CN107334764B - 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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- CN107334764B CN107334764B CN201710589075.1A CN201710589075A CN107334764B CN 107334764 B CN107334764 B CN 107334764B CN 201710589075 A CN201710589075 A CN 201710589075A CN 107334764 B CN107334764 B CN 107334764B
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- 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
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
- A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
-
- 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)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- Health & Medical Sciences (AREA)
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- Public Health (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The present invention provides a kind of medical composition and its uses for the treatment of cancer, belong to drug field.The pharmaceutical composition includes maackiain and pharmaceutically acceptable carrier or auxiliary material.Active constituent maackiain in this pharmaceutical composition has excellent active anticancer, it plays effect by cancer cell specific induction of apoptosis and retardance Cancer Cell cycle, available for preparing clinical anti-cancer drug, prevention or treatment for cancer provide new treatment means and thinking.
Description
Technical field
The present invention relates to drug field, in particular to a kind of medical composition and its use for the treatment of cancer.
Background technology
Shagspine peashrub bark stem and leaf also known as pappus caragana, medicinal part are legume shagspine peashrub bark stem and leaf (Caragana
Jubata (Pall.) Poir.) root and branches and leaves.The shagspine peashrub bark stem and leaf place of production is distributed mainly on Tibet, Qinghai, the Inner Mongol, Sichuan etc.
Ground.Shagspine peashrub bark stem and leaf acrid flavour, hardship, puckery, cold nature, Return liver, spleen, kidney channel, have clearing heat and detoxicating, dispelling wind and eliminating dampness, it is promoting blood circulation and removing obstruction in channels, disappear
The effect of swollen analgesic.It is conventionally used to the diseases such as treatment acute mastitis, boil swelling and pain, injury from falling down, rheumatism arthralgia and myalgia.According to traditional theory
With clinical application experience, shagspine peashrub bark stem and leaf also can be used for the modern diseases such as treatment hypertension and virus infection.The present invention is to terrible arrow
Caragana carries out in-depth study.
Invention content
The first object of the present invention is to provide a kind of pharmaceutical composition for the treatment of cancer, which includes height
Beautiful Chinese scholartree element, can be used in preventing and treating cancer.
The second object of the present invention is to provide a kind of pharmaceutical composition in the drug of prevention or treating cancer is prepared
Purposes, prevention or treatment for cancer provide new treatment means and thinking, while also develop maackiain and terrible arrow golden pheasant
The new medical value of youngster.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of pharmaceutical composition for the treatment of cancer, pharmaceutical composition include maackiain and pharmaceutically acceptable load
Body or auxiliary material.
A kind of purposes of pharmaceutical composition in the drug of prevention or treating cancer is prepared, pharmaceutical composition include Koryo Chinese scholartree
Element and pharmaceutically acceptable carrier or auxiliary material.
Compared with prior art, beneficial effects of the present invention for example including：
The present invention by the study found that maackiain has excellent active anticancer, by cancer cell specific induction of apoptosis and
Block Cancer Cell cycle to play effect, available for preparing clinical anti-cancer drug, prevention or treatment for cancer provide new control
Treatment means and thinking, while also develop maackiain and the new medical value of shagspine peashrub bark stem and leaf.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the extraction flow chart of shagspine peashrub bark stem and leaf extract in embodiment 1；
Fig. 2 is the separation process figure of maackiain in embodiment 2；
Fig. 3 is influence of the shagspine peashrub bark stem and leaf extract to HepG2, Hep3B, HEK-293 cell viability in experimental example one；
Fig. 4 is influence of the maackiain to HepG2, Hep3B, HEK-293 cell viability in experimental example one；
Fig. 5 is the influence of shagspine peashrub bark stem and leaf extract and maackiain to HepG2 cellular morphologies in experimental example two；
Fig. 6 be in experimental example three shagspine peashrub bark stem and leaf extract and maackiain to the shadow of HepG2 and Hep3B cell cycles
It rings；
Fig. 7 is influence of the shagspine peashrub bark stem and leaf extract to HepG2 cell DNAs in experimental example four；
Fig. 8 is shagspine peashrub bark stem and leaf extract in experimental example five and maackiain to protein expression in HepG2 apoptotic cells
Influence；
Fig. 9 is shagspine peashrub bark stem and leaf extract in experimental example six to the tumor inhibition effect of H22 tumor-bearing mices；
Figure 10 is influence of the shagspine peashrub bark stem and leaf extract to the immune organ and index of immunity of mouse in experimental example six；
Figure 11 is influence of the shagspine peashrub bark stem and leaf extract to the apoptosis of tumor cells of H22 tumor-bearing mices in experimental example six,
Middle figure A is blank group, and figure B is cis-platinum group, and figure C is MA dosage 25mg/kg groups, and figure D is MA dosage 50mg/kg groups,
Figure E is MA dosage 100mg/kg groups；
Figure 12 is shagspine peashrub bark stem and leaf extract in experimental example six to egg related to apoptosis in the tumour cell of H22 tumor-bearing mices
White influence；
In more than figure, normal control represent normal group, and CDDP represents cis-platinum positive drug group, and CJ-EtOH is represented
Shagspine peashrub bark stem and leaf extract, MA represent maackiain.
