CN105481936A - Novel oleanane type triterpene compound and preparation method and medical application thereof - Google Patents
Novel oleanane type triterpene compound and preparation method and medical application thereof Download PDFInfo
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Abstract
The invention discloses a novel oleanane type triterpene compound and a preparation method and medical application thereof and provides a structure of the compound and a medicine composition containing the compound and a preparation method and application of the medicine composition. The oleanane type triterpene compound is reported for the first time, is novel in structure and can be obtained through extraction, separation and purification conducted on dry rhododendron dauricum. It is verified through in vitro tests that the compound can be used for being developed into medicine for treating gliomas by inhibiting cell proliferation and inducing cell apoptosis to give play to the inhibition effect of human brain malignant glioblastoma U251 cells.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, what be specifically related to obtain from the Rhododendron dauricum middle separation of drying a kind ofly has oleanane type triterpene compounds for the treatment of neurospongioma effect and preparation method thereof.
Background technology
Rhododendron dauricum is the dry leave of Ericaceae rhododendron Folium Cuculus polioephalus, is commonly called as azalea, to reach son fragrant etc.Autumn, winter adopt, and dry, and are distributed in the ground such as Heilungkiang, Jilin, the Inner Mongol, are born on ridge, hillside and sylvan life acid soil.Begin to be loaded in " herbal medicine handbook is commonly used in northeast ", its taste is pungent, bitter, cold in nature, the effect of have cough-relieving, eliminating the phlegm, and be mainly used in anxious (slowly) the property bronchitis for the treatment of and asthma, and antibechic, expectorant effect is better.Be that the preparation that raw material is made mainly contains with Rhododendron dauricum: anticough-asthma syrup, azalea sheet, compound is Rhododendron dauricum syrup, only instant powder for asthma and cough, compound is Rhododendron dauricum capsule, compound is Rhododendron dauricum slice.
In recent years, relevant scholar had carried out large quantifier elimination to Rhododendron dauricum contained chemical composition, therefrom isolation identification tens of kinds of compounds.Wherein main Types has flavonoid, volatile oil, coumarins, phenolic acids, triterpenes etc.
Modern pharmacological research shows Rhododendron dauricumly have antibechic, phlegm-dispelling functions of relievining asthma, and step-down, diuretic properties, analgesic activity, to central inhibitory action etc.
Summary of the invention
The object of this invention is to provide a kind of obtain from the Rhododendron dauricum middle separation of drying a kind of there is oleanane type triterpene compounds for the treatment of neurospongioma effect and preparation method thereof.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: (a) is by the Rhododendron dauricum pulverizing of drying, extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 75% ethanol elution, 10 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 85:1,40:1,20:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), 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 70%, collect 8 ~ 10 column volume elutriants, elutriant 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.
Pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the gliomatous medicine of preparation treatment.
The application of described pharmaceutical composition in the gliomatous medicine of preparation treatment.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
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 the change of U251 cell hole apoptosis rate after compound (I) process.
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: Rhododendron dauricum (10kg) that (a) is dry pulverizes, (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 (398g) 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 (163g); C in () step (b), 75% ethanol elution medicinal extract 200-300 order purification on normal-phase silica gel is separated, successively with volume ratio be 85:1 (8 column volumes), the methylene chloride-methanol gradient elution of 40:1 (8 column volumes), 20:1 (8 column volumes), 12:1 (10 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (49g) 200-300 order purification on normal-phase silica gel is separated further 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 (5 column volumes) obtains 3 components; E in () step (d), component 2 (25g) the reverse phase silica gel ODS-C18 of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 8 ~ 10 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I) (44mg).
