CN107434764A - It is a kind of to be used to treat new triterpene compound of melanoma and preparation method thereof and medical usage - Google Patents
It is a kind of to be used to treat new triterpene compound of melanoma and preparation method thereof and medical usage Download PDFInfo
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- C07C59/40—Unsaturated compounds
- C07C59/76—Unsaturated compounds containing keto groups
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Abstract
The invention discloses a kind of new triterpene compound for being used to treat melanoma and preparation method thereof and medical usage.The compound is a kind of novel triterpene compound of structure, extracting and developing can purify to obtain from dry radix glehniae to report first.In vitro test proves that the compound can significantly inhibit melanoma cell growth, and cell number declines with compound (I) concentration in dependence.Compound (I) may provide a kind of potential means for the treatment of melanoma, can be used for developing into the medicine for the treatment of melanoma.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, and in particular to isolated one kind has treatment melanin from dry radix glehniae
Triterpene compound of knurl effect and preparation method thereof.
Background technology
The root of straight ladybell can be divided into two kinds of adenophora tetraphylla (RadixAdenophorae) and radix glehniae (Radix Glehniae), wherein radix glehniae
It is the traditional Chinese medicine in China for samphire glehnia littoralis (Glehnia littoralis Fr.Schmidt ex Miq.) dry root,
Its cool in nature, slightly sweet flavor, the return lung spleen liver heart, have clear tonifying lung cloudy, the fiery QI invigorating of system, tonifying spleen lung yin and middle drop is inverse, self-restraint liver-yin,
Solve Yu Qianyang, clear nourishing heart the moon and relieving restlessness and other effects of calming the nerves.
Radix glehniae is distributed mainly on Shandong Province of China, Hebei, Guangdong, Fujian, Liaoning, Jiangsu and zhejiang and other places, and Taiwan also has point
Cloth.Exist according to the bright chapter of Cao《Pseudo- medicinal strip is revised and enlarged to distinguish》In " pressing radix glehniae, Shandong Rizhao, Laiyang, each county in Haiyang all go out " record,
It is exactly the important locality of radix glehniae from ancient times to show Shandong, and quality is superior.At present, Hebei Anguo, Inner Mongol Chifeng Niu great Ying
Son and the 3 big producing regions that Laiyang Shandong Province is radix glehniae, wherein Hebei yield is maximum, and the Inner Mongol and Shandong are taken second place, and mainly for export.
Radix glehniae is herbaceos perennial, wild in seashore sandy beach；Cultivate in beach sandy soil, thin sand and soil and sandy loam more.It is high
5~20cm, complete stool have taupe fine hair, and main root is elongated, cylinder, seldom branch.Shandong radix glehniae bar is elongated, Beijing opera is white,
Texture is delicate, smell micro-perfume；The radix glehniae feature in Hebei is short and thick, relatively rough shaped like pen, crust；And Inner Mongol the root of straight ladybell
Bar length is delicate in vain without wooden fork, skin.《Pharmacopeia》In with regard to the commercial specification of radix glehniae have clear and definite standard, basic demand is dried food and nuts.In thin
Strip cylinder, removes cork.Surface yellow-white.Matter is hard and crisp.Section skin zone yellowish white, there is geelhout barycenter.It is micro- to have
Fragrance, taste micro-sweet.With regard to its long, upper mid diameter, whether there is reed head, damage by worms, go mouldy etc. is divided into 3 grades by radix glehniae.
Research in recent years shows, the chemical composition of radix glehniae mainly includes volatile oil, glucosides, Coumarins etc., also containing starch,
The compositions such as triterpenic acid, stigmasterol, phosphatide, amino acid.
Modern pharmacological research shows that radix glehniae has immunoregulation effect, protective effect, Hepatocyte protection, antitumor action etc..
The content of the invention
Isolated from dry radix glehniae a kind of have the three for the treatment of melanoma effect it is an object of the invention to provide a kind of
Terpenoid and preparation method thereof.