Specific embodiment
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, the condition suggested according to normal condition or manufacturer carry out.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 for the treatment of cancer, the pharmaceutical composition include maackiain and
Pharmaceutically acceptable carrier or auxiliary material.
Wherein, the structural formula of maackiain is：
Maackiain can be obtained commercially, can also be used from medicinal material shagspine peashrub bark stem and leaf or in his medicinal material
Extraction.
Further, which includes shagspine peashrub bark stem and leaf extract, contains Koryo in shagspine peashrub bark stem and leaf extract
Chinese scholartree element.Research has shown that shagspine peashrub bark stem and leaf extract also has the activity for inhibiting cancer cell, especially inhibits the work of liver cancer cells
Property, available for preventing or treating various cancers, especially liver cancer.
Further, the method for isolated maackiain includes from medicinal material shagspine peashrub bark stem and leaf：
Step S1:Using alcohol-water mixed solution system or alcoholic solution as extractant, medicinal material ghost arrow is extracted using solvent extraction method
Caragana obtains shagspine peashrub bark stem and leaf extract.
Wherein, solvent extraction method includes appointing in infusion process, percolation, heating reflux method, microwave oscillation method or decocting method
Meaning is a kind of.Optionally, solvent extraction method is infusion process.
Wherein, extractant is alcohol-water mixed solvent system, and optionally, extractant is the second that volume fraction is 60~95%
Alcoholic solution；Or it is 75~95% ethanol solution.
Optionally, 95% ethanol solution is used with solid-liquid ratio 1：8~10 impregnation shagspine peashrub bark stem and leafs 2~4 days.It is optional
, it impregnates 1~3 time, every time 2~4 days.
Step S2:After extracting the shagspine peashrub bark stem and leaf extract with chloroform, chloroform extract is obtained；Using silica gel chromatographic column
The chloroform extract is detached, gradient elution is carried out with chlorofonn-ethylacetate mixed solvent system.
More specifically, extraction step is：By shagspine peashrub bark stem and leaf extract according to volume ratio 1：After 10 add water to be suspended, with 2
The chloroform extraction of~3 times of volumes, after organic phase is concentrated under reduced pressure, obtains chloroform extract.
With silica gel chromatograph post separation chloroform extract, the wherein mesh number of silica gel is 100~200 mesh.Eluant, eluent is chloroform-first
Alcohol mixed solvent, with chloroform：The volume ratio of methanol is 98：2 start to elute, and gradually increase the ratio of methanol to chloroform：Methanol
Volume ratio is 6：4.
The fraction containing maackiain is obtained after silica gel post separation, then the fraction is divided using exclusion chromatography
From maackiain finally is prepared using preparative high performance liquid chromatography.
Present embodiment also provides a kind of purposes of pharmaceutical composition in the drug of prevention or treating cancer is prepared, the medicine
Compositions include maackiain and pharmaceutically acceptable carrier or auxiliary material.
Inventor research shows that, maackiain and shagspine peashrub bark stem and leaf extract are respectively provided with excellent active anticancer, into one
Step is the study found that it plays active anticancer by promoting cancer cell-apoptosis and retardance Cancer Cell cycle, available for clinical anti-
Cancer medication.
Further, inventor confirms through test cell line, and maackiain and shagspine peashrub bark stem and leaf extract are to pass through influence
Mitochondrial apoptosis signal transduction pathway carrys out cancer cell specific induction of apoptosis.