Structural identification: brown amorphous powder; HR-ESIMS shows [M+Na]
+for m/z523.3416, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
31h
48o
5, degree of unsaturation is 8.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 500MHz): H-1 (1.57, m), H-1 (2.39, m), H-2 (2.10, m), H-2 (2.52, m), H-5 (1.94, m), H-6 (1.46, m), H-6 (1.51, m), H-7 (1.18, m), H-7 (1.42, m), H-9 (1.82, dd, J=10.9, 6.6), H-11 (1.92, m), H-11 (2.01, m), H-12 (5.41, t, J=3.5), H-15 (1.64, d, J=14.0), H-15 (2.68, d, J=14.1), H-18 (2.51, dd, J=14.1, 3.4), H-19 (1.15, m), H-19 (1.50, m), H-21 (1.41, m), H-21 (1.52, m), H-22 (1.14, m), H-22 (2.26, m), H-23 (1.81, s), H-24 (4.85, s), H-24 (5.06, s), H-25 (1.08, s), H-26 (1.06, s), H-27 (1.11, s), H-28 (3.39, d, J=10.9), H-28 (3.92, d, J=10.9), H-29 (3.08, br, s, 2H), H-30 (0.87, s), 3-OMe (3.54, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 125MHz): 35.1 (CH
2, 1-C), 29.3 (CH
2, 2-C), 174.8 (C, 3-C), 147.9 (C, 4-C), 38.2 (CH, 5-C), 26.4 (CH
2, 6-C), 32.3 (CH
2, 7-C), 40.2 (C, 8-C), 38.5 (CH, 9-C), 41.1 (C, 10-C), 23.7 (CH
2, 11-C), 124.6 (CH, 12-C), 141.9 (C, 13-C), 48.3 (C, 14-C), 44.2 (CH
2, 15-C), 213.1 (C, 16-C), 54.2 (C, 17-C), 46.3 (CH, 18-C), 43.0 (CH
2, 19-C), 36.4 (C, 20-C), 30.3 (CH
2, 21-C), 25.5 (CH
2, 22-C), 22.2 (CH
3, 23-C), 113.7 (CH
2, 24-C), 20.3 (CH
3, 25-C), 17.5 (CH
3, 26-C), 27.1 (CH
3, 27-C), 70.6 (CH
2, 28-C), 73.7 (CH
2, 29-C), 19.3 (CH
3, 30-C), 51.1 (CH
3, 3-OMe), carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains hydroxyl (3376cm
-1), ester group (1742cm
-1), carbonyl (1708cm
-1) and alkene (1624cm
-1) group.
1h-NMR stave this compound bright contains five methyl signals [δ H0.87 (s, Me-30), 1.06 (s, Me-26), 1.08 (s, Me-25), 1.11 (s, Me-27), 1.81 (s, Me-23)], a methoxyl group [δ H3.54 (s, 3-OMe)], an olefinic methene proton signal [δ H4.85 (s, H-24), 5.06 (s, H-24)], two containing Oxymethylene proton signal [δ H3.08 (br, s, 2H, H-29), 3.39 (d, J=10.9Hz, H-28), 3.92 (d, J=10.9, H-28)], and an olefinic methyne [δ H5.41 (t, J=3.5Hz, H-12)].
13cNMR composes display 31 resonance carbon signal, comprises six methyl, 12 methylene radical (olefinic methylene radical, two containing Oxymethylene), four methynes (an olefinic methyne), and nine quaternary carbons (two carbonyls, two alkene quaternary carbons).Above-mentioned data show that this compound is 3,4-open loop-oleanane type triterpene compounds.In HMBC spectrum, Me-23 (δ H1.81) and H
2-24 (δ H4.85 and 5.06) and C-4 (δ C147.9) and C-5 (δ C38.2); Me-25 (δ H1.08) and C-1 (δ C35.1), C-5 (δ C38.2), C-9 (δ C38.5) and C-10 (δ C41.1); H
2-1 (δ H1.57 and 2.39), H
2the above-mentioned inference of the relevance verification of-2 (δ H2.10 and 2.52) and 3-OMe (δ H3.54) and C-3 (δ C174.8), also shows that C-3 position is connected with a carboxymethyl simultaneously.In HMBC spectrum, H
2-15 (δ H1.64 and 2.68), H
2with the dependency of C-16 (δ C213.1) ,-28 (δ H3.39 and 3.92) and H-22 (δ H1.14) show that C-16 is ketone carbonyl.In HMBC spectrum, hydroxyl-methylene proton signal δ H3.08 and methyl carbon (δ C19.3), C-19 (δ C43.0), the dependency of C-20 (δ C36.4) and C-21 (δ C30.3), and in NOESY spectrum with the dependency of Me-30 (δ H0.87) and H-12 (δ H5.41), H-18 (δ H2.51) shows that C-29 is hydroxy methylene, C-30 is methyl.In addition, C-28 is also hydroxy methylene; In HMBC spectrum, H
2-28 (δ H3.39 and 3.92) and C-16 (δ C213.1), C-17 (δ C54.2), C-18 (δ C46.3) and C-22 (δ C25.5), and the above-mentioned inference of relevance verification of proton signal δ H3.47 and H-18 (δ H2.51) in NOESY spectrum.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
