CN105748497A - Pharmaceutical composition for treating glioma - Google Patents
Pharmaceutical composition for treating glioma Download PDFInfo
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- IDADOMDFNZGHME-UNMCSNQZSA-N CC1(C)OC=C2c(c(OC)ccc3C(C4=O)=O)c3O[C@@]4(C3=CC=CCC3)O[C@H]12 Chemical compound CC1(C)OC=C2c(c(OC)ccc3C(C4=O)=O)c3O[C@@]4(C3=CC=CCC3)O[C@H]12 IDADOMDFNZGHME-UNMCSNQZSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of medicine and particularly relates to a pharmaceutical composition for treating glioma.The pharmaceutical composition comprises a component A, a component B, a component C and a component D, wherein the component A is diterpenoids (I) extracted from callicarpa nudiflora, the component B is withanolides (II) extracted from the dried whole herb of asiatic plantain, the component C is triterpenoid saponins echinoside A purchased from the market, and the component D is flavonoids (IV) extracted from the overground part of vervain.In-vitro experiment prove that the pharmaceutical composition can inhibit the human brain malignant glioblastoma U251 cells by inhibiting cell proliferation and inducing cell apoptosis and can be developed into medicine for treating the glioma.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of for treating gliomatous pharmaceutical composition.
Background technology
Glioma is called for short glioma, also referred to as glioma, is modal primary central nervous system tumor, accounts for the half of all intracranial primary tumo(u)rs, and broad sense refers to the tumor of all neuroepithelial origin, and narrow sense refers to the tumor coming from all kinds of glial cell.It is divided into astrocytoma, few branched oligodendrocyte tumor, ependymoma, Mixed Gliomas, choroid plexus tumor, uncertain neuroepithelial tissue tumor of originating, neuron and neuron neuroglia mixed tumor, pinus parenchymal tumor, embryonal tumors, neuroblastoma tumor according to World Health Organization (WHO) (WHO) classification schemes of 1999.
At present, the main treatment means of glioma is operative treatment, but operative treatment risk is high.And can first give hormone therapy to glioblastoma at present, with dexamethasone medicine effect.Dexamethasone medicine is except can alleviating cerebral edema, and has the effect suppressing growth of tumour cell, symptom also can be made to alleviate, then row operative treatment again.And to there being the patient of epilepsy, preoperative and postoperative should give Antiepileptic Drugs.Gliomatous complementary therapy Embolization therapy: include physical property embolus and Chemoembolization two kinds: the former itself blocks supply artery of the tumor and promote thrombosis, the latter is vasoactive wall endotheliocyte then, bring out thrombosis, thus reaching to reduce the purpose of meningioma blood confession；Two methods are all as preoperative complementary therapy, and are only limited to the meningioma that external carotid artery blood supply is master.Physics embolus includes the embolus that various different materials is fabricated to, the most desirable with silicone rubber barium agent bead (diameter 1mm).But it have been found that for a lot of glioma diseases, not only lack effective medicine, and medicament selection is little.
Summary of the invention
It is an object of the invention to provide a kind of novel for treating gliomatous pharmaceutical composition.
The above-mentioned purpose of the present invention is achieved by the techniques below scheme:
One is used for treating gliomatous pharmaceutical composition, it by component A, component B, form C and component D and form, component A is the diterpene compound (I) with following structural formula:
Component B is the liquor-saturated eggplant lactone compound (II) with following structural formula:
Component C is triterpene saponin componds echinosideA, structure formula III:
Component D is the flavone compound (IV) with following structural formula:
Wherein, it is the weight of 100% according to total amount,
Component A is: 52-63%；
Component C is: 22-25%；
Component B is: 8-12%
Component D sum is: 8-12%
Component B and component D sum are no less than 18%.
Further, the preparation of component A diterpene compound (I) comprises following operating procedure: the dry aerial parts of Callicarpa nudiflora are pulverized by (a), extract with 70～80% 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 in () step (a), n-butyl alcohol takes thing macroporous resin remove impurity, first with 6 column volumes of 10% ethanol elution, then with 8 column volumes of 70% ethanol elution, collect 70% eluent, and concentrating under reduced pressure obtains 70% ethanol elution concentrate；In (c) step (b) 70% ethanol elution concentrate with purification on normal-phase silica gel separate, successively with volume ratio be 40:1,20:1,10:1 and 5:1 methylene chloride-methanol gradient elution obtain 4 components；D in () step (c), component 4 separates further by purification on normal-phase silica gel, successively with volume ratio be 8:1,5:1 and 2:1 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 70%, collecting 8～12 column volume eluents, eluent concentrating under reduced pressure obtains pure diterpene compound (I)；
Component D flavone compound (IV) separates from Herba Verbenae and obtains, concrete separating step:
A, the dry aerial parts of Herba Verbenae are pulverized, extract with 70～80% alcohol heat reflux, united extraction liquid, be 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；
Acetic acid ethyl ester extract macroporous resin remove impurity in B, step A, first with 10 column volumes of 10% ethanol elution, then with 12 column volumes of 75% ethanol elution, collects 75% ethanol elution, and 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 80:1,40:1,20:1,10:1 and 1:1 methylene chloride-methanol gradient elution obtain 5 components；
In D, 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；
The reverse phase silica gel that in E, step D, component 2 is bonded by octadecylsilane separates, and with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collects 9～13 column volume eluents, and eluent concentrating under reduced pressure obtains pure flavone compound (IV)；
The percentage concentration of above ethanol each means concentration expressed in percentage by volume.
