CN107334765B - A kind of medical composition and its use for the treatment of cancer - Google Patents
A kind of medical composition and its use for the treatment of cancer Download PDFInfo
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- CN107334765B CN107334765B CN201710577162.5A CN201710577162A CN107334765B CN 107334765 B CN107334765 B CN 107334765B CN 201710577162 A CN201710577162 A CN 201710577162A CN 107334765 B CN107334765 B CN 107334765B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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Abstract
The present invention provides a kind of medical composition and its uses for the treatment of cancer, are related to cancer medication field.At least one of this pharmaceutical composition, including Eupatorium adenophorum lactone, 23,4,6,11 tetrahydrochysene cadinane of acetyl group, 7 ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.No matter three kinds of active constituents in this pharmaceutical composition are single use or are used in combination, and all have good active anticancer, play effect by cancer cell specific induction of apoptosis and retardance Cancer Cell cycle, can be used for preparing clinical anti-cancer drug.
Description
Technical field
The present invention relates to cancer medication fields, in particular to a kind of medical composition and its use for the treatment of cancer.
Background technology
Composite family feverwort Eupatorium adenophorum Eupatorium adenophorum Spreng. are commonly called as " lose horse grass ", " black
Stem grass ", " stinkweed ", " overlord's grass " etc., have powerful bioactivity.Eupatorium adenophorum has the record used as traditional herbal medicine,
The southern regions of the Yunnan Province is civil to take its cauline leaf is to boil water to wash, and controls athlete's foot and rice field eczema, also has and rub affected part with leaf, can stop blooding, relieve pain, anti-inflammatory.
It is less to the pharmacological research of Eupatorium adenophorum at present.Therefore in order in order to fully develop the potential medical value of Eupatorium adenophorum, control into
Species are invaded, are changed harmful to treasure, the present invention carries out in-depth study to Eupatorium adenophorum.
Invention content
The first object of the present invention is to provide a kind of pharmaceutical composition for the treatment of cancer, the activity in the pharmaceutical composition
Ingredient origin can be used in prevention or treating cancer in Eupatorium adenophorum.
The second object of the present invention be to provide a kind of pharmaceutical composition prepare prevent or the drug for the treatment of cancer in
Purposes provides new treatment means and thinking for the prevention or treatment of cancer, while also developing the new medicinal valence of Eupatorium adenophorum
Value.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of pharmaceutical composition for the treatment of cancer comprising Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- tetrahydrochysenes Du
At least one of loose alkane -7- ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.
A kind of pharmaceutical composition prepare prevent or the drug for the treatment of cancer in purposes comprising Eupatorium adenophorum lactone,
2- acetyl group -3,4, at least one of 6,11- tetrahydrochysene cadinane -7- ketone or carat dimension alcohol and pharmaceutically acceptable load
Body or auxiliary material.
Compared with prior art, beneficial effects of the present invention for example including：
The present invention isolates and purifies to obtain the active constituent with anticancer function from Eupatorium adenophorum:Eupatorium adenophorum lactone, 2-
Acetyl group -3,4,6,11- tetrahydrochysene cadinane -7- ketone, carat tie up alcohol, and no matter these three active constituents are single use or combine
It uses, all has good active anticancer, effect is played by cancer cell specific induction of apoptosis and retardance Cancer Cell cycle, can be used
In preparing clinical anti-cancer drug.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the flow chart that Eupatorium adenophorum petroleum ether extract is prepared in the embodiment of the present invention 1；
Fig. 2 is the flow chart of prepare compound A, B, C in the embodiment of the present invention 2；
Fig. 3 is compound A, B, C in experimental example one of the present invention and Eupatorium adenophorum petroleum ether extract to hepatocellular carcinoma H22
With the influence diagram of normal liver cell LO2 cell viabilities, wherein ordinate be tumour cell percent inhibition, abscissa be to
Concentration；
Fig. 4 is compound A, B, C and Eupatorium adenophorum petroleum ether extract cytotoxic activity IC50 in experimental example one of the present invention
Value, wherein ordinate is IC50 values；
Fig. 5 is Eupatorium adenophorum petroleum ether extract in experimental example two of the present invention to the influence diagram of HepG2 cellular morphologies；
Fig. 6 be in experimental example three of the present invention Eupatorium adenophorum petroleum ether extract to the influence diagrams of HepG2 cell cycles,
In, the horizontal seat side of flow cytometry figure is fluorescence intensity, and ordinate is cell quantity；The abscissa of histogram is that administration is dense
Degree, ordinate is the accounting of sub G1 phases；
Fig. 7 is Eupatorium adenophorum petroleum ether extract in experimental example four of the present invention to the influence diagram of HepG2 cell DNAs；
(A) is that Eupatorium adenophorum petroleum ether extract turns HepG2 mitochondrial apoptosis signals in experimental example five of the present invention in Fig. 8
The influence of guiding path correlative protein expression；(B) it is that Eupatorium adenophorum petroleum ether extract is logical to HepG2 cell JAK2/STAT3 signals
The influence on road.