This experiment adopts human brain malignant glioblastoma U251 cell strain, purchased from American Type Culture Collection.DMEM high glucose medium purchased from American Hyclone company; Sodium-chlor, sodium hydroxide, Repone K, chlorination oxygen, sodium hydroxide, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, methyl alcohol are purchased from chemical plant, Nanjing.Compound (I) is made by oneself, and HPLC normalization method purity is greater than 98%.Glycine, Tris, Tween20, dimethyl sulfoxide (DMSO) (DMEM) purchased from American Sigma company, Aimexin-V/PI Xi Bao Coverlet dies test kit purchased from American Invitrogen company, cell pyrolysis liquid, 0.25% trypsinase, CellCountingKit-8 test kit, penicillin, Streptomycin sulphate, Hoechst33258, nucleus dyestuff DAPI, PMSF, Westernblot gel reagent preparation box, Nuclear extract extracts test kit, coomassie brilliant blue staining liquid, standard protein, 5X protein denaturation damping fluid box, the super quick luminescent solution of ECL, developing fixing test kit, X-ray film, nitrocellulose filter, anti-fluorescent quenching mounting liquid grinds purchased from the green skies, Jiangsu biology the institute that makes internal disorder or usurp.Foetal calf serum is purchased from Hangzhou folium ilicis chinensis biotechnology company; Rabbit anti-human P65 antibody purchased from American NOVUS company; Rabbit anti-human HistonH3 antibody purchased from American CST company; Goat antirabbit HRP bis-anti-purchased from American SantaCruz company; Goat antirabbit fluorescence two anti-purchased from American EarthOx company.
-20 DEG C of cryogenic refrigerators (Haier Qingdao),-80 DEG C of cryogenic refrigerators (Haier Qingdao), cryogenic refrigerator (Haier Qingdao), supercentrifuge (flying crane Shanghai), culture dish/plate (the Coming U.S.), centrifuge tube (the Coming U.S.), cell culture incubator (the Thermo U.S.), inverted microscope (Olympus Japan), fluorescence inverted phase contrast microscope (ZEISS Germany), multipurpose thermostatic water bath (leap Shanghai), electronic balance instrument (Shanghai secret scientific instrument), Bechtop (ARITECH Japan), micropipet (Dragon Finland), microplate reader (Bio-RAD Germany), flow cytometer (BDAccuriC6
The U.S.).
Two, test method
1, cell cultures and going down to posterity
People glioblastoma cell line U251 cultivates in the DMEM high glucose medium containing 10% foetal calf serum, 1% penicillin, 1% Streptomycin sulphate, in 37 DEG C, and 5%CO
2cellar culture in incubator, gave replacing fresh culture every 2 days.After about cytogamy to 80%, absorb substratum, and wash 3 times with the Sterile phosphate phthalate buffer of preheating, then the cell dissociation buffer 1mL added in culture dish containing 0.25% race's proteolytic enzyme and 0.02%EDTA digests about 2 minutes, observation of cell under light microscopic, visible cell bounces back gradually and becomes circle, intercellular spaces broadens, now remove cell dissociation buffer, add perfect medium 4mL and stop digestion, 1mL rifle head is is softly blown and beaten and is made cell suspension, centrifugal 5 minutes of low speed centrifuge 1000rpm, absorb supernatant liquor, add perfect medium resuspended, after soft piping and druming evenly, rule of thumb go down to posterity by 1:3 ~ 1:5, cell after going down to posterity is placed in incubator and continues training, go down to posterity about weekly 2 times, logarithmic phase is in keep cell.Method for cell count: cell counting count board wiped clean, and cover glass is covered on tally; Cell suspension is slowly instilled tally along cover glass edge, light Microscopic observation, cell is transparent, refractivity is well viable cell, count the viable count in 4 block plaid.Viable count mean value × extension rate × 10 in every cell count=each grid
4.