As preferably, extracting, with alcohol heat reflux, the concentration of alcohol all adopted described in step (a) and step A is 75%.
As preferably, macroporous resin described in step (b) and step B is AB-8 type macroporous adsorbent resin.
One is used for treating gliomatous pharmaceutical composition, comprises the aforementioned pharmaceutical compositions of therapeutically effective amount and pharmaceutically acceptable carrier.
As preferably, be selected from a kind of of pharmaceutically acceptable carrier or mixing that both are above: lactose, sucrose, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate, stearic acid, cellulosic lower alkyl ether, corn starch, potato starch, natural gum, fatty acid, glycerol monostearate.
As preferably, effective dose every day of the first pharmaceutical composition is 0.001-0.006g/kg human body.
There is advantages that
One, pharmaceutical composition is by suppressing cell proliferation and inducing cell apoptosis to play the inhibitory action to human brain malignant glioblastoma U251 cell, and this pharmaceutical composition is compared simple component A and simple component D and suppressed human brain malignant glioblastoma U251 to be remarkably reinforced.Visible, component A, B, C and D combination there is synergism.
Two in use, pharmaceutical composition can directly use, or can be equipped with pharmaceutically acceptable carrier make medicine form use.Pharmaceutical composition compares pure material diterpene compound (I); because introducing component D also there is the effect that protection is neural; the side effect of human body to be reduced by relatively; therefore advantageously in preparing into the gliomatous medicine for the treatment of with carrier, the clinical medicine in treatment glioma provides a kind of new selection.
Accompanying drawing explanation
Fig. 1 is diterpene compound (I) structural formula；
Fig. 2 is that diterpene compound (I) calculates ECD and experiment ECD figure；
Fig. 3 is the structural formula of liquor-saturated eggplant lactone compound (II)；
Fig. 4 is liquor-saturated eggplant lactone compound (II) two dimension1H-1HCOSY composes；
Fig. 5 be flavone compound (IV) structural formula；
Fig. 6 is that flavone compound (IV) calculates ECD and experiment ECD figure.
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: one is used for treating gliomatous pharmaceutical composition, according to weight percent meter, it by 55% component A, 23% component B, 10% form C and 12% component D and form, component A is the diterpene compound (I) with following structural formula:
Component B is the liquor-saturated eggplant lactone compound (II) with following structural formula:
Component C is triterpene saponin componds echinosideA, structure formula III:
Component D is the flavone compound (IV) with following structural formula:
Wherein, in component A, the separation preparation of diterpene compound (I) and structural identification are as follows:
Main material, reagent source and instrument type:
Ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
A the dry aerial parts (10kg) of () Callicarpa nudiflora are pulverized, (25L × 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste (6L), extract with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) successively, respectively obtain petroleum ether extract, acetic acid ethyl ester extract (377g) and n-butyl alcohol extract；Acetic acid ethyl ester extract AB-8 macroporous resin remove impurity in (b) step (a), first with 6 column volumes of 10% ethanol elution, again with 8 column volumes of 70% ethanol elution, collecting 70% eluent, concentrating under reduced pressure obtains 70% ethanol elution concentrate (129g)；C in () step (b), 70% ethanol elution concentrate purification on normal-phase silica gel separates, obtain 4 components with the methylene chloride-methanol gradient elution that volume ratio is 40:1 (8 column volumes), 20:1 (8 column volumes), 10:1 (8 column volumes) and 5:1 (10 column volumes) successively；D in () step (c), component 4 (24g) separates further by purification on normal-phase silica gel, obtain 3 components with the methylene chloride-methanol gradient elution that volume ratio is 8:1 (8 column volumes), 5:1 (10 column volumes) and 2:1 (8 column volumes) successively；E reverse phase silica gel that in () step (d), component 2 (15g) is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collecting 8～12 column volume eluents, eluent concentrating under reduced pressure obtains pure diterpene compound (I) (42mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z449.1608, can obtain molecular formula in conjunction with nuclear-magnetism feature is C24H26O7, degree of unsaturation is 12.