CN107417744A - A kind of sucrose derivative and preparation method thereof and the purposes as cancer therapy drug - Google Patents
A kind of sucrose derivative and preparation method thereof and the purposes as cancer therapy drug Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- C07H1/00—Processes for the preparation of sugar derivatives
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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Abstract
The present invention relates to pharmaceutical technology field, sucrose derivative that the novel hydroxyl of an isolated class formation is esterified by isovaleric acid specifically from composite family Ainsliaea platymiscium Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.) and preparation method thereof, and the application in cancer therapy drug is prepared.The invention provides a kind of sucrose derivative, and its chemical constitution is shown in formula I：
Description
Technical field
It is specifically isolated from the Ainsliaea of composite family Ainsliaea platymiscium Yunnan the present invention relates to pharmaceutical technology field
Sucrose derivative that the novel hydroxyl of one class formation is esterified by isovaleric acid and preparation method thereof, and in cancer therapy drug is prepared
Using.
Background technology
Composite family Ainsliaea category (Ainsliaea DC) plant is distributed mainly on the Asia southeast, about 70 kinds of the whole world, China
Have 44 kinds, 4 mutation, in addition to a kind originates in northeast, remaining originate in the Yangtze river basin and its on the south each provinces and regions (China of the Chinese Academy of Sciences
Flora editorial board Chinese Plants will [M] Beijing of volume 79:Science Press, 1996:23.).Planted from the category
There is preferable cell toxicant to live to Partial tumors cell for isolated terpene, sesquiterpene dimers and sequiterpene tripolymer in thing
Property.For example, 5 terpenoid mokko lactone isolated from mapler leaf Ainsliaea (A.acerifolia),
Betulonic acid, betulinic acid, zaluzanin C and glucozaluzanin C can be significant non-specific right
Anti-human human umbilical vein endothelial cell, ovarian cancer cell SK-OV-3, melanoma SK-MEL-2, central nerve neuroma XF498 and
5 kinds of human tumor cell lines such as Human colorectal cancer cells HCT15 (Choi SZ, Yang MC, Choi SU, et al.Cytotoxic
terpenes and lignans from the roots of Ainsliaea acerifolia[J]
.Arch.Pharm.Res.,2006,29(3):203-208.), from the platymiscium major part Ainsliaea (A.macrocephala) and
In middle pasture Ainsliaea (A.fulvioides) isolated sesquiterpene dimers gochnatiolide A and
Gochnatiolide B are to human A549 cell lines, people's colon-cancer cell LOVO, human T cell leukemia cell 6T-CEM, human milk
Adenocarcinoma cell MDA-MB-435 have preferable cytotoxic activity (Wang Rong, Tang Yingxi, Shang little Ya Ainsliaeas Phytochemistry into
Divide and pharmacology activity research progress [J] Chinese medicines, 2012,7 (35):1171-1175.), times therefrom got in the glabrous ainsliaea herb of pasture
Hemiterpene tripolymer ainsliatrimer A and ainsliatrimer B LOVO thin to human colon cancer and leukaemia CEM have
There is very strong inhibitory action (Wang Y, Shen YH, Jin HZ, et al.Ainsliatrimers A and B, the first
two guaianolide trimers from Ainsliaea fulvioides[J].Org.Lett.,2008,10(24):
5517-5520.), these all show the platymiscium on the basis of based on Chinese medicine and autonomic drug in exploitation new type antineoplastic medicine
It is upper that there is huge application prospect.
Seminar where the present inventor, is also directed to the research of the platymiscium always, and achievement in research has been applied and obtained
Chinese patent CN200810035858.6, authorize publication No. CN101318946B；And CN200810035859.0, Granted publication
Number CN101318966B；This two pieces patent is related to extracts isolated dimerization sesquiterpenoid-bis- from major part Ainsliaea
Poly- Ainsliaea terpene A, Ainsliaidimer B, Ainsliaidimer C, are used equally for preparing anti-inflammatory and antineoplastic.In addition, I
Also apply and obtain Chinese patent application CN200810038569.1, authorize publication No. CN101284004B, this part patent
It is related in therefrom pasture Ainsliaea and extracts isolated dimeric sesquiterpene class compound-Gochnatiolide A and Gochnatiolide B,
There is good inhibitory action to a variety of human tumor cells, and also have significant inhibitory action to inflammation.
Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.), planted for composite family Ainsliaea category perennial herb
Thing, it is China's endemic plant, is mainly distributed on the ground such as Yunnan, Guizhou.The plant is claimed " oatax ", alias copper with all herbal medicine
Pin Wheeling, hang upside down flower, synthetism adder tongue herb, record in《National Chinese herbal medicine compilation》、《Kunming conventional herbal medicine among the people》With《China is originally
Grass》, acrid flavour；It is bitter；It is mild-natured, it is usually used in wind-damp dispelling, relaxing muscles and tendons bone, symplectic bone, cures mainly traumatic injury, fractures, rheumatism arthralgia and myalgia.Mesh
Before, the research for Yunnan Ainsliaea chemical composition is less, only a small amount of relevant report, therefrom isolated compound, including
Sequiterpene, triterpene, steroidal, (Li Jinjie, Wang Ali, institute is precious, waits Yunnan for the compound such as flavones and the organic phenolic acid class of small molecule
Triterpenes components [J] CHINA JOURNAL OF CHINESE MATERIA MEDICAs in glabrous ainsliaea herb, 2013,38 (22):3918–3922；Tian Liang diversifolious hemiphragma herb or roots and cloud
Chemical constitution study [D] the China Concord Medical Science University of southern Ainsliaea, 2004.).
Up to the present, we not yet have found to report for the activity research of Yunnan Ainsliaea.Therefore we are to Yunnan rabbit
The chemical composition of rumor has carried out system research, to therefrom extract the isolated compound with antitumor activity.