2, CCK-8 detects cells survival rate
The U251 cell of taking the logarithm vegetative period, makes single cell suspension and counts with the 0.25% trysinization piping and druming cell containing EDTA, with perfect medium adjustment cell concn about 2 × l0
4individual/mL, is inoculated in 96 well culture plates by cell, every hole 100 μ L, is placed in 37 DEG C, 5%CO
2cultivate 12 hours in incubator, absorb former substratum, different treatment is given: blank group (equivalent perfect medium) by grouping, compound (I) group establishes 1,2,4mmol/L, often group establishes three repeating holes, the aseptic each hole of PBS closed perimeter, is placed in 37 DEG C, 5%CO again
2in incubator, take out 96 orifice plates after cultivating 24,48,72 hours respectively, absorb each group of original substratum, be replaced by 100 μ LDMEM nutrient solutions, background group is acellular only has DMEM nutrient solution.Every hole adds 10 μ LCCK-8 solution, is placed in incubator and continues cultivation 2 hours.Measure every hole absorbance (OD value) by full-automatic microplate reader, wavelength is set in 450mn place, gets the average in the multiple hole of often group.Above-mentioned experimental procedure repeats 3 times, calculating mean value.Cells survival rate calculation formula is as follows: cells survival rate=(experimental group OD value-background group OD value)/(control group OD value-background group OD value) × 100%, draws growth-inhibiting graphic representation, and the Epidemiological Analysis that takes statistics.
3, Hoechst33258 dyeing
The green skies company Hoechst33258 staining fluid of experiment Bian, during apoptosis, can see that the nucleus of apoptosis is fine and close dense dye, or is the fine and close dense dye of chunky shape.According to cells survival rate result, choose 2mmol/L compound (I) effect 48 hours conditions as Hoechst33258 dyeing.By cell dissociation, centrifugal, counting, plant in 24 orifice plates after adjustment cell concn, be placed in 37 DEG C, 5%CO
2cultivate 12 hours in incubator, absorb original substratum, give different treatment by grouping: blank group (equivalent perfect medium), compound (I) group establishes 1,2,4mmol/L, the aseptic each hole of PBS closed perimeter, is placed in 37 DEG C, 5%CO again
2in incubator, continue cultivation and take out 24 orifice plates after 48 hours, absorb each group of original substratum, aseptic PBS washes 3 times, and every hole adds 0.5mL poly formic acid fixed cell 15 minutes, and PBS washes 3 times, each hole adds 100 μ LHoechst33258 staining fluids, dye under room temperature dark surrounds 10min, removes staining fluid, and PBS shaking table rocks flushing 3 times, each 5 minutes, wash most liquid, every hole drips an anti-fluorescent quenching liquid, takes pictures and the change of observation of cell karyomorphology under the fluorescent microscope of 340nm wavelength.
3, Flow cytometry cell follows and dies
According to cells survival rate result, choose the particular point in time of dying as detection cell pillbox for 48 hours.Get and be in logarithmic phase and U251 cell in good condition, had digestive transfer culture routinely, be inoculated in 37 DEG C, 5%CO in 6 orifice plates
2hatch 12h in incubator, give different treatment by grouping: blank group (equivalent perfect medium), compound (I) group establishes 1,2,4mmol/L, be placed in 37 DEG C, 5%CO
2continue cultivation in incubator after 48 hours, be washed till by original nutrient solution in 5mL centrifuge tube, PBS washs attached cell 2 times, adds the appropriate trypsin digestion cell containing EDTA.Incubated at room, to when piping and druming can make attached cell blow and beat gently, exhausts pancreatin cell dissociation buffer as far as possible.Add original cell culture fluid of collection, slightly mix, transfer in 5mL centrifuge tube, centrifugal 5 minutes of 1000g, abandons supernatant, collecting cell, with PBS re-suspended cell counting gently.Get 50,000 resuspended cells, centrifugal 5 minutes of 1000g, abandons supernatant, adds 100 μ LAimexinV-FITC in conjunction with liquid re-suspended cell gently.Add 2mLAnnexinV-FITC, gently mixed hook.Room temperature lucifuge brightness educates 10 minutes.Centrifugal 5 minutes of 1000g, abandons supernatant, adds 2 μ L iodate third and instigates staining fluid, gently mixed hook, and ice bath lucifuge places 15min.Carry out flow cytomery immediately, AmexinV-FITC is green fluorescence, and PI is red fluorescence.Record AnnexinV-FITC+PI+ and AnnexinV-FITC+PI-is as apoptotic cell, and each counting 10000 cells, calculate each group of apoptosis rate.
4, statistical procedures
Adopt SPSS16.0 to carry out statistical analysis, test all data and all represent the result repeating for 3 times to test, represent with mean scholar standard deviation.The variance analysis adopting Factorial Design data is compared between the group of survival rate, Bian one-way analysis of variance is compared between the group of apoptosis rate, inspection level be a=0.05, P<0.05 for there being significant difference, P<0.01 is that pole has significant difference.