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6, 600MHz): H-1 (5.58, s), H-3 (2.18, m), H-3 (1.96, m), H-6 (5.47, d, J=6.0), H-7 (5.12, d, J=6.0), and H-11 (6.92, s), H-15 (6.74, d, J=2.4), H-16 (7.58, d, J=2.4), H-17 (2.45, s), H-18 (1.11, s), and H-19 (1.11, s), H-20 (1.47, s), 1-OCOCH3(1.91, s), 6-OCOCH3(2.14, s)；Carbon-13 nmr spectra data δC(ppm, DMSO-d6, 125MHz): 91.7 (CH, 1-C), 201.3 (C, 2-C), 43.7 (CH2, 3-C), 30.1 (C, 4-C), 99.7 (C, 5-C), (81.2 CH, 6-C), 79.6 (CH, 7-C), 128.6 (C, 8-C), (140.1 C, 9-C), 46.4 (C, 10-C), 104.2 (CH, 11-C), (155.3 C, 12-C), 131.0 (C, 13-C), 131.9 (C, 14-C), (105.8 CH, 15-C), 145.8 (CH, 16-C), 16.8 (CH3, 17-C), 29.3 (CH3, 18-C), 24.2 (CH3, 19-C), 29.1 (CH3, 20-C), 171.1 (C, 1-OCOCH3), 20.7 (CH3, 1-OCOCH3), 172.8 (C, 6-OCOCH3), 21.9 (CH3, 6-OCOCH3)；Carbon atom labelling is referring to Fig. 1.Infrared spectrum shows that this compound contains aromatic carbon (3012nm and 1613nm).1HNMR spectrum 4 methyl signals δ H1.11 (H-18 and H-19) of display, 1.47 (H-20), 2.45 (H-17).Olefinic proton signals δ H6.74 (H-15, d, J=2.4) and 7.58 (H-16, d, J=2.4Hz) that two intercouple show that this compound contains a fused furan ring.13CNMR shows 24 carbon signals, including 6 aromatic series carbon signal δ C104.2, and 128.6,131.0,131.9,140.1 and 155.3.These data show, this compound is the diterpene-kind compound condensed with furan nucleus.In HMBC spectrum, (δ H6.92, s) with C-12 (δ C155.3) and C-13 (δ C131.0) for H-11；H3-17 (δ H2.45, s) with C-8 (δ C128.6), C-13 (δ C131.0), the relevance verification of C-14 (δ C131.9) and C-15 (δ C105.8) phenyl ring and this inference of furan nucleus conjugation.Additionally, with the dependency of corresponding acetoxyl group (δ C171.1, δ H1.91 and δ C172.8, δ H2.14), C-1 (δ C91.7) and C-6 (δ C81.2) shows that C-1 and C-6 position is respectively connected with an acetoxyl group.In HMBC spectrum, the dependency of H-7 and C-5 illustrates that this compound contains oxetanes part.In NOESY spectrum, H-1 and H-6 and H3The dependency of-20 shows that H-1 and H-6 is beta 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 it is shown in figure 1, spatial configuration is determined by ECD test further, theoretical value and experiment value basically identical (Fig. 2).
Liquor-saturated eggplant lactone compound (II) in component B separates preparation and structural identification is as follows:
Main material, reagent source:
Ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Aβ25-35It is purchased from sigma company of the U.S..MTT (nitroblue tetrazolium) is purchased from Amresco company of the U.S..LDH mensuration test kit is purchased from Nanjing and builds up biological company limited.Acridine orange (AO) is purchased from sigma company of the U.S..Ethidum Eremide (EB) is purchased from sigma company of the U.S..RNase enzyme is purchased from sigma company of the U.S..E.C. 3.4.21.64 is purchased from sigmaAnnexin company of the U.S..V-FITC cell apoptosis detection kit is purchased from Nanjing KaiJi Biology Science Development Co., Ltd.D-MEM/F12 culture medium dry powder is purchased from GibcoL company of the U.S..Horse serum is purchased from hycLon company of the U.S..Hyclone is purchased from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering company limited.Poly-D-lysine (PLL) is purchased from sigma company of the U.S..Compound (I) is made by oneself, and HPLC normalization purity is more than 98%.
Instrument type: low-temperature and high-speed centrifuge, U.S. Heraeus；CO2 gas incubator, U.S. SHELL/JB；Superclean bench, Suzhou Decontamination Equipment Plant；The vigorous MK3 microplate reader of thunder, Finland Thermolabsystems；Fluorescence microscope, Germany Leica；Flow cytometer EpicsXL, CouLter company of the U.S.；Electrophoresis system, Liuyi Instruments Plant, Beijing；The vigorous MK3 microplate reader of thunder, Finland Thermolabsystems；Fluorescence microscope, Germany Leica；Flow cytometer EpicsXL, CouLter company of the U.S..