The content of the invention
It is an object of the invention to the novel and active significant natural products of isolated structure, tool from the Ainsliaea of Yunnan
Body is the sucrose derivative that a kind of hydroxyl is esterified by isovaleric acid；It is a further object of the present invention to provide the system of this sucrose derivative
Preparation Method；The third object of the present invention is the medical usage for providing this sucrose derivative, is specifically preparing antineoplastic
Application in thing.
The present inventor has obtained a kind of hydroxyl and has been esterified by isovaleric acid in the system research to Yunnan Ainsliaea chemical composition
Sucrose derivative.Through external cytotoxic activity it is demonstrated experimentally that the compound in the present invention has to human A549 cell lines
Significant cytotoxic activity.Further study on mechanism show such compound by inducing cell cycle arrest and apoptosis come
Suppress the growth of A549 cells, it is by mitochondria pathway and ROS paths that it, which induces A549 Apoptosis,.Therefore, in the present invention
Sucrose derivative have the function that as potential antineoplastic.
The first aspect of the present invention provides a kind of sucrose derivative, and its chemical constitution is shown in formula I：
In Formulas I, R1~R8Group is respectively selected from：Straight chain is contained in hydrogen atom, or isovaleryl, substitution or unsubstituted C1~6
Or the acyl group of side chain, the substituted or unsubstituted acyl group containing five yuan or hexa-atomic aromatic rings；
Described acyl group quantity is 4~5, and remaining is that (described acyl group is isovaleryl, substitution or unsubstituted to hydrogen atom
Acyl in the acyl group containing straight or branched of C1~6, the substituted or unsubstituted acyl group containing five yuan or hexa-atomic aromatic rings
Base).
In Formulas I, R1~R8Group can be with identical or different.
A kind of sucrose derivative of the present invention, parent nucleus are a sucrose molecule, and its hydrolysate is a molecule D- Portugals
Grape sugar and a molecule D-Fructose.
A kind of sucrose derivative of the present invention, it is a kind of sucrose derivative that hydroxyl is esterified by isovaleric acid.
Described 4~5 acyl groups, it is that with the hydroxyl on sucrose esterification occurs for isovaleric acid in Formulas I.
In Formulas I, R1~R8Group is preferred：Hydrogen atom, or isovaleryl.
Preferred compounds of the invention, its R1~R8Combination it is as follows：
Compound 1:R1=R2=R4=R6=R7=-COCH2CH(CH3)2,R3=R5=R8=H；
Compound 2:R1=R2=R6=R7=R8=-COCH2CH(CH3)2,R3=R4=R5=H；
Compound 3:R1=R2=R3=R6=R7=-COCH2CH(CH3)2,R4=R5=R8=H；
Compound 4:R1=R3=R4=R6=R7=-COCH2CH(CH3)2,R2=R5=R8=H；
Compound 5:R1=R4=R6=R7=-COCH2CH(CH3)2,R2=R3=R5=R8=H；
Compound 6:R1=R2=R4=R7=-COCH2CH(CH3)2,R3=R5=R6=R8=H；
Compound 7:R1=R2=R6=R7=-COCH2CH(CH3)2,R3=R4=R5=R8=H；
Compound 8:R1=R2=R4=R6=-COCH2CH(CH3)2,R3=R5=R7=R8=H.
In a preferred embodiment of the invention, compound 2：1′,4,4′,6,6′-pentakis-O-(3-
Methylbutanoyl)-β-D-fructofuranosyl α-D-glucopyranoside, ainsloside B (rabbits are named as
Rumor sugar B), for the most preferred compound of the present invention.
Above compound 1-8 is isolated from the Ainsliaea of Yunnan, it is also possible to which the method for chemical synthesis obtains.
The second aspect of the present invention, there is provided the preparation side for a kind of sucrose derivative that above-mentioned hydroxyl is esterified by isovaleric acid
Method, the method that preparation is specially separated from the Ainsliaea of Ainsliaea platymiscium Yunnan.
The method of the present invention comprises the following steps：
The drying herb of Ainsliaea platymiscium Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.) is crushed
Ethanol heating and refluxing extraction is used afterwards, and the medicinal extract that extract solution obtains after being concentrated under reduced pressure is water-dispersible, using the method point of liquid-liquid extraction
Petroleum ether, chloroform, the extract part of four kinds of opposed polarities of ethyl acetate and n-butanol are not prepared, with upper bit with one
It is secondary or the conventional chromatograph packing materials such as silica gel, macroporous absorbent resin, ODS, Sephadex LH-20 are used for multiple times as stationary phase, with stone
The common solvents such as oily ether, chloroform, ethyl acetate, acetone, methanol, acetonitrile, water are matched somebody with somebody with single or various combination to be used for washing
De- agent or recrystallization solvent, separate and purify a kind of sucrose that the hydroxyl being prepared in the present invention is esterified by isovaleric acid and derive
Thing.
In the method for the present invention, preferably, distinguish refluxing extraction twice from 95% and 80% ethanol, merge extraction
Liquid, the temperature of heating and refluxing extraction is 80 DEG C.
The method of described liquid-liquid extraction is conventional extraction processes.
Described petroleum ether extract part, using petroleum ether-ethyl acetate system (200:1–0:1) gradient elution.
Described chloroform extract part, using chloroform-methanol (100:0–0:1) gradient elution.
Described ethyl acetate extract part, using petroleum ether-ethyl acetate (100:1–0:1) gradient elution.
Described n-butanol extract part, using chloroform-methanol (100:1–0:1) gradient elution.
Described eluant, eluent, preferably chloroform-methanol 50:1.