The above-mentioned purpose of the present invention is achieved by following technical scheme：
Compound (I) with following structural formula,
The preparation method of described compound (I), includes following operating procedure：(a) dry radix glehniae is crushed, used
80~90% alcohol heat reflux extract, and merge extract solution, are concentrated into no alcohol taste, successively with petroleum ether, ethyl acetate and water saturated
Extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract；(b) in step (a)
Acetic acid ethyl ester extract is cleaned with macroreticular resin, first with 10% ethanol elution, 6 column volumes, then with 75% ethanol elution 10
Column volume, 75% ethanol eluate is collected, be concentrated under reduced pressure to obtain 75% ethanol elution thing medicinal extract；(c) 75% second in step (b)
Alcohol elution medicinal extract is separated with purification on normal-phase silica gel, is successively 80 with volume ratio:1、55:1、30:1、15:1 and 1:1 dichloromethane-first
Alcohol gradient elution obtains 5 components；(d) component 4 is further separated with purification on normal-phase silica gel in step (c), uses volume ratio successively
For 20:1、15:1 and 10:1 methylene chloride-methanol gradient elution obtains 3 components；(e) in step (d) component 2 with ten
The reverse phase silica gel separation of eight alkyl silanes bonding, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collects 8~10
Individual column volume eluent, eluent are concentrated under reduced pressure to give pure compound (I).
Further, the macroreticular resin is AB-8 type macroporous absorbent resins.
Further, the concentration of alcohol used with alcohol heat reflux extraction is 85%.
A kind of pharmaceutical composition, wherein the described compound (I) and pharmaceutically acceptable carrier containing therapeutically effective amount.
Application of the described compound (I) in the medicine for preparing treatment melanoma.
Application of the described pharmaceutical composition in the medicine for preparing treatment melanoma.
It when the compounds of this invention is used as medicine, can directly use, or be used in the form of pharmaceutical composition.
The pharmaceutical composition contains the compounds of this invention (I) of therapeutically effective amount, and remaining is pharmaceutically acceptable, to people
Nontoxic and inert with animal pharmaceutical acceptable carrier and/or excipient.
Described pharmaceutical acceptable carrier or excipient is one or more selected from solid, semisolid and liquid diluent, filler and medicine
Tetramune assistant agent.The pharmaceutical composition of the present invention is used in the form of per weight dose.Medicine of the present invention can be by oral
Or the form of injection is applied to the patient for needing to treat.For it is oral when, tablet, sustained release tablets, controlled release tablet, glue can be made into
Capsule, dripping pill, micropill, supensoid agent, emulsion, powder or granule, oral liquid etc.；During for injecting, the water of sterilizing can be made into
Property or oily solution, aseptic powder injection, liposome or emulsion etc..
Brief description of the drawings
Fig. 1 is compound (I) structural formula；
Fig. 2 is the theoretical ECD values of compound (I) compared with testing ECD values；
Fig. 3 is BrdU Immunofluorescence test Cell proliferation results.
Embodiment
The essentiality content of the present invention is further illustrated with reference to embodiment, but the scope of the present invention is not limited with this.To the greatest extent
Pipe is explained in detail with reference to preferred embodiment to the present invention, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent substitution, without departing from the spirit and scope of technical solution of the present invention.
Embodiment 1：Compound (I) separation prepares and structural identification
Reagent source：Ethanol, petroleum ether, ethyl acetate, n-butanol, dichloromethane are pure to analyze, purchased from Shanghai Ling Feng chemistry
Reagent Co., Ltd, methanol, analysis is pure, purchased from Jiangsu Han Bang chemical reagent Co., Ltd.
Preparation method：(a) dry radix glehniae (8kg) is crushed, (25L × 3 time) is extracted with 85% alcohol heat reflux,
Merge extract solution, be concentrated into no alcohol taste (3L), successively with petroleum ether (3L × 3 time), ethyl acetate (3L × 3 time) and
The extraction of water saturated n-butanol (3L × 3 time), respectively obtain petroleum ether extract, acetic acid ethyl ester extract (355g) and just
Butanol extract；(b) acetic acid ethyl ester extract is cleaned with AB-8 types macroreticular resin in step (a), is first washed with 10% ethanol
6 column volumes are taken off, then with 75% ethanol elution, 10 column volumes, collect 75% ethanol eluate, be concentrated under reduced pressure to obtain 75% second
Alcohol eluate medicinal extract (136g)；(c) 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in step (b), uses volume successively
Than for 80:1 (8 column volumes), 55:1 (8 column volumes), 30:1 (6 column volumes), 15:1 (8 column volumes) and
1:The methylene chloride-methanol gradient elution of 1 (5 column volumes) obtains 5 components；(d) component 4 (31g) in step (c)
Further separated with purification on normal-phase silica gel, be successively 20 with volume ratio:1 (8 column volumes), 15:1 (10 column volumes) and 10:1
The methylene chloride-methanol gradient elution of (6 column volumes) obtains 3 components；(e) component 2 (11g) is used in step (d)
The reverse phase silica gel separation of octadecylsilane bonding, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collects 8-10
Individual column volume eluent, eluent are concentrated under reduced pressure to give pure compound (I) (228mg).