Preparation method:
A the dry herb (8kg) of () Herba Plantaginis is pulverized, (25L × 3 time) are extracted with 85% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste (6L), extract with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) successively, respectively obtain petroleum ether extract, acetic acid ethyl ester extract (333g) and n-butyl alcohol extract；Acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in (b) step (a), successively with 20% ethanol (8L) and 80% (12L) ethanol elution, collecting 80% ethanol elution, concentrating under reduced pressure obtains 80% ethanol elution thing extractum (152g)；C in () step (b), 80% ethanol elution extractum purification on normal-phase silica gel separates, obtain 5 components with the methylene chloride-methanol gradient elution that volume ratio is 90:1 (7 column volumes), 60:1 (7 column volumes), 30:1 (8 column volumes), 15:1 (7 column volumes) and 1:1 (5 column volumes) successively；D in () step (c), component 4 (38g) separates further by purification on normal-phase silica gel, obtain 3 components with the methylene chloride-methanol gradient elution that volume ratio is 20:1 (10 column volumes), 12:1 (8 column volumes) and 5:1 (6 column volumes) successively；E reverse phase silica gel that in () step (d), component 2 (11g) is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 65%, collecting 8-11 column volume eluent, eluent concentrating under reduced pressure obtains pure liquor-saturated eggplant lactone compound (II) (26mg).
Structural identification:
Colourless powder；HR-ESIMS shows [M+Na]+For m/z493.2604, can obtain molecular formula in conjunction with nuclear-magnetism feature is C28H38O6, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δH(ppm,DMSO-d6null,500MHz): H-2 (2.99，dd，J=18.8，3.3)，H-2(2.49，dd，J=18.8，2.2)，H-3(4.29，dd，J=3.3，2.2)，H-4(3.69，d，J=9.4)，H-6(3.37，d，J=3.2)，H-7(2.14，dt，J=14.6，3.5)，H-7(1.29，dd，J=14.6，11.3)，H-8(1.62，m)，H-9(1.90，m)，H-11(1.18，m)，H-11(0.94，dd，J=13.2，4.0)，H-12(1.88，m)，H-12(1.22，m)，H-14(1.05，m)，H-15(1.64，m)，H-15(1.17，m)，H-16(1.68，m)，H-16(1.36，m)，H-17(1.12，m)，H-18(0.66，s)，H-19(4.19，d，J=9.0)，H-19(3.86，d，J=9.0)，H-20(1.98，m)，H-21(0.97，d，J=6.7)，H-22(4.35，dt，J=13.3，3.5)，H-23(2.42，brdd，J=17.0，13.3)，H-23(1.92，m)，H-27(1.88，s)，H-28(1.94，s)，6-OH(3.16，d，J=9.4)；Carbon-13 nmr spectra data δC(ppm,DMSO-d6, 125Hz): 208.0 (C, 1-C), 42.8 (CH2, 2-C), 74.3 (CH, 3-C), 69.7 (CH, 4-C), 60.8 (C, 5-C), 56.9 (CH, 6-C), 29.8 (CH2, 7-C), 28.8 (CH, 8-C), 36.9 (CH, 9-C), 50.1 (C, 10-C), 22.1 (CH2, 11-C), 39.3 (CH2, 12-C), 43.2 (C, 13-C), 54.8 (CH, 14-C), 24.2 (CH2, 15-C), 27.3 (CH2, 16-C), 52.3 (CH, 17-C), 11.8 (CH3, 18-C), 63.5 (CH2, 19-C), 38.9 (CH, 20-C), 13.5 (CH3, 21-C), 78.4 (CH, 22-C), 29.7 (CH2, 23-C), 149.1 (C, 24-C), 122.1 (C, 25-C), 167.2 (C, 26-C), 12.6 (CH3, 27-C), 20.7 (CH3, 28-C)；Carbon atom labelling is shown in Fig. 3.1H-1HCOSY spectrum (Fig. 4) shows that liquor-saturated eggplant lactone compound (II) is containing-CH2CH (OR)-part-structure [δ H2.99 (dd, J=18.8,3.3Hz, H-2 β), 2.49 (dd, J=18.8,2.2Hz, H-2 α), 4.29 (dd, J=3.3,2.2Hz, H-3)]；In HMBC spectrum, H-3 and C-1 (δC208.0), H-2 β and C-4 (δC69.7), and H-3 and C-5 (δC60.8) dependency further demonstrate that said structure speculates.These data show, liquor-saturated eggplant lactone compound (II) meets, containing other circulus, the requirement that degree of unsaturation is 10.In liquor-saturated eggplant lactone compound (II), the coupling between H-3 and H-4 shows there is about dihedral angle of 90 ° between the two.C-19 (δ in liquor-saturated eggplant lactone compound (II)C63.5)、H-19[δH4.19 (d, J=9.0Hz), 3.86 (d, J=9.0Hz)] and H-3 (δH4.29) chemical shift shows one oxo bridge of existence between C-3 and C-19.C-19 (δ in HMBC spectrumC63.5) and H-3 (δH4.29) and C-3 (δC74.3) and H-19 α (δH4.19) the above-mentioned inference of relevance verification.Comprehensive HMBC,1H-1The two-dimensional spectrums such as HCOSY, ROESY and pertinent literature, it is determined that this compound structure is as shown in Figure 3.
In component D, flavone compound (IV) separation preparation and structural identification are as follows:
Medical material and 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.The dry aerial parts of Herba Verbenae are purchased from Hui nationality's Chinese Medicinal Materials Markets, Guangdong, the place of production.