The compounds of this invention 1-8 preparation method：After taking the drying herb (15.0kg) of Yunnan Ainsliaea to crush, with 95%
With 80% ethanol be respectively heated refluxing extraction twice after, merge extract solution；Gained medicinal extract warp after extract solution is concentrated under reduced pressure
Moisture dissipate after using petroleum ether, chloroform, ethyl acetate, n-butanol according to polarity order from small to large to successively to moisture
Scattered medicinal extract carries out liquid-liquid extraction, respectively obtains four petroleum ether, chloroform, ethyl acetate and n-butanol opposed polarities
Extract part；Take the medicinal extract (400g) of ethyl acetate extract, the silica gel column chromatography through 80-100 mesh, with petroleum ether-ethyl acetate
System 100:1,50:1,30:1,20:1,10:1,5:1,0:1 is eluted, and 7 components are obtained by the difference of gradient：A
(36g；100:1),B(46g；50:1),C(58g；30:1),D(43g；20:1),E(57g；10:1),F(49g；5:1),G(37g；
0:1)；Component C is by petroleum ether-ethyl acetate system 30:1 gradient elution obtains, the component through mesolow reversed-phase column chromatography with
MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-90%；90-100%；100% presses gradient not
It is same to obtain 7 components：C-1(3g；40-50%), C-2 (15g；50-60%), C-3 (6g；60-70%), C-4 (2g；70–
80%), C-5 (7g；80-90%), C-6 (6g；90-100%), C-7 (5g；100%)；Silica gel of the component C-4 through 200-300 mesh
Column chromatography, with chloroform-methanol 200:1；100:1；50:1；30:1；10:1；5:1 gradient elution separates, wherein three chloromethanes
Alkane-methanol 50:1 gradient elution cut (300mg), (21.0mg of compound 3 is obtained after purification by Sephadex LH-20；Three
Chloromethanes-methanol 20:1), chloroform-methanol 30:1 gradient elution obtains 2 (12.0mg；Chloroform-methanol 20:And 4 1)
(7.0mg；Chloroform-methanol 20:1)；C-2 is through RP-MPLC with MeOH-H2O 40-50%；50-60%；60-70%；70–
80%；80-100% gradient elutions obtain 5 component C-21 (2.3g by different collect of gradient；40-50%), C-22
(1.8g；50-60%), C-23 (2.9g；60-70%), C-24 (2.3g；70-80%), C-25 (3.1g；80-100%)；Component
Silica gel column chromatographies of the C-21 through 200-300 mesh, with petroleum ether-ethyl acetate 50:1；40:1；30:1；20:1；10:1；1:1 gradient
Elution, wherein petroleum ether-ethyl acetate 20:1 gradient elution obtains the (9.3mg of compound 5；Chloroform-methanol 20:1)
With 7 (7.1mg；Chloroform-methanol 20:1), petroleum ether-ethyl acetate 10:1 gradient elution obtains the (11.2mg of compound 6；Three
Chloromethanes-methanol 20:1), petroleum ether-ethyl acetate 1:1 gradient elution obtains the (6.4mg of compound 8；Chloroform-methanol 20:
1)；Component C-23 is through RP-MPLC with MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-90%；90–
100%；100% gradient elution, wherein 70-80%MeOH gradient elutions obtain the (10.5mg of compound 1；Chloroform-methanol
20:1)。
The third aspect of the present invention, there is provided above-mentioned hydroxyl is preparing anti-swell by a kind of sucrose derivative that isovaleric acid is esterified
Application in tumor medicine.
Described tumour includes but is not limited to adenocarcinoma of lung, colon cancer, breast cancer, liver cancer etc..
A kind of sucrose derivative that described hydroxyl is esterified by isovaleric acid refers to have significantly to human A549 cell lines
A kind of compound of cytotoxic activity.
Described medicine is a kind of sucrose derivative being esterified with the hydroxyl of the present invention by isovaleric acid and its can pharmaceutically be connect
The Pharmaceutical composition received, or the Pharmaceutical composition formed using it as active component, including combined with pharmaceutical carrier, for preparing
Antitumor related medicine；Can by oral, non-bowel, intracerebral direct administration, nasal cavity brain-targeted drug delivery, gene engineering research, by
Body mediate transport method is administered or other topical routes；Form of administration can be tablet, dispersible tablet, lozenge, oral disintegrating tablet, sustained release
Piece, granule, pill, capsule, emulsion, solution, suspension, injection, instillation, powder-injection or aerosol etc. pharmaceutically may be used
The formulation of receiving.
Beneficial effects of the present invention and meaning are：Active ingredient of Chinese herbs in the present invention be from the Ainsliaea of Yunnan and
, it has substantial amounts of medicinal history, resource very abundant China is among the people, and its congener Frangrant Ainsliaea Herb has realized people
Work is cultivated.Our research finds to can be used as potential antineoplastic, the new therapy field of exploitation traditional Chinese medicine, to driving cloud
The development tool of southern regional economy is of great significance.
Brief description of the drawings
Fig. 1 is for the structure of compound 2 and to A549 cell growth inhibitions；The wherein structure of (A) compound 2；(B)
Concentration is 1 μM, 3 μM, and 10 μM of compound 2 is acted on after 24,48,72h to A549 cell inhibitory rates；(C) chemical combination of various concentrations
Thing 2 acts on the change of A549 karyomorphisms after 24h；
Fig. 2 is that compound 2 being capable of inducing cell G1Phase retardation；Experimental data represents with average ± standard error,
Using one-way analysis of variance (One-Way ANOVA), Duncan test are examined, P<0.05 is significant difference (*, P<
0.05vs.Vehicle)；
Fig. 3 is that the compound 2 of various dose induces the situation of A549 Apoptosis；The wherein compound 2 of (A) various concentrations
After handling A549 cells, the representative picture of Apoptosis, wherein X- and Y- axles represent Annexin V-FITC and PI dye respectively
Color；(B) the apoptosis trend statistics of A549 cells；Experimental data is represented with average ± standard error, using single factor test variance point
Analyse (One-Way ANOVA), Duncan test are examined, P<0.05 is significant difference (*, P<0.05；**,P<
0.01vs.Vehicle)；
Fig. 4 is influence of the various concentrations compound 2 to A549 mitochondrial membrane potential in anoxic；Wherein (A) various concentrations chemical combination
The mitochondrial membrane potential streaming figure of A549 cells after thing 2 acts on；(B) A549 cell mitochondrials after various concentrations compound 2 acts on
The loss of film potential；Experimental data is represented with average ± standard error, using one-way analysis of variance (One-Way
ANOVA), Duncan test are examined, P<0.05 is significant difference (*, P<0.05vs.Vehicle)；
Fig. 5 is the content of A549 reactive oxygen species ROS after various concentrations compound 2 acts on；Wherein (A) is fluidic cell
Instrument detects DCFH-DA fluorescence intensities；(B) it is A549 reactive oxygen species ROS contents.