In the above figure, Norma indicates normal group, and CDDP indicates that cis-platinum positive drug group, EA-PE indicate Eupatorium adenophorum oil
Ether extract, A indicate that 2- acetoxy-3s, 4,6,11- tetrahydrochysene cadinane -7- ketone, B indicate that carat ties up alcohol, and C indicates Eupatorium adenophorum
Lactone.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Present embodiment provides a kind of pharmaceutical composition comprising：Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- four
At least one of hydrogen cadinane -7- ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.
Wherein, the structural formula of 2- acetyl group -3,4,6,11- tetrahydrochysene cadinane -7- ketone (compound A) is：
Wherein, the structural formula of carat dimension alcohol (compound B) is：
Wherein, the structural formula of Eupatorium adenophorum lactone (compound C) is：
Further, pharmaceutical composition includes Eupatorium adenophorum petroleum ether extract, wherein Eupatorium adenophorum petroleum ether extract
Preparation method include：Eupatorium adenophorum is extracted using alcohol-water mixed solution, by obtained purple stem boneset extract oil
Ether extraction to get.This extracting method is simple for process, and paste-forming rate is high, and the extract active anticancer extracted is good, is suitble to scale
Production.
Present embodiment also provides a kind of preparation method of the pharmaceutical composition for the treatment of cancer comprising：
Step S1:Eupatorium adenophorum is extracted using alcohol-water mixed solution, by obtained purple stem boneset extract oil
Ether extracts, and obtains Eupatorium adenophorum petroleum ether extract.
Further, it is extracted using infusion process.Optionally, with 95% ethanol solution with solid-liquid ratio be 1：7~9 dippings are purple
Stem Herba Lycopi 2~4 days.Optionally, it impregnates 1~3 time, every time 2~4 days.
Step S2:Using silica gel column chromatography, petroleum ether extraction is detached with petroleum ether-ethyl acetate mixed solvent gradient elution
Object obtains Eupatorium adenophorum lactone and mixed fraction.
Optionally, gradient elution is carried out using petroleum ether-ethyl acetate mixed solution, obtains Eupatorium adenophorum lactone and three
It is a to evaporate section (F1-F3), section is evaporated as mixed fraction (F1) using first.Wherein, petroleum ether and ethyl acetate preferentially use 60~90 DEG C
The solvent of boiling range specification.
Step S3:Silica gel column chromatography is used again, and mixed fraction is detached with acetone-petroleum ether mixed solvent gradient elution,
Obtain 2- acetyl group -3,4,6,11- tetrahydrochysenes cadinane -7- ketone and carat dimension alcohol.
Further, it using 2% acetone-petroleum ether as eluant, eluent, using silica gel column chromatography separation mixed fraction (F1), obtains
3 evaporate section (F1.1-F1.3), continue that carat dimension alcohol and Eupatorium adenophorum lactone is prepared using thin layer chromatography or HPLC methods.
Present embodiment also provide a kind of pharmaceutical composition prepare prevent or the drug for the treatment of cancer in purposes, it is described
Pharmaceutical composition includes that Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- tetrahydrochysenes cadinane -7- ketone or carat are tieed up in alcohol extremely
A kind of few and pharmaceutically acceptable carrier or auxiliary material.
Inventor is studies have shown that the ligroin extraction of Eupatorium adenophorum and above-mentioned three kinds of active constituents have excellent anticancer
Activity, further study show that, play anticancer by promoting cancer cell-apoptosis approach and blocking the effect of Cancer Cell cycle
It is active, it can be used for clinical anti-cancer medication.
Further, inventor confirms through test cell line, the ligroin extraction of the Eupatorium adenophorum and above-mentioned three kinds of activity
Ingredient is to induce the cancer cell-apoptosis by influencing JAK-STAT signal paths.
JAK-STAT signal paths are a signal transduction pathways stimulated by cell factor of discovered in recent years, are participated in
Many important biological processes such as proliferation, differentiation, apoptosis and the immunological regulation of cell.Wherein, the activity of STAT3 is in cell
Serve during canceration key.Many tumour derived cell strains are required for stat protein (especially STAT3) to keep turning
Phenotype after change, these tumour derived cell strains include liver cancer, breast cancer, lung cancer, gastric cancer, neuroblastoma, nasopharyngeal carcinoma,
Prostate cancer or carcinoma of urinary bladder.