Preparation method: the dry aerial parts (8kg) of Herba Verbenae are pulverized by (a), (25L × 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste (6L), extract with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) successively, concentration, respectively obtains petroleum ether extract, acetic acid ethyl ester extract (315g) and n-butyl alcohol extract；B in () step (a), acetic acid ethyl ester extract dissolved in purified water is to 2L, medical absorbent cotton filters, filtrate separates with AB-8 type macroporous resin (1.5kg), successively with 10% ethanol (10L) and 75% (12L) ethanol elution, collecting 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing extractum (155g)；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 80:1 (10 column volumes), 40:1 (8 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 (36g) 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 (8 column volumes) and 10:1 (5 column volumes) successively；E reverse phase silica gel ODS-C18 that in () step (d), component 2 (12g) is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collecting 9～13 column volume eluents, eluent concentrating under reduced pressure obtains pure flavone compound (IV) (25mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z401.1006, can obtain molecular formula in conjunction with nuclear-magnetism feature is C22H18O6, degree of unsaturation is 14.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6, 400MHz): H-5 (7.99, d, J=8.7), H-6 (6.65, d, J=8.5), H-2 ', 6 ' (7.31, m), H-3 ', 5 ' (7.27, m), H-4 ' (7.31, m), H-5 " (8.01; s), 2 "-Me (1.31, s), 2 "-Me (1.31; s), and 7-OMe (3.89, s)；Carbon-13 nmr spectra data δC(ppm, DMSO-d6null，100MHz): 131.8 (C，2-C)，193.7(C，3-C)，190.3(C，4-C)，113.1(C，4a-C)，126.9(CH，5-C)，104.7(CH，6-C)，163.7(C，7-C)，105.6(C，8-C)，158.1(C，8a-C)，136.3(C，1’-C)，127.8(CH，2’,6’-C)，127.3(CH，3’,5’-C)，127.5(CH，4’-C)，101.8(C，2”-C)，91.7(CH，3”-C)，110.9(C，4”-C)，142.7(CH，5”-C)，21.8(CH3, 2 " and-Me), 21.8 (CH3, 2 " and-Me), 55.2 (CH3, 7-OMe)；Carbon atom labelling is referring to Fig. 5.Infrared spectrum shows that this compound contains aromatic carbon (2964nm, 1695nm and 1591nm).13CNMR spectrum shows 22 carbon signals, comprise three methyl (δ C21.8,2 "-Me；21.8,2 " and-Me and 55.2,7-OMe), nine methines (δ C126.9,5-C；104.7,6-C；127.8,2 ', 6 '-C；127.3,3 ', 5 '-C；127.5,4 '-C；91.7,3 " and-C；142.7,5 " and-C), ten quaternary carbons (δ C131.8,2-C；193.7,3-C；190.3,4-C；113.1,4a-C；163.7,7-C；105.6,8-C；158.1,8a-C；136.3,1 '-C；101.8,2 " and-C；110.9,4 " and-C).Nuclear magnetic data shows that this compound is flavone compound.In NOESY spectrum, H-6 (δ H6.65, d, J=8.5Hz) with methoxyl group proton (δ H3.89, dependency s), and H-5 (δ H7.99, d, J=8.7Hz) and the dependency of H-6, it was shown that C-7 (δ C163.7) position is connected with a methoxyl group.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this flavone compound as shown in Figure 4, and spatial configuration is determined by ECD test further, theoretical value and experiment value basically identical (Fig. 6).
It is disclosed in CN102614209A that component C belongs at publication number, can also buy acquisition in the market.
Embodiment 2: the pharmaceutical composition pharmacological action test in embodiment 1
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 chloride, sodium hydroxide, potassium chloride, chlorination oxygen, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, methanol are purchased from chemical plant, Nanjing.Glycine, Tris, Tween20, dimethyl sulfoxide (DMEM) purchased from American Sigma company；Aimexin-V/PI cell is died test kit purchased from American Invitrogen company；Cell pyrolysis liquid, 0.25% trypsin, CellCountingKit-8 test kit, penicillin, streptomycin, Hoechst33258, nucleus dyestuff DAPI, PMSF, Westernblot gel reagent preparation box, Nuclear extract extraction test kit, coomassie brilliant blue staining liquid, standard protein, 5X protein denaturation buffer box, the super quick luminescent solution of ECL, developing fixing test kit, X-ray film, nitrocellulose filter, anti-fluorescent quenching mounting liquid grind, purchased from the green skies, Jiangsu biology, the institute that makes internal disorder or usurp.Hyclone is purchased from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims 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), high speed centrifuge (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), superclean bench (ARITECH Japan), micropipettor (Dragon Finland), microplate reader (Bio-RAD Germany), flow cytometer (the BDAccuriC6 U.S.).