Structural identification：Colourless Oblique Crystal (methanol), 140~142 DEG C of fusing point；HR-ESIMS is shown [M+Na]+For m/z 421.2714,
It is C that can obtain molecular formula with reference to nuclear-magnetism feature26H38O3, degree of unsaturation 8.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6,
500MHz)：H-1 (2.04, m), H-1 (2.29, m), H-3 (2.15, m), H-3 (2.41, m), H-5
(1.58, m), H-6 (1.53, m), H-6 (1.61, m), H-7 (1.87, m), H-7 (2.08, m), H-11
(1.89, m), H-11 (2.06, m), H-12 (1.36, m), H-12 (2.00, m), H-15 (2.27, m),
H-15 (2.31, m), H-16 (1.42, m), H-16 (1.50, m), H-18 (0.86, s), H-19 (1.05, s),
H-20 (2.25, m), H-21 (0.92, d, J=6.5), H-22 (1.93, br, d, J=14.5), H-22 (2.67,
Br, d, J=14.5), H-28 (1.08, s), H-29 (1.05, s), H-30 (4.45, br, s), H-30 (4.69,
Br, s)；Carbon-13 nmr spectra data δC(ppm, DMSO-d6, 125MHz)：52.2(CH2, 1-C), 209.8 (C,
2-C), 56.1 (CH2, 3-C), 38.9 (C, 4-C), 50.4 (CH, 5-C), 20.1 (CH2, 6-C), 25.8
(CH2, 7-C), 135.4 (C, 8-C), 147.5 (C, 9-C), 35.3 (C, 10-C), 26.2 (CH2, 11-C),
32.3(CH2, 12-C), 67.7 (C, 13-C), 155.0 (C, 14-C), 26.6 (CH2, 15-C), 37.4 (CH2,
16-C), 48.6 (C, 17-C), 18.1 (CH3, 18-C), 18.3 (CH3, 19-C), 34.8 (CH, 20-C),
15.8(CH3, 21-C), 35.9 (CH2, 22-C), 179.5 (C, 23-C), 26.2 (CH3, 28-C), 20.7 (CH3,
29-C), 103.8 (CH2, 30-C)；Carbon atom is marked referring to Fig. 1.Infrared spectrum shows that the compound contains carbonyl (1706cm-1)
With double bond (1646cm-1)。1H-NMR modal datas show four unimodal methyl signals [δ H0.86 (Me-18), 1.05 (Me-19),
1.05 (Me-29) and 1.08 (Me-28)], a bimodal methyl signals [δ H0.92 (d, J=6.5Hz, Me-21)], and
One olefinic methene proton signal [δ H4.45 (br, s, H-30) and 4.69 (br, s, H-30)].13C H NMR spectroscopies are shown
26 resonance carbon signals, including five methyl, ten methylene (an olefinic methylene), two methines, and nine
Quaternary carbon (two carbonyls, three alkene quaternary carbons).Characteristic signal quaternary carbon signal (δ C67.7) and two double bond carbon signals (δ C135.4,
147.5,103.8,155.0) it is lanosterol triterpene compound to show the compound.Pass through1H-1H COSY spectrum in five from
Rotation system H2-1/H2- 3, H-5/H2-6/H2- 7, H2-11/H2- 12, H2-30/H2-15/H2- 16, H3-21/H-20/H2- 22, and
Me-18, Me-19, Me-21, Me-28, Me-29 and H in HMBC spectrums2- 30 correlation, the compound can be derived
Planar structure it is as shown in Figure 1.Spatial configuration is further tested by ECD and determined, theoretical value and the basically identical (figure of experiment value
2)。
Embodiment 2：Compound (I) pharmacological action is tested
First, material and instrument
People's A375 melanoma cell strains are given by Third Military Medical University.Compound (I) is made by oneself, and preparation method is shown in embodiment 1,
HPLC normalization purity is more than 98%.DMEM culture mediums, 0.25% pancreatin are purchased from Gibco companies of the U.S..MTT(3-(4,5-
Dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides；Methyl thiazoly tetrazolium assay), DMSO (dimethyl sulfoxide (DMSO)), BrdU be purchased from
Sigma Co., USA.The anti-BrdU polyclonal antibodies of mouse are purchased from American AB cam companies.Mountain sheep anti mouse secondary antibody is purchased from U.S. Cell
Signaling companies.Annexin V-FITC/PI cell apoptosis detection kits are purchased from Promega companies of the U.S..