Below by taking hepatocellular carcinoma as an example, it is discussed in detail：
The risk factor of many hepatocellular carcinomas, including the infection of HBV, HCV and the steatosis of liver can promote
Into the activation of STAT3.The transmittance process of JAK-STAT signal paths is relatively easy.Signal transduction process is as follows：Cell factor with
Corresponding receptor causes the dimerization of acceptor molecule after combining, this makes close to each other with the jak kinase of coupled receptors and passes through
Interactive tyrosine phosphorylation effect and activate.Phosphorylation modification occurs for the tyrosine residue after JAK activation on catalytic receptor, after
And the tyrosine site of these phosphorylations and the amino acid sequence of surrounding form " docking site " (docking site), contain simultaneously
There is the stat protein of SH2 structural domains to be enrolled into this " docking site ".Finally, kinases JAK catalyzed combinations are on receptor
Phosphorylation modification occurs for stat protein, and the stat protein of activation is entered in the form of dimer in nucleus to be combined with target gene,
The transcription of controlling gene.
STAT3, often by persistent activation, is indispensable in the generation of hepatocellular carcinoma in hepatocellular carcinoma
, it is the main tumorigenesis transcription factor of hepatocellular carcinoma.The phosphorylation and its downstream gene Bcl-2, Bcl-xl of STAT3,
The up-regulation of CyclinD1, VEGF promote the proliferation of hepatocellular carcinoma, vascularization, invasion transfer and immunologic escape, and activate
STAT3 be also essential for the survival of hepatocellular carcinoma cell.Therefore, STAT3 is treated as hepatocellular carcinoma
A very important molecular targeted target.
Inventor the study found that Eupatorium adenophorum petroleum ether extract and three kinds of active constituents can reduce JAK2 albumen with
The phosphorylation level of STAT3 albumen inhibits tumor correlated albumen expression downstream that is, by regulating and controlling JAK-STAT signal paths.
At the same time, inventor also found, the pharmaceutical composition be by influence mitochondrial apoptosis signal transduction pathway come
Cancer cell specific induction of apoptosis.The expression and regulation and control of bcl-2 families are to influence the key factor of mitochondrial apoptosis signal transduction
One of, main function site is on mitochondrial membrane.After cell is stimulated by dead signal, the rush apoptosis egg in bcl-2 families
Raw conformation variation is issued in albumen enzyme effect in vain, is displaced on organelle film from cytoplasm, especially on mitochondrial outer membrane, and with
Anti-apoptotic proteins interaction on organelle film and in film, makes anti-apoptotic proteins lose the inhibiting effect to apoptosis, causes thin
Born of the same parents' device function loses and the various releases for promoting antiapoptotic factors, eventually leads to cancer cell-apoptosis.
Bcl-2 and Bax is most representative inhibition apoptosis and promotion apoptogene in bcl-2 families respectively, and
Bcl-2 is the expression of target gene albumen of STAT3 classics.In normal liver tissue, Bcl-2 is often expressed as feminine gender, and Bax expression is positive
Property.But in malignant tumours such as such as liver cancer, lung cancer, gastric cancer, breast cancer, neuroblastoma, nasopharyngeal carcinoma, prostate cancer and carcinomas of urinary bladder
In, the up-regulation and Bax that all observed Bcl-2 are lowered, and cell differentiation is lower, and this trend is more obvious.
Inventor is the study found that the composition can lower the expression of Bcl-2 albumen, the expression of up-regulation Bax albumen
Level, to reduce the ratio of Bcl-2/Bax.
The change of Bcl-2/Bax ratios eventually leads to Apoptosis caused by caspase families.Caspase families are one
The cysteine proteinase in class energy specific recognition aspartic acid site participates in multiple processes of antiapoptotic signals transduction.
In the apoptosis pathway that mitochondrial mediates, the amplification of Caspase families apoptosis involvement signal and apoptosis effect pass through enzymatic grade
The mode of connection reaction amplifies apoptotic signal, and is catalyzed apoptosis correlation effect albumen, eventually leads to the apoptosis of cell.Such as in Bcl-2
Under the regulation and control of family, mitochondria release apoptosis activation factor 1 (Apaf1), Apaf1 combination cell pigment C (cytoC), ATP, and
By conformation variation and autoactivation, so that the ends Apaf1N is formed a kind of surface texture, can activate Caspase 8/9, then into
One step activates Caspase 3/6/7, and final Caspase 3/6/7 is catalyzed a series of apoptosis related substrates, causes Apoptosis.