Two, test method
1, cell is cultivated and goes down to posterity
People glioblastoma cell line U251 cultivate containing 10% hyclone, 1% penicillin, 1% streptomycin DMEM high glucose medium in, in 37 DEG C, 5%CO2Cellar culture in incubator, gave replacing fresh culture every 2 days.After cell fusion is to about 80%, absorb culture medium, and wash 3 times with the Sterile phosphate phthalate buffer of preheating, then in culture dish, add the cell dissociation buffer 1mL containing 0.25% race's protease and 0.02%EDTA digest about 2 minutes, observation of cell under light microscopic, visible cell bounces back gradually and becomes round, intercellular spaces broadens, now remove cell dissociation buffer, add complete medium 4mL and terminate digestion, 1mL rifle head is softly blown and beaten and is made cell suspension, centrifugal 5 minutes of low speed centrifuge 1000rpm, absorb supernatant, add complete medium resuspended, after soft piping and druming uniformly, 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, to keep cell to be in exponential phase.Method for cell count: cell counting count board wiped clean, and coverslip is covered on counting chamber；Cell suspension is slowly dropped into counting chamber, light Microscopic observation along coverslip edge, and cell is transparent, refractivity is well living cells, counts the viable count in 4 block plaid.Viable count meansigma methods × extension rate × 10 in every cell number=each grid4。
2, CCK-8 detects cells survival rate
Take the logarithm the U251 cell of trophophase, make single cell suspension with the 0.25% trypsinization piping and druming cell containing EDTA and count, adjusting cell concentration about 2 × l0 with complete medium4Individual/mL, is inoculated in 96 well culture plates by cell, every hole 100 μ L, is placed in 37 DEG C, 5%CO2Incubator is cultivated 12 hours, absorb former culture medium, different disposal is given: blank group (equivalent complete medium) by packet, pharmaceutical composition group sets 1,2,4mmol/L, simple component A diterpene compound (I) 2mmol/L sets one group, simple component D flavone compound (IV) 2mmol/L sets one group, six groups altogether.Often group sets three repeating holes, the aseptic each hole of PBS closed perimeter, is again placed in 37 DEG C, 5%CO2In incubator, taking out 96 orifice plates after cultivating 24,48,72 hours respectively, absorb each group of original culture medium, be replaced by 100 μ LDMEM culture fluid, background group is acellular only has DMEM culture fluid.Every hole adds 10 μ LCCK-8 solution, is placed in incubator and continues to cultivate 2 hours.Measuring every hole absorbance (OD value) by full-automatic microplate reader, wavelength is set in 450mn place, takes the average in the multiple hole of often group.Above-mentioned experimental procedure repeats 3 times, calculates meansigma methods.Cells survival rate computing formula is as follows: cells survival rate=(experimental group OD value-background group OD value)/(matched group OD value-background group OD value) × 100%, draws growth inhibited curve chart the credit analysis that takes statistics.
3, Hoechst33258 dyeing
Experiment, with green skies company Hoechst33258 dyeing liquor, during apoptosis, can be appreciated 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 the 48 hours conditions as Hoechst33258 dyeing of pharmaceutical composition effect in 2mmol/L embodiment 1.By cell dissociation, centrifugal, counting, plant in 24 orifice plates after adjusting cell concentration, be placed in 37 DEG C, 5%CO2Incubator is cultivated 12 hours, absorb original culture medium, different disposal is given: blank group (equivalent complete medium) by packet, pharmaceutical composition group sets 1,2,4mmol/L, simple component A diterpene compound (I) 2mmol/L sets one group, simple component D flavone compound (IV) 2mmol/L sets one group, six groups altogether.The aseptic each hole of PBS closed perimeter, is again placed in 37 DEG C, 5%CO2In incubator, take out 24 orifice plates after continuing cultivation 48 hours, absorb each group of original culture medium, aseptic PBS washes 3 times, and every hole adds the fixing cell of 0.5mL poly formic acid 15 minutes, and PBS washes 3 times, each hole adds 100 μ LHoechst33258 dyeing liquors, dye under room temperature dark surrounds 10min, goes dyeing liquor, PBS shaking table to rock flushing 3 times, each 5 minutes, wash most liquid, a droplet anti-fluorescent quenching liquid of every hole dropping, take pictures under the fluorescence microscope of 340nm wavelength and the change of observation of cell karyomorphology.
3, Flow cytometry cell follows and dies
According to cells survival rate result, choose the particular point in time died as detection cell pillbox 48 hours.Take and be in exponential phase and U251 cell in good condition, had digestive transfer culture routinely, be inoculated in 6 orifice plates 37 DEG C, 5%CO2Incubator is hatched 12h, gives different disposal by packet: by pharmaceutical composition 1,2,4mmol/L, simple component A diterpene compound (I) 2mmol/L, simple component D flavone compound (IV) 2mmol/L, be placed in 37 DEG C, 5%CO2After continuing cultivation in incubator 48 hours, being washed till in 5mL centrifuge tube by original culture fluid, PBS washs attached cell 2 times, adds the appropriate trypsin digestion cell containing EDTA.Incubated at room is to, when piping and druming can make attached cell blow and beat gently, exhausting pancreatin cell dissociation buffer as far as possible.Add the original cell culture fluid collected, slightly mix, transfer in 5mL centrifuge tube, centrifugal 5 minutes of 1000g, abandons supernatant, collects cell, with PBS re-suspended cell gently and count.Take 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 instigate dyeing liquor, gently mixed hook, and ice bath lucifuge places 15min.Carrying 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 10000 cells of counting, calculate each group of apoptosis rate every time.