Embodiment
In conjunction with embodiment, the invention will be further described, but the implementation of the present invention is not limited to that.
Embodiment 1：Ansloside B (2) preparation
1 sample source
The herb of composite family Ainsliaea platymiscium Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.).
2 extraction isolation and purification methods
After taking the drying herb (15.0kg) of Yunnan Ainsliaea to crush, added respectively with 80L 95% and 80% ethanol
Circumfluence distillation twice after, merge extract solution；Gained medicinal extract uses petroleum ether after moisture dissipates after extract solution is concentrated under reduced pressure
(15L), chloroform (15L), ethyl acetate (15L), n-butanol (15L) are according to polarity order from small to large to successively to water
Scattered medicinal extract carries out liquid-liquid extraction, respectively obtains four petroleum ether, chloroform, ethyl acetate and n-butanol opposed polarities
Extract part；Take the medicinal extract (400g) of ethyl acetate extract, the silica gel column chromatography through 80-100 mesh, with petroleum ether-acetic acid second
Ester system 100:1,50:1,30:1,20:1,10:1,5:1,0:1 is eluted, and 7 components are obtained by the difference of gradient：A
(36g；100:1),B(46g；50:1),C(58g；30:1),D(43g；20:1),E(57g；10:1),F(49g；5:1),G(37g；
0:1)；Component C is by petroleum ether-ethyl acetate system 30:1 gradient elution obtains, the component through mesolow reversed-phase column chromatography with
MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-90%；90-100%；100% presses gradient not
It is same to obtain 7 components：C-1(3g；40-50%), C-2 (15g；50-60%), C-3 (6g；60-70%), C-4 (2g；70–
80%), C-5 (7g；80-90%), C-6 (6g；90-100%), C-7 (5g；100%)；Silica gel of the component C-4 through 200-300 mesh
Column chromatography, with chloroform-methanol 200:1；100:1；50:1；30:1；10:1；5:1 gradient elution separates, wherein three chloromethanes
Alkane-methanol 30:1 gradient elution obtains the (12.0mg of compound 2；Chloroform-methanol 20:1).
The Structural Identification of 3 compounds 2
Using Modern spectroscopy technology (IR,1H-NMR,13C-NMR,DEPT,1H-1H COSY,HSQC,HMBC,NOESY,
HRESIMS) the structure of authenticating compound 2, as shown in formula II：
Ansloside B(2):Colorless oil；IR(KBr)νmax 3446,2960,2929,2871,1741,1662,
1468,1371,1296,1252,1188,1095,1011cm-1；HRESIMS(positive)m/z785.3952[M+Na]+
(calcd 785.3936)；1H-NMR(500MHz,CDCl3)δ:5.55 (1H, d, J=3.5Hz, H-1), 5.16 (1H, d, J=
7.0Hz, H-4'), 4.87 (1H, t, J=9.5Hz, H-4), 4.32 (2H, m, H-1'), 4.31 (2H, m, H-6'), 4.25 (1H,
T, J=9.5Hz, H-3'), 4.19 (1H, m, H-5), 4.15 (1H, m, H-6), 4.08 (1H, m, H-5'), 3.88 (1H, t, J=
9.5Hz,H-3),3.66(1H,m,H-2),2.21-2.26(10H,m,CH2×5),2.06-2.14(5H,m,CH×5),0.92-
0.98(30H,m,CH3×10)；13C-NMR(125MHz,CDCl3)δ:173.0 (C=O), 172.9 (C=O), 172.7 (C=
), O 172.6 (C=O), 172.2 (C=O), 104.3 (C-2'), 92.3 (C-1), 78.0 (C-5'), 77.4 (C-4'), 77.2
(C-3'),72.3(C-2),72.2(C-3),70.2(C-4),68.9(C-5),64.1(C-6'),63.0(C-1'),62.1(C-
6),42.7-43.2(CH2×5),25.4-25.7(CH×5),22.3(CH3×10).
Embodiment 2：The determination of Aglycone and sugared structure in compound 2
The 6.0mg of compound 2 is dissolved in 5%KOH-H2In O (5mL), 95 DEG C of backflow 3h are heated.Reactant mixture hydrochloric acid acid
Change to PH=4.0, respectively with chloroform and extracting n-butyl alcohol three times, be dried under reduced pressure, obtain chloroform layer 2.1mg.Dissolved with deuterochloroform
Afterwards, H NMR spectroscopy is surveyed, is accredited as isovaleric acid.
Isovaleric acid:1H NMR(in CDCl3,500MHz)δH2.23 (2H, d, J=7.5Hz), 2.12 (1H,
M), 0.99 (6H, d, J=7.0Hz)；13C NMRδC 178.8(-COOH),43.0(CH2),25.5(CH),23.3(CH3),22.3
(CH3).
The n-butanol layer of the basic hydrolysis product of compound 2, adds a small amount of methanol to be dissolved in 2M HCl-H2In O (5mL), 95 DEG C add
Heat backflow 4h, after water layer is dried under reduced pressure, then is dissolved with water, directly with ELSD detectors with extracting n-butyl alcohol three times
HPLC is analyzed：YMC-Pack NH2Analyze chromatographic column (5 μm, 12nm, 4.6 × 250mm), 75% acetonitrile-water Gradient elution, stream
Fast 1mL/min, the μ L of sample size 3, is compareed with saccharide, determines the composition of sugar unit, D-Glucose [tR=16.308min,
[α]23 D+53(c0.113,H2O)], D-Fructose [tR=13.116min), [α]23 D-149(c 0.118,H2O)]。
Embodiment 3：Cytotoxic activity is tested
1. experiment material
Human lung adenocarcinoma cell (A549) purchase is given birth to from Shanghai Inst. of Life Science, CAS biochemistry and cell
Wu Xue research institutes；
Compound a nsloside B (2), embodiment 1 are made.