The expression and regulation and control of bcl-2 families are one of key factors for influencing mitochondrial apoptosis signal transduction, are led
Action site is wanted on mitochondrial membrane.After cell is stimulated by dead signal, the pro apoptotic protein in bcl-2 families is in protease
Effect is lower to occur conformation variation, is displaced on organelle film from cytoplasm, especially on mitochondrial outer membrane, and on organelle film
Anti-apoptotic proteins interaction in film, makes anti-apoptotic proteins lose the inhibiting effect to apoptosis, organelle function is caused to be lost
Become estranged it is various promote antiapoptotic factors releases, eventually lead 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, breast cancer, lung cancer, gastric 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 maackiain and shagspine peashrub bark stem and leaf extract can raise the expression water of Bax albumen
It is flat, the expression of Bcl-2 albumen is slightly lowered, so as 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 of mitochondrial mediation, the amplification of Caspase families apoptosis involvement signal and apoptosis effect pass through enzymatic grade
The mode of connection reaction amplifies apoptotic signal, and be 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, Apaf1N ends is made to form 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.
Inventor is the study found that maackiain and shagspine peashrub bark stem and leaf extract can raise the expression water of Caspase-3
It is flat, so as to amplify apoptotic signal by way of enzymatic cascade reaction, eventually lead to the apoptosis of cancer cell.
Further, research finds that the pharmaceutical composition is 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 inactivates when having gene alteration more, causes cell cycle monitoring point functional defect, final result is that cell obtains uncontrolled proliferation
Ability leads oncogenic generation.
Inventor the study found that maackiain and shagspine peashrub bark stem and leaf extract can block the cell cycle of cancer cell,
It is made to be stuck in the G2/M phases, and can induce this quasi-cancer cell that apoptosis occurs.
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, for the treatment of liver cancer.
Further, it is of the invention in order to make the drug rapidly, continuously and the discharge active component 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.Preparation comprising the composition can be tablet, soft capsule, ebonite
Capsule, oral liquid, 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 composition, including：Shagspine peashrub bark stem and leaf extract and pharmaceutically acceptable
Carrier or auxiliary material.
The preparation method of wherein shagspine peashrub bark stem and leaf extract includes：
As shown in Figure 1, the dried powder 10kg of shagspine peashrub bark stem and leaf root is taken, by solid-liquid ratio 1:8 (1kg medicinal materials correspond to the molten of 8L
Agent) with the alcohol dipping 3 days that volumetric concentration is 95%, filter to take filtrate.Filter residue is extracted twice using same operation, filtering
Merge the filtrate extracted three times, be concentrated under reduced pressure to give shagspine peashrub bark stem and leaf extract 763g.
Embodiment 2
The present embodiment provides a kind of pharmaceutical composition, including：Maackiain and pharmaceutically acceptable carrier are auxiliary
Material.
The preparation method of wherein maackiain includes：
A. the method with reference to embodiment 1 prepares shagspine peashrub bark stem and leaf extract.
B. as shown in Fig. 2, taking the shagspine peashrub bark stem and leaf extract of 300g by volume 1:10 add water to be suspended, with 3 times of volumes three
Chloromethanes extracts sample, and extract liquor is concentrated under reduced pressure, and recycling design is evaporated, and obtains 63g chloroform extraction objects.
C. the chloroform extraction object of 60g is taken, through silica gel chromatograph post separation, with chloroform-ethyl acetate mixed solvent
System carries out gradient elution, obtains the fraction containing maackiain.The fraction is through Sephadex LH-20 pillar layer separations, to contain
The methanol solution elution of 0.1% formic acid, TLC detection trackings merge, and obtain 4 parts of A, B, C, D.Take part B (1.6g) system
Standby type efficient liquid phase is detached, and gradient elution, isolated maackiain 330mg are carried out by mobility of first alcohol and water.
Experimental example
With reference to the test of pesticide effectiveness to the shagspine peashrub bark stem and leaf extract (CJ-EtOH) that is provided in the embodiment of the present invention 1~2 and
Maackiain (MA) carries out evaluating drug effect.
