts the alcohol concn adopted is 80%.
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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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