Constant temperature CO2Incubator, general refrigerator, -80 degree refrigerators are U.S.'s Forma Products.Superclean bench (China
Suzhou cleaning project company).Inverted microscope, inverted fluorescence microscope (olympus companies).Electro-heating standing-temperature cultivator (in
Shanghai leap medical apparatus and instruments factory of state).Automatic ELIASA (Japanese Wako companies).UV detector (U.S. Beckman
Company).J6-HC supercentrifuges (Beckman companies of the U.S.).Low-temperature trace centrifuge (German Eppendorf companies).
Vibrate shaking table (Forma companies of the U.S.).
2nd, test method
1st, cell culture
1.1 cell recovery
The A375 melanoma cells frozen in liquid nitrogen container are taken out, are put into rapidly in 37 DEG C of warm water, gently shaking makes it
Melt (about lmin) as early as possible.Then cell suspension is suctioned out, is added to added with the centrifuge tube of 2mL culture mediums, gently
Piping and druming mixes, 800r/min, centrifuges 5min, discards upper strata culture medium.With the mould G/ streptomysins containing 10%FBS and 1%
DMEM culture mediums 8mL cell suspension is made after, be seeded in 10cm culture plates.It is subsequently placed in 5%CO2, 37 DEG C of perseverances
Cultivated in warm incubator.
1.2 passage
Micro- Microscopic observation cell fusion degree can be passed on when reaching 80%-90%.First cell is washed with 2mL PBS 2 times, so
0.25% pancreatin lmL is added afterwards.It is gently horizontal to shake culture plate, make the surface of digestion liquid energy covering cell, be subsequently placed in 37 DEG C
About lmin is digested in incubator.It is placed under microscope and sees that cell rounding bounces back, is then rapidly added the culture medium that 1mL contains FBS
Terminate digestion.Gently blow and beat to cell and be uniformly dispersed, then according to cell density according to 1:2 or 1:3 passages, are re-seeded into
In new culture plate, 5%CO is placed in2, cultivate in 37 DEG C of constant incubators.
1.3 cell cryopreservation
1 disk is taken to be in the cell of exponential phase, pancreatin digests and is collected into centrifuge tube, 1000r/min centrifugation 5min, abandons
Supernatant.Then lmL frozen stock solutions are added, cell is gently dispelled and cell suspension is made, cell suspension is added to 1.5mL jelly
Deposit in pipe.In the title of cryopreservation tube subscript clear-cells, holding time, the name of preserver.Cryopreservation tube is first put into 4 DEG C of refrigerators,
Place about 30min.Then -20 DEG C of refrigerators are placed in, place about 2h.Then it is put in -80 DEG C of ultra low temperature freezers and stands overnight.The
The cell frozen was placed in liquid nitrogen container in two days and preserved for a long time.
2nd, cell counting draws cell growth curve
(1) take the logarithm growth period A375 melanoma cells, count, adjustment cell density is 2 × 104/mL。
(2) by cell suspension inoculation in 12 orifice plates, 1.5mL/ holes.
(3) after 12h, cell attachment is treated, changes 1mL plasma-free DMEM mediums, and respectively with 5 μm of ol/L compounds (I)
(for the compound (I) of DMSO dilutions, control DMSO concentration is below 1/1000, it is ensured that to cell with DMSO
It is harmless) handle, every group of cell sets 3 parallel holes, is placed in 5%C02,37 DEG C of constant incubators and is cultivated.
(4) terminate and cultivate respectively at 0h, 24h, 48h, 72h, 96h, collect each group cell, add 0.25% trypsase
Digestion, centrifuge, be resuspended.
(5) cell count：The μ L of cell suspension 10 are drawn, isometric platform phenol blue area is added and divides living cells, 10 μ L is drawn and mixes
Close liquid gently to add in cell counting count board groove, ensure tight and bubble.Under inverted microscope, four, periphery block plaid is counted
Interior cell number, substitute into formula：Archaeocyte suspension concentration (cell number/mL)=(cell number/4 of 4 block plaids) × 104×
Extension rate.