The study found that the composition can increase the activation levels of Caspase-9 and Caspase-3, to pass through enzymatic
The mode of cascade reaction amplifies apoptotic signal, eventually leads to the apoptosis of cancer cell.
Further, which prevented or treating cancer by blocking Cancer Cell cycle.
Cell cycle regulating is another important means that body maintains cell state.Cancerous tumor cell is in addition to resisting apoptosis mistake
Journey, the proliferative capacity abnormal toward contact performance.Such as p53, BRCA1, Rb, p16, p15 and p53, the Downstream regulatory gene of BRCA1
If p21, Gadd45 are the important components of cell cycle monitoring point.But in most tumors generating process, these suppression cancer bases
It is inactivated because there is gene alteration, it is that cell obtains uncontrolled proliferation to cause cell cycle monitoring point functional defect, final result more
Ability leads oncogenic generation.
Inventor is the study found that Eupatorium adenophorum petroleum ether extract and three kinds of active constituents can block the thin of cancer cell
Born of the same parents' period makes it be stuck in G2 phases or M phases, and can induce the apoptosis of this two quasi-cancer cell.
Thus illustrate, which can be used for the treatment of cancer, such as liver cancer, breast cancer, lung cancer, gastric cancer, god
Through blastoma, nasopharyngeal carcinoma, prostate cancer or carcinoma of urinary bladder.Optionally, it is used for the treatment of liver cancer.
Further, of the invention in order to make drug discharge active component rapidly, continuously and in a very long time
Pharmaceutical composition can be manufactured according to those conventional methods in the art are disclosed in.The administration of the drug of present embodiment
Approach can be oral, nasal inhalation or parenteral administration.Including the preparation of the composition can be tablet, soft capsule, ebonite
Capsule, oral solution, pill, suppository, powder, particle, emulsion, syrup, aerosol, sterile injectable preparation and sterilized powder etc..
In the present invention, it is physiologically may be used that term " pharmaceutically acceptable ", which refers to the compound when compound is to human administration,
Receive, and the allergic reactions such as gastrointestinal disturbance, dizziness or these similar anaphylactoid systemic anaphylaxis will not occur.
In the present invention, " pharmaceutically acceptable carrier or auxiliary material " includes but not limited to：Adhesive (such as microcrystalline cellulose
Element, alginates, gelatin and polyvinylpyrrolidone), filler (such as starch, sucrose, glucose and anhydrous lactic acid), disintegrant
(such as cross-linked pvp, crosslinked carboxymethyl fecula sodium, croscarmellose sodium and low-substituted hydroxypropyl cellulose), lubricant
(magnesium stearate, aluminum stearate, talcum, polyethylene glycol, sodium benzoate), wetting agent (such as glycerine), surfactant (such as hexadecane
Alcohol) and sorbefacient, corrigent, sweetener, diluent, coating agent etc..
The feature and performance of the present invention are described in further detail with reference to embodiments：
Embodiment 1
The present embodiment provides a kind of pharmaceutical compositions comprising Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- tetrahydrochysenes
At least one of cadinane -7- ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.
Preparation method includes：
As shown in Figure 1, Eupatorium adenophorum complete stool dried powder 10kg is taken, by solid-liquid ratio 1:8 (1kg medicinal materials correspond to the solvent of 8L)
The alcohol dipping 3 days for being 95% with volumetric concentration, filters to take filtrate, to filter residue using same operation extraction twice, filtering is closed
And the filtrate extracted three times, it is concentrated under reduced pressure to give Eupatorium adenophorum medicinal extract 806g.By volume 1:10 add water to be suspended, with three times volume
Petroleum ether extraction sample, petroleum ether extraction liquid are concentrated under reduced pressure, and recycling design is evaporated, and obtains Eupatorium adenophorum petroleum ether extract
182g。
As shown in Fig. 2, taking 100g Eupatorium adenophorum petroleum ether extracts, chromatographed through silicagel column (200-300 mesh), petroleum ether
(60~90 DEG C)-ethyl acetate gradient, obtains compound A：2- acetoxy-3s, 4,6,11- tetrahydrochysene cadinane -7- ketone
(3.362g) evaporates section (F1-F3) with 3；F1 through silica gel column chromatography, 2% acetone-petroleum ether elute 3 evaporate section (F1.1-
F1.3)；F1.2 carries out preparing thin-layer chromatography using 5% Acetone-Benzene as solvent to be obtained 6 and evaporates section (F1.2.1-F1.2.6), F1.2.6
It carries out preparing thin-layer chromatography as solvent using 3% acetone-dichloromethane and obtains compound B：Carat dimension alcohol (85mg)；F1.2.5 is through silicon
Plastic column chromatography, 2% Acetone-Benzene elute to obtain compound C：Eupatorium adenophorum lactone (28mg).