2. cytotoxic activity screens and study on mechanism method
(1) mtt assay detection cytotoxic activity
Collect the A549 cells of logarithmic phase growth, adjustment concentration of cell suspension to 1 × 10596 orifice plates are inoculated in behind individual/hole,
Per the μ L of pore volume 100,37 DEG C, 5%CO2Under the conditions of cultivate, after cell attachment add various concentrations testing compound continue
Cultivate 24h.After culture terminates, 10 μ L 5mg/mL MTT solution is added per hole, continues to be incubated 4 hours, centrifugation discards nutrient solution,
Add 100 μ L DMSO per hole to dissolve formed first a ceremonial jade-ladle, used in libation product, using the light absorption value in each hole at ELIASA measure 570nm.
(2) m- dose dependent detection during cell inhibitory rate
Mtt assay：The A549 cells of culture are after pancreatin digests with 6 × 103Individual/mL concentration is inoculated in 96 orifice plates, often
Hole adds the μ L of cell suspension 100, is placed in 37 DEG C, 5%CO2In incubator after 24h, the given the test agent solution prepared is added
Ansloside B (1,3,10 μM), blank group add DMSO (0.1%), 10 μ L/ holes, and set three multiple holes, are placed in 37 DEG C, and 5%
CO2In incubator after effect 24h, 48h, 72h.5mg/mL MTT solution 10 μ L are added per hole, are discarded after acting on 4h at 37 DEG C
Clear liquid, 100 μ L/ hole DMSO are added, are placed in incubator, detect the OD under 570nm wavelength after to be dissolved with full-automatic ELIASA
Value.Inhibiting rate and IC are calculated according to the following formula50：Inhibiting rate (%)=(control group OD values-experimental group OD values)/control group OD
Value × 100%.Again to add the concentration of compound as abscissa, inhibiting rate (%) is ordinate, is calculated using direct diagram method
Go out IC50。
(3) Apoptosis detects
Using the situation of the double dyeing detection Apoptosis of Annexin V-FITC/PI.The A549 cells of culture pass through pancreatin
With 2 × 10 after digestion5Individual/mL concentration is inoculated in 6 orifice plates, and cell suspension 2mL is added per hole, is placed in 37 DEG C, 5%CO2Incubator
After interior 24h, for experimental group, the given the test agent solution ansloside B (1,3,10 μM) prepared are added, for positive right
According to group, 10 μM of adriamycin is added, for blank group, adds DMSO (0.1%), is placed in 37 DEG C, 5%CO2Effect 24h in incubator
Afterwards, cell being collected to be placed in centrifuge tube, PBS (containing 2%FBS) washs 2 times (2000r/min centrifuges 5min), eliminates liquid as far as possible,
200 μ L binding buffer suspension cells are added, sequentially add 5 μ L annexin V (10 μ g/mL) and 10 μ L PI (20 μ
G/mL), flow cytomery is used immediately after being placed in room temperature dark place 10min.
(4) decoration methods of Hoechst 33258 detection Apoptosis
Using the situation of the decoration methods of Hoechst 33258 detection Apoptosis.The A549 cells of culture digest by pancreatin
After be inoculated in 6 orifice plates, per hole add cell suspension 2mL, be placed in 37 DEG C, 5%CO2In incubator after 24h, for experimental group, add
Enter the given the test agent solution ansloside B (1,3,10 μM) prepared, for positive controls, add 10 μM of adriamycins,
For blank group, add DMSO (0.1%), be placed in 37 DEG C, 5%CO2In incubator after effect 24h, add Fresh more than 4%
Polyformaldehyde room temperature fixes 15min, and PBS is washed 2 times, adds Hoechst 33258 (50 μ g/mL) and is placed in 37 DEG C of dark place dyeing
30min, PBS are washed, suspension cell, with the form of fluorescence microscopy nucleus.
(5) cell cycle is detected
Using the situation of cell cycle detection kit detection cell cycle.The A549 cells of culture are after pancreatin digests
6 orifice plates are inoculated in, cell suspension 2mL is added per hole, is placed in 37 DEG C, 5%CO2In incubator after 24h, for experimental group, add
The given the test agent solution ansloside B (1,3,10 μM) prepared, for positive controls, 10 μM of adriamycin is added, it is right
In blank group, add DMSO (0.1%), be placed in 37 DEG C, 5%CO2In incubator after effect 24h, collect cell and be placed in centrifuge tube,
PBS (containing 2%FBS) washs 2 times (2000r/min centrifuges 5min), eliminates liquid as far as possible, adds cold 4 DEG C of absolute ethyl alcohol overnight,
PBS is washed 2 times, adds 1mL PI staining solutions (sodium citrate containing 3.8mM and 50 μ g/mL PI), adds 50 μ L ribalgilases
Solution A (100 μ g/mL ribonuclease As), is placed in after the 30min of room temperature dark place immediately with each cell week of flow cytomery
The DNA content of phase.
(6) mitochondrial membrane potential detects
Using the situation of mitochondrial membrane potential detection kit detection mitochondrial membrane potential.The A549 cells of culture pass through pancreas
6 orifice plates are inoculated in after enzymic digestion, cell suspension 2mL is added per hole, is placed in 37 DEG C, 5%CO2In incubator after 24h, for experiment
Group, the given the test agent solution ansloside B (1,3,10 μM) prepared are added, for positive controls, add adriamycin
10 μM, for blank group, add DMSO (0.1%), be placed in 37 DEG C, 5%CO2In incubator after effect 24h, 10 μM of rhodamines are added
123 (Rhodamine 123) are examined with flow cytometer immediately after being placed in the dyeing of room temperature dark place 30min, PBS washing and suspension cell
Survey.