Three, result and conclusion
1, compound (I) is on the impact of U251 cells survival rate
Compound (I) suppresses the display of U251 cell survival growth CCK-8 experimental result, after different concns compound (I) effect U251 cell, along with activity increases, action time lengthens, U251 cells survival rate obviously declines, the main effect F=945.8 (P<0.01) of variance analysis display compound (I) concentration factor; Action time factor main effect F=302.67 (P<0.01), the interaction F=45.7 (P<0.01) of concentration, time factor.Associative list 1, can find that the restraining effect of compound (I) to U251 cells survival rate is time, dose-dependently, namely compound (I) concentration is higher, and action time is longer, and its restraining effect is stronger.By calculating the IC of compound (I) effect 24h, 48h, 72h
50value is respectively, 3.67mmol/L, 1.37mmol/L, 1.09mmol/L.
2, compound (I) to be died morphologic impact on U251 cell pillbox
Normal group and compound (I) treatment group cell dye through Hochest33258, the visible cellular control unit nuclear morphology rule of fluorescence microscopy Microscopic observation, and in light blue uniform coloring; And through 2mmol/L compound (I) process after 48 hours, compared with control group, pyknosis in various degree appears in nucleus, color is comparatively bright, the dense dye of stained dense, and nuclear fragmentation is obvious, and visible typical apoptotic body.Change prompting compound (I) of above Cell Image Analyzer has induction U251 cells apoptosis.
3, compound (I) is on the impact of U251 apoptosis rate
Control group and each treatment group row Flow cytometry after AnnexinV-FITC/PI dyeing, result shows: control group, 1mmol/L compound (I), 2mmol/L compound (I) and 4mol/L compound (I) process 48 hours group natural death of cerebral cells rates are respectively 3.30 ± 0.98,14.97 ± 2.67,27.53 ± 5.51 and 55.90 ± 5.1%.Compared with control group, the apoptosis rate of each treatment group cell significantly increases (P<0.01).The results are shown in Figure 3.
Conclusion: compound (I) plays the restraining effect to human brain malignant glioblastoma U251 cell by antiproliferative effect and cell death inducing.These clinical drug research being found to be compound (I) treatment glioma provide foundation.
U251 cells survival rate (mean ± standard deviation) after table 1 different concns compound (I) process effect 24 ~ 72h
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:9 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, 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 (7)
1. there is the compound (I) of following structural formula,
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: (a) is by the Rhododendron dauricum pulverizing of drying, extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 75% ethanol elution, 10 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 85:1,40:1,20:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), 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 70%, collect 8 ~ 10 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: in step (a), extracts, united extraction liquid with 80% alcohol heat reflux.
4. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the gliomatous medicine of preparation treatment.
7. the application of pharmaceutical composition according to claim 5 in the gliomatous medicine of preparation treatment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294727A (en) * | 2015-10-09 | 2016-02-03 | 杭州启澄科技有限公司 | Novel clerodane diterpenoid compound, preparation method of clerodane diterpenoid compound and medical application of novel clerodane diterpenoid compound |
| CN105294818A (en) * | 2015-12-07 | 2016-02-03 | 西宁意格知识产权咨询服务有限公司 | New triterpenoid as well as preparation method and medical application thereof |
| CN115057907A (en) * | 2022-08-18 | 2022-09-16 | 中国中医科学院中药研究所 | Tripterine coumarin derivative and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105198845A (en) * | 2015-10-09 | 2015-12-30 | 吴正锋 | Novel diterpene compound for treating glioma |
-
2016
- 2016-01-29 CN CN201610066457.1A patent/CN105481936A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105198845A (en) * | 2015-10-09 | 2015-12-30 | 吴正锋 | Novel diterpene compound for treating glioma |
Non-Patent Citations (2)
| Title |
|---|
| 付晓丽等: "满山红化学成分的研究", 《中草药》 * |
| 桑树荣等: "满山红中三萜类成份的研究", 《中国林副特产》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294727A (en) * | 2015-10-09 | 2016-02-03 | 杭州启澄科技有限公司 | Novel clerodane diterpenoid compound, preparation method of clerodane diterpenoid compound and medical application of novel clerodane diterpenoid compound |
| CN105294818A (en) * | 2015-12-07 | 2016-02-03 | 西宁意格知识产权咨询服务有限公司 | New triterpenoid as well as preparation method and medical application thereof |
| CN115057907A (en) * | 2022-08-18 | 2022-09-16 | 中国中医科学院中药研究所 | Tripterine coumarin derivative and preparation method and application thereof |
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Application publication date: 20160413 |