Embodiment 2
The present embodiment provides a kind of pharmaceutical compositions comprising Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- tetrahydrochysenes
At least one of cadinane -7- ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.
Preparation method includes：
Eupatorium adenophorum complete stool dried powder 10kg is taken, by solid-liquid ratio 1:9 (1kg medicinal materials correspond to the solvent of 9L) use volumetric concentration
For 90% alcohol dipping 4 days, filtrate is filtered to take, primary using same operation extraction to filter residue, filtering merges extracts twice
Filtrate, be concentrated under reduced pressure to give Eupatorium adenophorum medicinal extract 780g.By volume 1:9 add water to be suspended, and petroleum ether extraction is accumulated with tetraploid
Sample, petroleum ether extraction liquid are concentrated under reduced pressure, and recycling design is evaporated, and obtains Eupatorium adenophorum petroleum ether extract 176g.
100g Eupatorium adenophorum petroleum ether extracts are taken, are chromatographed through silicagel column (200-300 mesh), petroleum ether (60~90 DEG C)-
Ethyl acetate gradient obtains compound A：2- acetoxy-3s, 4,6,11- tetrahydrochysene cadinane -7- ketone (3.24g) with 3
Evaporate section (F1-F3)；F1 through silica gel column chromatography, 2% acetone-petroleum ether elute 3 evaporate section (F1.1-F1.3)；F1.2 is with 3% the third
Ketone-benzene is that solvent carries out preparing thin-layer chromatography and obtains 6 and evaporate section (F1.2.1-F1.2.6), and F1.2.6 is with 6% acetone-dichloromethane
Alkane is that solvent carries out preparing thin-layer chromatography and obtains compound B：Carat dimension alcohol (82mg)；F1.2.5 is through silica gel column chromatography, and 2% the third
Ketone-benzene elutes to obtain compound C：Eupatorium adenophorum lactone (24mg).
Embodiment 3
The present embodiment provides a kind of pharmaceutical compositions comprising Eupatorium adenophorum lactone, 2- acetyl group -3,4,6,11- tetrahydrochysenes
At least one of cadinane -7- ketone or carat dimension alcohol and pharmaceutically acceptable carrier or auxiliary material.
Preparation method includes：
Eupatorium adenophorum complete stool dried powder 10kg is taken, by solid-liquid ratio 1:7 (1kg medicinal materials correspond to the solvent of 7L) use volumetric concentration
For 98% alcohol dipping 2 days, filter to take filtrate, to filter residue using same operation extraction twice, filtering merges extracts three times
Filtrate, be concentrated under reduced pressure to give Eupatorium adenophorum medicinal extract 812g.By volume 1:11 add water to be suspended, with two volumes petroleum ether extraction
Sample, petroleum ether extraction liquid are concentrated under reduced pressure, and recycling design is evaporated, and obtains Eupatorium adenophorum petroleum ether extract 186g.
100g Eupatorium adenophorum petroleum ether extracts are taken, are chromatographed through silicagel column (200-300 mesh), petroleum ether (60~90 DEG C)-
Ethyl acetate gradient obtains compound A：2- acetoxy-3s, 4,6,11- tetrahydrochysene cadinane -7- ketone (3.43g) with 3
Evaporate section (F1-F3)；F1 through silica gel column chromatography, 2% acetone-petroleum ether elute 3 evaporate section (F1.1-F1.3)；F1.2 is with 5% the third
Ketone-benzene is that solvent carries out preparing thin-layer chromatography and obtains 6 and evaporate section (F1.2.1-F1.2.6), and F1.2.6 is with 3% acetone-dichloromethane
Alkane is that solvent carries out preparing thin-layer chromatography and obtains compound B：Carat dimension alcohol (87mg)；F1.2.5 is through silica gel column chromatography, and 2% the third
Ketone-benzene elutes to obtain compound C：Eupatorium adenophorum lactone (29mg).