(6) number of viable cells is calculated, draws cell growth curve.
3rd, mtt assay detection cell propagation
(1) take the logarithm the A375 cells in growth period, digest, centrifuge, be resuspended, count, adjust cell in cell suspension
Density is 2 × 104/mL。
(2) by each group cell suspension inoculation in 96 well culture plates, 200 μ L are per hole, 3 parallel holes of every group of cell, simultaneously
If blank control wells (only add culture medium).
(3) 12h changes 200 μ L plasma-free DMEM mediums after cell attachment, and respectively with 5 μm of ol/L compounds
And DMSO processing (I).
(4) culture was terminated respectively at 0,1,2,3,4 day.
(5) the μ L of Methyl thiazoly tetrazolium assay (MTT) 20 that concentration is 5mg/mL are added to every hole to continue to cultivate cell 4h.
(6) culture medium abandoned in each aperture is inhaled, 200 μ L DMSO is added, 10min is vibrated in microoscillator, makes crystallization
Thing fully dissolves.
(7) returned to zero with blank control wells, the absorption photometric value (OD values) at each hole 570nm determined using automatic ELIASA,
Ability of cell proliferation is represented with corresponding OD values, each group takes the average value of 3 parallel holes, using the time as transverse axis, with each extinction
Angle value is that the longitudinal axis draws cell growth curve.
4th, BrdU Immunofluorescence tests cell is bred
(1) creep plate is prepared：Creep plate is put into immersion 24h in 75% alcohol to disinfect.
(2) sterile creep plate is placed in 24 well culture plates, the A375 melanoma cells in growth period of taking the logarithm, digestion, from
The heart, is made cell suspension, and 2 × 104Cell/ holes, are inoculated in the hole for being placed with creep plate.
(3) 12h changes 1mL plasma-free DMEM mediums after cell attachment, and respectively with 5 μm of ol/L compounds (I)
With DMSO processing.
(4) after 72h, 10 μ l BrdU (1mg/mL), 37 DEG C of culture 1h are added in the medium.
(5) 24 orifice plates are taken out, 5min × 2 time are washed with cold PBS, 4% paraformaldehyde fixes cell, room temperature 30min.
(6) PBS washes 5min × 3 time.
(7) 2mol/LHCl processing, after room temperature 10min, 37 DEG C of 20min.
(8) the cell penetrating processing in 1%Triton × -100, washes 5min × 3 time.
(9) 10% lowlenthal serums are closed, room temperature, lh.
(10) BrdU monoclonal antibodies (1 are added:300 dilutions), 37 DEG C of 1.5h.
(11) l × PBST, which shakes, washes 5min × 3 time.
(12) BrdU secondary antibodies (1 are added:300), after room temperature lucifuge 1h, l × PBST is washed 3 times.
(13) DAPI dyeing liquors dye nucleus 15min.
(14) PBS washes 5min × 3 time.
(15) plus 1 drips anti-fluorescent quenching mounting liquid, neutral gum mounting, fluorescence microscopy Microscopic observation, photograph.
5th, statistical analysis
Using the statistical analysis softwares of SPSS 17.0, data point are carried out using two independent samples t tests and one-way analysis of variance
Analysis, data X ± S expressions, P<0.05 is statistically significant.
3rd, result and conclusion
Display is observed and counted to cellular morphology, and DMSO control groups ratio, compound (I) processing A375 cells 24h, 48h,
After 72h, viable count is significantly reduced up to 44.5%.1 be the results are shown in Table (with control group ratio*P<0.05,**P<0.01, similarly hereinafter).
MTT results show, compound (I) can significantly inhibit A375 cells propagation, OD values be in concentration (5 μm of ol/L,
10 μm of ol/L, 20 μm of ol/L) up to 27.3%, difference is statistically significant for dependence decline.It the results are shown in Table 2.
Further confirm that compound (I) can suppress melanoma cells and breed by BrdU immunofluorescence dyeings, 5 μm of ol/L
Compound (I) handled A375 cells after 3 days, compared with compareing (26.07 ± 2.59%), experimental group BrdU positive cells
Percentage (7.55 ± 2.76%) significantly reduces (P=0.000).As a result Fig. 3 (* * P are seen<0.01).