4, statistical procedures
Adopt SPSS16.0 to carry out statistical analysis, test all data and all represent the result repeating experiment 3 times, represent with mean scholar's standard deviation.Comparing the variance analysis adopting Factorial Design data between the group of survival rate, compare and use one factor analysis of variance between the group of apoptosis rate, inspection level is that < 0.05 for there being significant difference, and P < 0.01 has significant difference for pole for a=0.05, P.
Three, result and conclusion
1, the pharmaceutical composition impact on U251 cells survival rate
Pharmaceutical composition suppresses U251 cell survival growth CCK-8 experimental result to show, after variable concentrations pharmaceutical composition effect U251 cell, along with activity is high, action time lengthens, U251 cells survival rate is decreased obviously, the main effect F=956.9 (P < 0.01) of variance analysis display pharmaceutical composition concentration factor；Action time factor main effect F=401.67 (P < 0.01), concentration, time factor reciprocal action F=55.7 (P < 0.01).Associative list 1, it is possible to find the inhibitory action of U251 cells survival rate is time, dose dependent by pharmaceutical composition, and namely drug regimen substrate concentration is more high, and action time is more long, and its inhibitory action is more strong.And the inhibitory action of pharmaceutical composition is superior to simple component A diterpene compound (I) 2mmol/L group and simple component D flavone compound (IV) 2mmol/L group under the same terms.By calculating the IC of pharmaceutical composition effect 24h, 48h, 72h50It is worth respectively, 3.27mmol/L, 1.05mmol/L, 0.89mmol/L.
2, U251 cell pillbox is died morphologic impact by pharmaceutical composition
Cell in normal group, pharmaceutical composition process group and simple component A, D group dyes through Hochest33258, the visible cellular control unit nuclear morphology rule of fluorescence microscopy Microscopic observation, and in light blue uniform coloring；And after 2mmol/L pharmaceutical composition processes 48 hours, compared with matched group, pyknosis in various degree occurs in nucleus, color is comparatively bright, the dense dye of stained dense, and karyorrhexis is obvious, and visible typical apoptotic body.The change prompting pharmaceutical composition of above Cell Image Analyzer has induction U251 cells apoptosis.
3, the pharmaceutical composition impact on U251 apoptosis rate
Matched group and each process group be row Flow cytometry after AnnexinV-FITC/PI dyes, and result show: matched group, 1mmol/L pharmaceutical composition, 2mmol/L pharmaceutical composition, 4mmol/L pharmaceutical composition, 2mmol/L component A, 2mmol/L component D process 48 hours and organize natural death of cerebral cells rate respectively 3.30 ± 0.98,18.56 ± 2.60,37.55 ± 5.41,60.92 ± 5.5%, 27.54 ± 5.49 and 5.30 ± 0.88.Compared with matched group, the apoptosis rate of each process group cell dramatically increases (P < 0.01).
Conclusion: pharmaceutical composition is by suppressing cell proliferation and inducing cell apoptosis to play the inhibitory action to human brain malignant glioblastoma U251 cell, and this pharmaceutical composition is compared simple component A and simple component D and suppressed human brain malignant glioblastoma U251 to be remarkably reinforced.These find to provide foundation for the clinical drug research of medicine composite for curing glioma.
U251 cells survival rate (mean ± standard deviation) after table 1 variable concentrations pharmaceutical composition, component A and component D process effect 24～72h
Embodiment 3:
One is used for treating gliomatous pharmaceutical composition, comprises the pharmaceutical composition of above-described embodiment 1 of therapeutically effective amount and pharmaceutically acceptable carrier.And pharmaceutically acceptable carrier is selected from a kind of or mixing that both are above: lactose, sucrose, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate, stearic acid, cellulosic lower alkyl ether, corn starch, potato starch, natural gum, fatty acid, glycerol monostearate.Effective dose every day wanting pharmaceutical composition (not comprising pharmaceutically acceptable carrier) in above-described embodiment 1 is 0.001-0.006g/kg human body, can make the medicine of corresponding content as required, follow the doctor's advice and take during pharmacy.
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 (7)
1. one kind is used for treating gliomatous pharmaceutical composition, it is characterised in that: it by component A, component B, form C and component D and form, component A is the diterpene compound (I) with following structural formula:
Component B is the liquor-saturated eggplant lactone compound (II) with following structural formula:
Component C is triterpene saponin componds echinosideA, structure formula III:
Component D is the flavone compound (IV) with following structural formula:
Wherein, it is the weight of 100% according to total amount,
Component A is: 52-63%；
Component C is: 22-25%；
Component B is: 8-12%
Component D sum is: 8-12%
Component B and component D sum are no less than 18%.