Experimental example
With reference to the test of pesticide effectiveness to the Eupatorium adenophorum petroleum ether extract (EA-PE) that is provided in the embodiment of the present invention 1~3
Evaluating drug effect is carried out with three kinds of active constituents, wherein three kinds of active constituents are specially：Eupatorium adenophorum lactone (compound C), 2- second
Acyl group -3,4,6,11- tetrahydrochysene cadinane -7- ketone (compound A), carat dimension alcohol (compound B).
One Eupatorium adenophorum petroleum ether extract of experimental example and three kinds of active constituents are thin with normal hepatocytes to hepatocellular carcinoma H22
The influence of born of the same parents' LO2 cell viabilities
Logarithmic growth phase HepG2 cell strains, it is 5 × 10 to adjust cell concentration4A/mL is seeded to 96 holes per hole 0.2mL
After continuing culture for 24 hours, the sample to be tested being configured to by 1640 liquid containing 10% calf serum RPMI is added per hole respectively for culture plate
0.2mL, make sample concentration be respectively 5,10,25,50,100,150,200 μ g/mL continue culture for 24 hours with 48h；Each concentration one
3 hole of formula, while the blank control wells for setting the experiment contrast for being not added with test sample and being not added with sample and cell.After reaching administration time
The MTT 20mL that 5 μ g/mL are added per hole continue to be incubated 4h, are inhaled after arrival incubation time and abandon culture solution, 0.15mL is added per hole
DMSO shakes 30min, fully dissolves the purple formazan crystallization generated into the cell, is measured per hole absorbance A value under 550nm.
With administration in addition to administration time is for 24 hours, remaining operates same HepG2 for the culture of LO2 cells.
Growth inhibition ratio=[1- (experimental group A values-blank group A values)/(control group A value-blank of HepG2 and LO2 cells
Group A values)] × 100%.Use 19.0 calculation of half inhibitory concentration of SPSS (IC50).
As a result see Fig. 3, Fig. 4, the concentration range of compound A, B, C and Eupatorium adenophorum petroleum ether extract in 5-200 μ g/mL
It is interior, it shows that preferable vigor inhibits to hepatocellular carcinoma H22, also shows that apparent dose-effect, time-effect relationship, but to normal hepatocytes
Cell LO2 influences less (Fig. 3).Compound A, B, C and Eupatorium adenophorum petroleum ether extract are respectively to the IC50 of HepG2 for 24 hours
44.8, the IC50 of 31.8,27.1 and 25.1 μ g/mL, 48h is respectively 31.4,21.4,17.8 and 13.4 μ g/mL.Above-mentioned each chemical combination
Object and Eupatorium adenophorum petroleum ether extract hardly exhibit vigour inhibition to LO2 cells, and IC10 is all more than 200 μ g/mL.
The influence of experimental example two, Eupatorium adenophorum petroleum ether extract to HepG2 cellular morphologies
The HepG2 cells of logarithmic growth phase, with 5 × 104The concentration in a/hole is inoculated with into 6 orifice plates, and for 24 hours, training is abandoned in suction for culture
The Eupatorium adenophorum petroleum ether extract (sample concentration is respectively 10,30,50 μ g/mL) of various concentration is added in nutrient solution, and blank is added
Liquid group (physiological saline) is used as negative control group, continues culture for 24 hours, and after microscope light field observes cellular morphology, culture solution is abandoned in suction,
Fixer (methanol is added per hole：Glacial acetic acid=3:1) it inhales and abandons after, fixing, PBS is added and cleans twice, each 3min, absorbs liquid
Body air-dries.Hoechst33258 dyeing liquors are added and cover all cells.Cellular morphology is observed using fluorescence inverted microscope.
As a result see that good adherence quality, total more, boundary clear, cell is presented in Fig. 5, the HepG2 cells of two blank groups
Be evenly distributed, karyomorphism is normal, rounded or irregular ellipse, caryoplasm are evenly distributed, uniform fluorescence is presented in cell.And
There is different degrees of apoptosis after purple stem boneset extract is handled, with the increase of dosage in HepG2 cells, apoptotic cell feature by
Step is apparent.Low dose group cellular morphology disunity, attached cell quantity are reduced, and start apoptotic body occur；Middle dose group is adherent
Cell quantity is further reduced, and a small amount of apoptotic body, the graininess fluorescence of the visible densification of nucleus occurs；High dose group is adherent
Cell quantity significantly reduces, karyopyknosis, has more apoptotic body to occur.