(7) reactive oxygen species detect
Intracellular ROS content is detected using active oxygen ROS detection kits.The A549 cells of culture digest by pancreatin
After be inoculated in 6 orifice plates, per hole add cell suspension 2mL, be placed in 37 DEG C, 5%CO2In incubator after 24h, for experimental group, add
Enter the given the test agent solution ansloside B (1,3,10 μM) prepared, for positive controls, add adriamycin 10mM,
For blank group, add DMSO (0.1%), be placed in 37 DEG C, 5%CO2In incubator after effect 24h, cell, PBS washings 2 are collected
It is secondary, 37 DEG C of 20min are placed in after DMEM culture mediums (containing 10 μM of 2', the fat of 7' dichloro fluorescins second two) suspension cell, PBS is washed
Flow cytomery is used immediately after washing 2 times.
3. experimental result
From cytotoxic activity test result, IC of the compound 2 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth and is
3.3 μM, show notable cytotoxic activity.(positive control drug is adriamycin, IC50It is worth for 0.034 μM)
Cytotoxic activity of the compound 2 to human lung adenocarcinoma cell (A549) is tested using mtt assay, using A549 cells as mould
Type come study compound 2 suppress lung adenocarcinoma cell growth mechanism of action.Figure 1B shows suppression of the compound 2 to A549 cell growths
Make to use and there is concentration-time dependence.
After the compound 2 that 1,3,10 μM of various dose acts on A549 cells 24h, under the survival rates of A549 cells is obvious
Drop, influence of the compound 2 to A549 karyomorphisms, the chemical combination of various concentrations are tested using the decoration methods of Hoechst 33258
There occurs the change of nuclear morphology (Fig. 1 C) for many A549 cells after the effect of thing 2 24h.Above experimental result further demonstrates that chemical combination
Thing 2 is capable of the suppression growth of tumour cell of dose dependent.
The flow cytomery influence in the cell growth cycle of compound 2 is used after being dyed using PI.As shown in Figure 2 not
After the processing A549 cells of compound 2 24h of dosage, compound 2 being capable of dose dependent induction G1The increase of phase cell proportion
With S, G2The reduction of phase cell proportion.Above test result indicates that compound 2 can induce A549 cell-cycle arrests in G1Phase.
Have detected compound 2 whether being capable of inducing cell apoptosis using the double dyeing of Annexin V-FITC/PI.Such as Fig. 3 A and
There is the situation of Apoptosis after giving the processing of compound 2 24h of various concentrations in A549 cells shown in 3B, and with change
The increase Apoptosis of compound concentration is more obvious.
Flow cytometry analysis compound 2 is used after being dyed using Rhodamine 123 (Rhodamine 123) to mitochondrial membrane
The influence of potential change.After the compound 2 of various dose handles A549 cells 24h, the mitochondrial membrane potentials of A549 cells with
The increase of compound dosage and decline (Fig. 4).Above test result indicates that compound 2 may be induced carefully by mitochondria pathway
Born of the same parents' apoptosis.
Test influence of the compound 2 to reactive oxygen species ROS contents.A549 cells give the compound of various dose
After 2 processing 24h, flow cytomery DCFH-DA fluorescence intensities are used in DCFH-DA dyeing immediately.The energy of compound 2 as shown in Figure 5
Enough make the increase of A549 reactive oxygen species ROS contents.Above test result indicates that compound 2 may be induced by ROS paths
A549 Apoptosis.
In vitro cell experiment result shows that compound 2 passes through cell G1Phase blocks and apoptosis induction is played to A549 cells
The inhibitory action of growth.In addition, the induction of Apoptosis declines with mitochondrial membrane potential (MMP) and reactive oxygen species ROS
The increase of content is relevant.
Embodiment 4:It is prepared by Ansloside B tablets
Ansloside B 10g
Lactose 130g
Cornstarch 55g
Magnesium stearate 5g
Preparation method：Ansloside B, lactose and cornstarch are mixed, uniformly moistened with water, the mixing after moistening
Thing sieves and dried, re-sieving, adds magnesium stearate, then by mixture tabletting, every weight 200mg, ansloside B contents
For 10mg.
Embodiment 5:It is prepared by Ansloside B parenteral solutions
Ansloside B 2g
Glucose 50g
Preparation method：Ansloside B, glucose are dissolved in appropriate water for injection, resulting solution are filtered, sterile
Under the conditions of be fitted into infusion bottle (every bottle of 100mL), every bottle of 2mg of B containing ansloside.
Embodiment 6:It is prepared by Ansloside B freeze-dried powder injections
Ansloside B 2g
Mannitol 28g
Preparation method：Ansloside B, mannitol are dissolved in appropriate water for injection, resulting solution are filtered, in nothing
Load under the conditions of bacterium in cillin bottle (10mL cillin bottles, every bottle of 2mL), freeze, every 2mg of B containing ansloside.