2. according to claim 1 for treating gliomatous pharmaceutical composition, it is characterised in that:
The preparation of component A diterpene compound (I) comprises following operating procedure: the dry aerial parts of Callicarpa nudiflora are pulverized by (a), extract with 70～80% 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 in () step (a), n-butyl alcohol takes thing macroporous resin remove impurity, first with 6 column volumes of 10% ethanol elution, then with 8 column volumes of 70% ethanol elution, collect 70% eluent, and concentrating under reduced pressure obtains 70% ethanol elution concentrate；In (c) step (b) 70% ethanol elution concentrate with purification on normal-phase silica gel separate, successively with volume ratio be 40:1,20:1,10:1 and 5:1 methylene chloride-methanol gradient elution obtain 4 components；D in () step (c), component 4 separates further by purification on normal-phase silica gel, successively with volume ratio be 8:1,5:1 and 2:1 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 70%, collecting 8～12 column volume eluents, eluent concentrating under reduced pressure obtains pure diterpene compound (I)；
Component D flavone compound (IV) separates from Herba Verbenae and obtains, concrete separating step:
A, the dry aerial parts of Herba Verbenae are pulverized, extract with 70～80% alcohol heat reflux, united extraction liquid, be 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；
Acetic acid ethyl ester extract macroporous resin remove impurity in B, step A, first with 10 column volumes of 10% ethanol elution, then with 12 column volumes of 75% ethanol elution, collects 75% ethanol elution, and 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 80:1,40:1,20:1,10:1 and 1:1 methylene chloride-methanol gradient elution obtain 5 components；
In D, 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；
The reverse phase silica gel that in E, step D, component 2 is bonded by octadecylsilane separates, and with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collects 9～13 column volume eluents, and eluent concentrating under reduced pressure obtains pure flavone compound (IV)；
The percentage concentration of above ethanol each means concentration expressed in percentage by volume.
3. according to claim 2 for treating gliomatous pharmaceutical composition, it is characterised in that: extracting, with alcohol heat reflux, the concentration of alcohol all adopted described in step (a) and step A is 75%.
4. according to claim 2 for treating gliomatous pharmaceutical composition, it is characterised in that: macroporous resin described in step (b) and step B is AB-8 type macroporous adsorbent resin.
5. one kind is used for treating gliomatous pharmaceutical composition, it is characterised in that: comprise pharmaceutical composition described in the claim 1 of therapeutically effective amount and pharmaceutically acceptable carrier.
6. one according to claim 5 is used for treating gliomatous pharmaceutical composition, it is characterised in that: a kind of or both mixing above that pharmaceutically acceptable carrier is selected from: lactose, sucrose, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate, stearic acid, cellulosic lower alkyl ether, corn starch, potato starch, natural gum, fatty acid, glycerol monostearate.
7. one according to claim 5 is used for treating gliomatous pharmaceutical composition, it is characterised in that: effective dose every day of pharmaceutical composition described in claim 1 is 0.001-0.006g/kg human body.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN106397369A (en) * | 2016-09-09 | 2017-02-15 | 中国科学院西北高原生物研究所 | Novel labdane-type diterpenoid compound, preparation method and application thereof, pharmaceutical composition and application of pharmaceutical composition |
| CN111704544A (en) * | 2020-06-30 | 2020-09-25 | 海南师范大学 | Labdane diterpenoid compound and separation method and application thereof |
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| CN1823760A (en) * | 2005-12-28 | 2006-08-30 | 广州中医药大学热带医学研究所 | Use of artemisia apiacea kind medicine for treating gliosis |
| CN1924006A (en) * | 2006-09-21 | 2007-03-07 | 武汉大学 | Anti-glioma peptide of scorpion, preparation method and application thereof |
| CN101333239A (en) * | 2008-08-04 | 2008-12-31 | 中国人民解放军第四军医大学 | Anti-glioma compounds of triterpenoid saponin extracted from ardipusilloside |
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| CN1823760A (en) * | 2005-12-28 | 2006-08-30 | 广州中医药大学热带医学研究所 | Use of artemisia apiacea kind medicine for treating gliosis |
| CN1924006A (en) * | 2006-09-21 | 2007-03-07 | 武汉大学 | Anti-glioma peptide of scorpion, preparation method and application thereof |
| CN101333239A (en) * | 2008-08-04 | 2008-12-31 | 中国人民解放军第四军医大学 | Anti-glioma compounds of triterpenoid saponin extracted from ardipusilloside |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN106397369A (en) * | 2016-09-09 | 2017-02-15 | 中国科学院西北高原生物研究所 | Novel labdane-type diterpenoid compound, preparation method and application thereof, pharmaceutical composition and application of pharmaceutical composition |
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