The influence of experimental example three, Eupatorium adenophorum petroleum ether extract to the HepG2 cell cycles
Cell culture and medication are shown in experiment two.Attached cell is received together with floating cells after cell administration culture for 24 hours
Collection, is washed 2 times using PBS, is subsequently added into the 75% ethyl alcohol fixation pre-cooled and is prepared into cell suspending liquid, is put in -20 DEG C overnight
It preserves.Fixed cell suspension centrifugation, is washed, the RNase A that 1mg/mL is added at 37 DEG C are incubated 1h altogether, then using PBS
The PI dye liquors of final concentration of 50 μ g/mL are added, filter, using the DNA of flow cytomery cell, select argon laser for
Excitation light source, excitation wavelength are set as 488nm.With cis-platinum (CDDP) for positive control medicine, operate with Eupatorium adenophorum oil
Ether extract.
As a result see Fig. 6, under Eupatorium adenophorum petroleum ether extract administration group (10,30,50 μ g/mL) each dosage, the HepG2 periods
Significant change has occurred, increases with dosage and is constantly reduced in the G0/G1 phases, the G2/M phases are continuously increased, and occur before the G1 phases
The apoptotic peak (Sub G1) that degree continues to increase, shows that purple stem boneset extract can block the liver cancer cells G2/M phases, and energy
Induce both hepatoma cell apoptosis.
The influence of experimental example four, Eupatorium adenophorum petroleum ether extract to HepG2 cell DNAs
In apoptosis process, specificity cascade biochemical reaction can occur, wherein most characteristic is in endogenous nucleic acid
The activation of enzyme cutting, this enzyme may act on region between the nucleosome for connecting DNA, and DNA chain is cut into 180~200bp or its multiple
Several segments extracts DNA, through the visible scalariform electrophoresis pattern of agarose electrophoresis.
The HepG2 for testing logarithmic growth phase is inoculated with the concentration in 5 × 104/hole into 12 orifice plates, and culture for 24 hours, makes thin
Born of the same parents are adherent, and the sample liquid of various concentration is added in 12 orifice plates, is further cultured for 48h, collects cell afterwards, according to DNA extraction kit
Operating instruction extracts DNA in sample.Above-mentioned 20 μ L of the DNA sample of extraction liquid are taken respectively, and the Loading of 4 μ L is added
Buffer, the Marker of 5 μ L are analyzed into row agarose gel electrophoresis, and voltage is set as 50V, electrophoresis 1h, and gel imaging system is taken the photograph
As analysis result, launch wavelength 530nm.With cis-platinum (CDDP) for positive control medicine, operate with Eupatorium adenophorum petroleum ether
Extract.
As a result see that DNA ladder shape band (DNA Ladder) occur in Fig. 7, administration group, and with purple stem boneset extract
The increase of concentration, band is more and more clear, and the above results clearly show that purple stem boneset extract can induce the generation of HepG2 cells
Apoptosis makes the fracture of the DNA double chain Development pattern of its cell.
The influence of experimental example five, Eupatorium adenophorum petroleum ether extract to protein expression in HepG2 apoptotic cells
When Apoptosis, with a series of change of albumen zymetologys.Bcl-2 families are joined with the variation of Caspase family actives
Startup with mitochondrial apoptosis access and implementation procedure.Bcl-2, Bax, Caspase-3, Caspase-9 are that crucial participation divides
Son, being detected to it can react whether drug induces liver cancer cells to pass through mitochondria pathway apoptosis.
Cell culture and medication are shown in experiment two.Certain density sample is added in the attached cell of logarithmic growth phase
After acting on 48h, cell liquid is collected, cell pyrolysis liquid cracking, Ultrasonic Pulverization, boiling water bath 10min, using BCA is added in PBS washings
Determination of protein concentration kit measurement albumen concentration.Concentration glue and separation gel are prepared, using 12%SDS-PAGE gel electrophoresises point
It is closed overnight with 5% skimmed milk power solution according to western blotting method by protein delivery to pvdf membrane in glue from total protein
Afterwards, be separately added into primary antibody anti-Bax, anti-Bcl-2, anti-cleaved-Caspase-3, anti-Caspase-9,
Anti- β-actin are incubated at room temperature 3h, and TBST is washed three times, and the secondary antibody of horseradish peroxidase-labeled is added, and is incubated at room temperature 2h,
TBST is washed 3 times, after according to the method for ECL sequentially add luminous substrate, exposure, developing and fixing, analyzed using Image softwares
Band.
As a result see Fig. 8 (A), significant change occurs for the expression of some intracellular HepG2 important albumen, with purple stem
The increase of Herba Lycopi extract administration concentration, Bax, Caspase-3 expression rise, and the expression of Bcl-2 declines, Bcl-2/
The ratio of Bax is substantially reduced.This result shows that, purple stem boneset extract be by influence mitochondrial apoptosis access related egg
White expression, plays the effect of anti-liver cancer and anti-.