This experiment have detected the influence that compound (I) is bred to melanoma cells, and cell count and MTT analysis results are equal
Prove that compound (I) can significantly inhibit melanoma cell growth, and cell number and compound (I) concentration are under dependence
Drop.BrdU positive cell percentages are substantially less than control group after BrdU immunofluorescence dyeings display that compound (I) processing,
Illustrate that compound (I) can suppress A375 melanoma cells propagation.Compound (I) is probably effective target of melanoma
To medicine.
The compound of table 1 (I) dose-dependent inhibition A375 cells are bred
(cell counting, unit：×l04/mL)(N=3)
| Group | 0h | 24h | 48h | 72h | 96h |
| DMSO | 2.87±0.23 | 4.72±0.30 | 7.36±0.13 | 7.56±0.39 | 8.04±0.27 |
| (I) -5 μm of ol/L of compound | 2.87±0.23 | 3.87±0.36* | 3.48±0.32** | 3.40±0.34** | 2.54±0.41** |
| (I) -10 μm of ol/L of compound | 2.87±0.23 | 2.36±0.28** | 2.95±0.37** | 1.56±0.36** | 0.89±0.26** |
The compound of table 2 (I) dose-dependent inhibition A375 cells propagation (mtt assay, OD570) (N=3)
Embodiment 3
The preparation of tablet：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, it is 1 by itself and excipient weight ratio:9
Ratio adds excipient, pelletizing press sheet.
Embodiment 4
It is prepared by oral liquid：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely oral liquid preparation method oral liquid is made.
Embodiment 5
The preparation of capsule or granule：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as winestone
Acid or citric acid or formic acid or ethanedioic acid etc., the inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, by itself and excipient weight
Than for 1:9 ratio adds excipient, and capsule or granule is made.
Embodiment 6
The preparation of parenteral solution：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Lemon acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely plus water for injection, refined filtration,
Parenteral solution is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection：By the method for embodiment 1 first be made compound (I), and using organic acid for example tartaric acid or
Citric acid or formic acid or ethanedioic acid etc., the inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, are dissolved in sterile water for injection,
Stirring makes molten, is filtered with sterile suction funnel, then sterile refined filtration is sub-packed in ampoule, sterile after frozen drying to seal
Powder-injection.
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 technical scheme can be modified or equivalent substitution, without de-
From the essence and protection domain of technical solution of the present invention.
Claims (7)
1. the compound (I) with following structural formula,
2. the preparation method of the compound (I) described in claim 1, it is characterised in that include following operating procedure：(a)
Dry radix glehniae is crushed, extracted with 80~90% alcohol heat reflux, merges extract solution, is concentrated into no alcohol taste, uses oil successively
Ether, ethyl acetate and water saturated extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butanol extraction
Take thing；(b) acetic acid ethyl ester extract is cleaned with macroreticular resin in step (a), first with 10% ethanol elution, 6 column volumes,
Again with 75% ethanol elution, 10 column volumes, 75% ethanol eluate is collected, be concentrated under reduced pressure to obtain 75% ethanol elution thing medicinal extract；
(c) 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in step (b), is successively 80 with volume ratio:1、55:1、30:1、15:1
With 1:1 methylene chloride-methanol gradient elution obtains 5 components；(d) component 4 is further with purification on normal-phase silica gel in step (c)
Separation, it is successively 20 with volume ratio:1、15:1 and 10:1 methylene chloride-methanol gradient elution obtains 3 components；(e) step
Suddenly the reverse phase silica gel that component 2 is bonded with octadecylsilane in (d) separates, with the methanol aqueous solution that concentration expressed in percentage by volume is 75%
Isocratic elution, collects 8~10 column volume eluents, and eluent is concentrated under reduced pressure to give pure compound (I).
3. the preparation method of compound (I) according to claim 2, it is characterised in that：The macroreticular resin is AB-8
Type macroporous absorbent resin.
4. the preparation method of compound (I) according to claim 2, it is characterised in that：It is described to be carried with alcohol heat reflux
The concentration of alcohol used is taken as 85%.
A kind of 5. pharmaceutical composition, it is characterised in that：Compound (I) described in claim 1 wherein containing therapeutically effective amount
And pharmaceutically acceptable carrier.
6. application of the compound (I) in the medicine for preparing treatment melanoma described in claim 1.
7. application of the pharmaceutical composition in the medicine for preparing treatment melanoma described in claim 5.
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