Embodiment 7:Compound 1,3-8 preparation method
After taking the drying herb (15.0kg) of Yunnan Ainsliaea to crush, it is respectively heated back with the ethanol of 95% and 80%
After stream extraction twice, merge extract solution；Gained medicinal extract uses petroleum ether, three chloromethanes after moisture dissipates after extract solution is concentrated under reduced pressure
Alkane, ethyl acetate, n-butanol according to polarity order from small to large to carrying out liquid-liquid extraction to the medicinal extract that moisture dissipates successively, point
Petroleum ether, chloroform, the extract part of four opposed polarities of ethyl acetate and n-butanol are not obtained；Take ethyl acetate extract
Medicinal extract (400g), the silica gel column chromatography through 80-100 mesh, with petroleum ether-ethyl acetate system 100:1,50:1,30:1,20:1,
10:1,5:1,0:1 is eluted, and 7 components are obtained by the difference of gradient：A(36g；100:1),B(46g；50:1),C
(58g；30:1),D(43g；20:1),E(57g；10:1),F(49g；5:1),G(37g；0:1)；Component C is by petroleum ether-acetic acid second
Ester system 30:1 gradient elution obtains, and the component is through mesolow reversed-phase column chromatography with MeOH-H2O 40-50%；50-60%；
60-70%；70-80%；80-90%；90-100%；100% obtains 7 components by the difference of gradient：C-1(3g；40–
50%), C-2 (15g；50-60%), C-3 (6g；60-70%), C-4 (2g；70-80%), C-5 (7g；80-90%), C-6
(6g；90-100%), C-7 (5g；100%)；Silica gel column chromatographies of the component C-4 through 200-300 mesh, with chloroform-methanol
200:1；100:1；50:1；30:1；10:1；5:1 gradient elution separates, wherein chloroform-methanol 50:1 gradient elution cut
(300mg), (21.0mg of compound 3 is obtained after purification by Sephadex LH-20；Chloroform-methanol 20:1), three chloromethane
Alkane-methanol 30:1 gradient elution obtains 4 (7.0mg；Chloroform-methanol 20:1)；C-2 is through RP-MPLC with MeOH-H2O 40–
50%；50-60%；60-70%；70-80%；80-100% gradient elutions obtain 5 components by different collect of gradient
C-21(2.3g；40-50%), C-22 (1.8g；50-60%), C-23 (2.9g；60-70%), C-24 (2.3g；70-80%),
C-25(3.1g；80-100%)；Silica gel column chromatographies of the component C-21 through 200-300 mesh, with petroleum ether-ethyl acetate 50:1；40:
1；30:1；20:1；10:1；1:1 gradient elution, wherein petroleum ether-ethyl acetate 20:1 gradient elution obtains compound 5
(9.3mg；Chloroform-methanol 20:And 7 (7.1mg 1)；Chloroform-methanol 20:1), petroleum ether-ethyl acetate 10:1 gradient
Afford (the 11.2mg of compound 6；Chloroform-methanol 20:1), petroleum ether-ethyl acetate 1:1 gradient elution obtains chemical combination
(the 6.4mg of thing 8；Chloroform-methanol 20:1)；Component C-23 is through RP-MPLC with MeOH-H2O 40-50%；50-60%；60–
70%；70-80%；80-90%；90-100%；100% gradient elution, wherein 70-80%MeOH gradient elutions obtain compound
1(10.5mg；Chloroform-methanol 20:1).
Embodiment 8:The Structural Identification of compound 1,3-8
Using Modern spectroscopy technology (IR,1H-NMR,13C-NMR,DEPT,1H-1H COSY,HSQC,HMBC,NOESY,
HRESIMS) authenticating compound 1,3-8 structure, it is as follows：
Ainsloside A(1):Colorless oil；IR(KBr)νmax 3471,2960,2927,2873,1743,1658,
1468,1369,1296,1254,1188,1120,1014cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS
(positive)m/z 785.3939[M+Na]+(calcd 785.3936).
Ainsloside C(3):Colorless oil；IR(KBr)νmax 3485,3259,2960,2873,1741,1468,
1369,1296,1188,1115,1005cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS(positive)m/z
785.3950[M+Na]+(calcd 785.3936).
Ainsloside D(4):Colorless oil；IR(KBr)νmax 3477,2960,2933,2873,1741,1668,
1468,1369,1296,1254,1188,1120,1005cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS
(positive)m/z 785.3942[M+Na]+(calcd 785.3936).
Ainsloside E(5):Colorless oil；IR(KBr)νmax 3467,2960,2933,2873,1739,1468,
1369,1296,1255,1190,1120,1063,1005cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS
(positive)m/z 701.3365[M+Na]+(calcd 701.3360).
Ainsloside F(6):Colorless oil；IR(KBr)νmax 3460,2960,2931,2873,1741,1468,
1371,1296,1190,1099,1012cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS(positive)m/z
701.3362[M+Na]+(calcd 701.3360).
Ainsloside G(7):Colorless oil；IR(KBr)νmax 3408,2960,2933,2873,1743,1468,
1369,1296,1254,1188,1093,1003cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；HRESIMS
(positive)m/z 701.3371[M+Na]+(calcd 701.3360).
Ainsloside H(8):Colorless oil；IR(KBr)νmax 3456,2960,2929,2873,1743,1668,
1468,1369,1296,1254,1188,1101,1065,1012cm-1；1H and13C H NMR spectroscopy data, are shown in Tables 1 and 2；
HRESIMS(positive)m/z 701.3357[M+Na]+(calcd 701.3360).
Table 1 compound 1,3-81H NMR (500MHz) data (in ppm) (CDCl3)
Table 2 compound 1,3-813C NMR (125MHz) data (in ppm) (CDCl3)
Embodiment 9:The cytotoxic activity test of compound 1,3-8
Compound 1,3-8 are tested to the cytotoxic activity of A549 human lung adenocarcinoma cells, experiment material experiment using mtt assay
Method is the same as embodiment 3.
Experimental result：
IC of the compound 1 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 9.74 μM,
IC of the compound 3 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth and is>100 μM,
IC of the compound 4 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 19.93 μM,
IC of the compound 5 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 10.75 μM,
IC of the compound 6 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 60.06 μM,
IC of the compound 7 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 46.19 μM,
IC of the compound 8 to A549 human lung adenocarcinoma cell cytotoxic activities50It is worth for 66.00 μM.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (9)
1. a kind of sucrose derivative, its chemical constitution is shown in formula I：
In Formulas I, R1~R8Group is respectively selected from：
Hydrogen atom,
Isovaleryl,
Substitution or the acyl group containing straight or branched of unsubstituted C1~6,
The substituted or unsubstituted acyl group containing five yuan or hexa-atomic aromatic rings；
Described acyl group quantity is 4~5, and remaining is hydrogen atom.