The influence of experimental example six, Eupatorium adenophorum petroleum ether extract to HepG2 cell JAK2/STAT3 signal paths
JAK/STAT signal transduction pathways, proliferation, differentiation, apoptosis, immunological regulation, inflammation, the tumour of wide participation cell
Etc. a variety of physiology, pathologic process.Composing type STAT3 is formed by the STAT3 dimerizations of phosphorylation, is the activated form of STAT3,
Abnormal activation is mainly caused by the abnormal activation of its upstream element and expression.A variety of tyrosine kinase can be direct or indirect swash
STAT3 living, JAK2 is the main upstream element of STAT3 in JAK2/STAT3 signal transduction pathways, and the phosphorylation of JAK2 can swash
Whether STAT3 activity living, therefore, detection JAK2 can react drug by inhibiting JAK2/ with the phosphorylation degree of STAT3
STAT3 signal transduction pathway liver cancer apoptosis reducings.
Cell manipulation is shown in that experiment five, wherein primary antibody use anti-JAK2, anti-pTyr1007/ with western blotting method
1008-JAK2, anti-STAT3, anti-pTyr705-STAT3 and anti-β-actin.
As a result see Fig. 8 (B), the intracellular JAK2 and STAT3 phosphorylation levels of HepG2 are all as purple stem boneset extract is administered
The increase of concentration and reduce, illustrate purple stem boneset extract liver cancer apoptosis reducing pass through influence JAK2/STAT3 signal transductions
Access plays a role, and purple stem boneset extract may be by the STAT3 inhibiting effect activated to inhibit its upstream JAK2
Activation and caused by.
To sum up, purple stem boneset extract shows good effect in vitro in Anticancer Activities.Eupatorium adenophorum oil
Ether extract can effectively induce hepatoma cell strain that apoptosis occurs, and arresting cell cycle is in the G2/M phases, and to normal liver cell
It influences smaller.It finds that purple stem boneset extract can increase the expression of Bax in the Mechanism Study for promoting apoptosis to it, reduces
The level of Bcl-2 separately raises the level of Caspase-9, Caspase-3 to reduce the ratio of Bcl-2/Bax, and causes to lure
Guided cell apoptosis.
In the experimental result of apoptotic proteins, the reduction of Bcl-2 is found, and Bcl-2 is in JAK2/STAT3 signal paths
Typical response protein, therefore, inventor further explore purple stem boneset extract using JAK2/STAT3 signal paths as target spot
Promote the mechanism of apoptosis.As a result, it has been found that purple stem boneset extract can be in dose-dependently to reduce JAK2 and STAT3 phosphorylation water
It is flat, inhibit STAT3 activity may be by inhibiting upstream JAK2 activation and caused by.It can be seen that purple stem boneset extract
The great potentiality for being developed into anticancer drug.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (8)
1. a kind of purposes of pharmaceutical composition in preparing the drug of prevention or treating cancer, which is characterized in that the medicine group
The active constituent for closing object is Eupatorium adenophorum lactone, 2- acetyl group -3,4, and 6,11- tetrahydrochysene cadinane -7- ketone and carat tie up alcohol, described
Pharmaceutical composition also contains pharmaceutically acceptable carrier or auxiliary material.
2. purposes according to claim 1, which is characterized in that described pharmaceutical composition be by cancer cell specific induction of apoptosis come
Prevent or treat the cancer.
3. purposes according to claim 2, which is characterized in that described pharmaceutical composition is by influencing JAK-STAT signals
Access induces the cancer cell-apoptosis.
4. purposes according to claim 3, which is characterized in that described pharmaceutical composition be by lower JAK2 albumen with
The phosphorylation level of STAT3 albumen influences the JAK-STAT signal paths.
5. purposes according to claim 2, which is characterized in that described pharmaceutical composition is by influencing mitochondrial apoptosis letter
Number Signal Transduction Pathways induce the cancer cell-apoptosis.
6. purposes according to claim 5, which is characterized in that described pharmaceutical composition is by lowering Bcl-2 albumen
Expression and the expression for raising Bax albumen influence the mitochondrial apoptosis signal transduction pathway.
7. purposes according to claim 5, which is characterized in that described pharmaceutical composition be by raise Caspase-3,
The expression of Caspase-9 influences the mitochondrial apoptosis signal transduction pathway.
8. purposes according to claim 1, which is characterized in that described pharmaceutical composition be by block Cancer Cell cycle come
Prevent or treat the cancer.
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