A kind of 2. sucrose derivative according to claim 1, it is characterised in that R1~R8The combination of group is as follows：
Compound 1:R1=R2=R4=R6=R7=-COCH2CH(CH3)2,R3=R5=R8=H；
Compound 2:R1=R2=R6=R7=R8=-COCH2CH(CH3)2,R3=R4=R5=H；
Compound 3:R1=R2=R3=R6=R7=-COCH2CH(CH3)2,R4=R5=R8=H；
Compound 4:R1=R3=R4=R6=R7=-COCH2CH(CH3)2,R2=R5=R8=H；
Compound 5:R1=R4=R6=R7=-COCH2CH(CH3)2,R2=R3=R5=R8=H；
Compound 6:R1=R2=R4=R7=-COCH2CH(CH3)2,R3=R5=R6=R8=H；
Compound 7:R1=R2=R6=R7=-COCH2CH(CH3)2,R3=R4=R5=R8=H；
Compound 8:R1=R2=R4=R6=-COCH2CH(CH3)2,R3=R5=R7=R8=H.
3. a kind of preparation method of sucrose derivative as claimed in claim 1, comprises the following steps：
Added after the herb of rabbit ear platymiscium Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.) is crushed with ethanol
Circumfluence distillation, the medicinal extract that extract solution obtains after being concentrated under reduced pressure is water-dispersible, is prepared respectively using the method for liquid-liquid extraction
Petroleum ether, chloroform, the extract part of four kinds of opposed polarities of ethyl acetate and n-butanol, are made with upper bit with one or many
By the use of conventional chromatograph packing material silica gel, macroporous absorbent resin, ODS or Sephadex LH-20 as stationary phase, with petroleum ether, three chloromethanes
Alkane, ethyl acetate, acetone, methanol, acetonitrile or water with being used for eluant, eluent, are separated and purified and be prepared into single or various combination
To target compound.
4. the preparation method of a kind of sucrose derivative according to claim 3, it is characterised in that the ethanol selects 95%
With 80% ethanol, heating and refluxing extraction is each twice respectively, and merges extract solution, and the temperature of heating and refluxing extraction is 80 DEG C.
A kind of 5. preparation method of sucrose derivative according to claim 3, it is characterised in that described petroleum ether extraction
Position, using petroleum ether-ethyl acetate system 200:1–0:1 gradient elution；Described chloroform extract part, using trichlorine
Methane-methanol 100:0–0:1 gradient elution；Described ethyl acetate extract part, using petroleum ether-ethyl acetate 100:1–0:
1 gradient elution；Described n-butanol extract part, using chloroform-methanol 100:1–0:1 gradient elution.
A kind of 6. preparation method of sucrose derivative as claimed in claim 2, it is characterised in that the system of the compound 1-8
Preparation Method is as follows：
After taking the drying herb of Yunnan Ainsliaea (Ainsliaea yunnanensis Franch.) to crush, with 95% and 80%
Ethanol be respectively heated refluxing extraction twice after, merge extract solution；Gained medicinal extract dissipates through moisture after extract solution is concentrated under reduced pressure
Afterwards using leaching of the order to being dissipated successively to moisture of petroleum ether, chloroform, ethyl acetate, n-butanol according to polarity from small to large
Cream carries out liquid-liquid extraction, respectively obtains petroleum ether, chloroform, the extraction unit of four opposed polarities of ethyl acetate and n-butanol
Position；The medicinal extract of ethyl acetate extract is taken, the silica gel column chromatography through 80-100 mesh, with petroleum ether-ethyl acetate system 100:1,50:
1,30:1,20:1,10:1,5:1,0:1 is eluted, and 7 components are obtained by the difference of gradient：A 100:1,B 50:1,C
30:1,D 20:1,E 10:1,F 5:1,G 0:1；Component C is by petroleum ether-ethyl acetate system 30:1 gradient elution obtains,
The component is through mesolow reversed-phase column chromatography with MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-90%；
90-100%；100% obtains 7 components by the difference of gradient：C-1 40-50%, C-2 50-60%, C-3 60-
70%, C-4 70-80%, C-5 80-90%, C-6 90-100%, C-7 100%；
Silica gel column chromatographies of the component C-4 through 200-300 mesh, with chloroform-methanol 200:1；100:1；50:1；30:1；10:1；
5:1 gradient elution separates, wherein chloroform-methanol 50:1 gradient elution cut, by Sephadex LH-20 after purification
To compound 3, chloroform-methanol 30:1 gradient elution obtains compound 2 and compound 4；
Component C-2 is through RP-MPLC with MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-100% gradients are washed
It is de- to obtain 5 component C-21 40-50%, C-22 50-60%, C-23 60-70%, C-24 by different collect of gradient
70-80%, C-25 80-100%；Silica gel column chromatographies of the component C-21 through 200-300 mesh, with petroleum ether-ethyl acetate 50:1；
40:1；30:1；20:1；10:1；1:1 gradient elution, wherein petroleum ether-ethyl acetate 20:1 gradient elution obtains compound
5 and 7, petroleum ether-ethyl acetate 10:1 gradient elution obtains compound 6, petroleum ether-ethyl acetate 1:1 gradient elution
Compound 8；Component C-23 is through RP-MPLC with MeOH-H2O 40-50%；50-60%；60-70%；70-80%；80-90%；90–
100%；100% gradient elution, wherein 70-80%MeOH gradient elutions obtain compound 1.
A kind of 7. application of sucrose derivative in antineoplastic is prepared as claimed in claim 1 or 2.
A kind of 8. application of the sucrose derivative according to claim 7 in antineoplastic is prepared, it is characterised in that institute
The tumour stated is adenocarcinoma of lung, colon cancer, breast cancer, liver cancer.
9. application of a kind of sucrose derivative in antineoplastic is prepared according to claim 7 or 8, its feature exist
In tablet, granule, pill, capsule, emulsion, solution, suspension, note is made by pharmacy conventional method in medicine therein
Penetrate agent, instillation, powder-injection or aerosol.
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| NGEHJ.TOYANG, ET AL.,: "In vivo antiprostatetumorpotentialof Vernoniaguineensis Benth.(Asteraceae)tuber extract(VGDE) and the cytotoxicity of its major compound pentaisovaleryl sucrose.", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
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