Experimental example one, shagspine peashrub bark stem and leaf extract and maackiain are to HepG2, Hep3B, HEK-293 cell viability
It influences
It takes the logarithm tri- kinds of cell strains of growth period HepG2, Hep3B, HEK-293, it is 5 × 10 to adjust cell concentration4A/mL,
96 well culture plates are seeded to per hole 0.2mL, after continuing culture for 24 hours, are added respectively per hole by containing 10% calf serum RPMI 1640
The sample to be tested 0.1mL that liquid is configured to, sample concentration is made to be respectively 5,10,20,40,100,200mg/mL continues to cultivate；Each
One formula of concentration, 3 hole, at the same set the experiment contrast for being not added with test sample and be not added with sample and cell blank control culture 24,48,
After 72h, liquid is changed respectively 1 time, after cultivating 72h, the MTT 20mL for adding in 5mg/mL continue after cultivating 4h, and culture solution is abandoned in suction, per hole
0.15mLDMSO is added in, shakes 30min, the purple formazan crystallization generated into the cell is fully dissolved, is measured under 550nm per hole A
Value.Calculate growth inhibition ratio (%) and half-inhibition concentration (IC50).Wherein, the calculation formula of growth inhibition ratio is：
Growth inhibition ratio=[1- (experimental group A values-blank group A values)/(control group A value-blank group A values)] × 100%.
As a result see Fig. 3, Fig. 4, shagspine peashrub bark stem and leaf extract and maackiain are right in the concentration range of 5-200 μ g/mL
HepG2, Hep3B liver cancer cells show that certain vigor inhibits, and also show that apparent dose-effect relationship.Shagspine peashrub bark stem and leaf extracts
Object is respectively 42.7 and 48.2 μ g/mL (Fig. 3) to the IC50 of both tumour cells；Maackiain is to two kinds of tumour cells
IC50 is respectively 23.6 and 35.5 μ g/mL (Fig. 4).Shagspine peashrub bark stem and leaf extract and maackiain under the concentration of 200 μ g/mL
Inhibiting effect is hardly shown to HEK-293 cells.
The influence of experimental example two, shagspine peashrub bark stem and leaf extract and maackiain to HepG2 cellular morphologies
It takes the logarithm the HepG2 cells in growth period, 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
Nutrient solution adds in the shagspine peashrub bark stem and leaf extract (sample concentration is respectively 30,60,120 μ g/mL) of various concentration, adds in blank solution
Group (physiological saline) continues to cultivate 48h, culture solution is abandoned in suction, and fixer (methanol is added in per hole as negative control group：Glacial acetic acid
=3:1) it inhales and abandons after, fixing, add in PBS and clean twice, 3 minutes every time, absorb liquid, air-dry.Hoechst 33258 is added in contaminate
Color liquid covers all cells.Cellular morphology is observed using fluorescence inverted microscope.
As a result see Fig. 5, good adherence quality is presented in the HepG2 cells of two blank groups, and sum is more, boundary clear, cell
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 shagspine peashrub bark stem and leaf extract or maackiain processing, with the increase of dosage in HepG2 cells, wither
It is gradually apparent to die cell characteristic.Low dose group (30 μ g/mL) cellular morphology disunity, attached cell quantity reduce, start to occur
Apoptotic body；Middle dose group (60 μ g/mL) attached cell quantity is further reduced, and a small amount of apoptotic body occurs, nucleus can
See fine and close graininess fluorescence；High dose group (120 μ g/mL) attached cell quantity significantly reduces, and karyopyknosis has more
Apoptotic body occurs.
The influence of experimental example three, shagspine peashrub bark stem and leaf extract and maackiain 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
Add in the PI dye liquors of final concentration of 50 μ g/mL, filter, using the DNA of flow cytomery cell, select argon laser for
Excitation light source, excitation wavelength are set as 488nm.
As a result see Fig. 6, under shagspine peashrub bark stem and leaf extract or maackiain administration group (60,120 μ g/mL) each dosage,
Significant change has occurred in the HepG2 periods, and apoptotic peak occurs before the G1 phases, shows shagspine peashrub bark stem and leaf extract and height
Beautiful Chinese scholartree element not only influences the cell cycle of HepG2 and Hep3B, and can induce both hepatoma cell apoptosis.
The influence of experimental example four, shagspine peashrub bark stem and leaf 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.
Take the logarithm the HepG2 in growth period, with 5 × 104The concentration in a/hole is inoculated with into 12 orifice plates, and culture for 24 hours, pastes cell
Wall adds in the sample liquid of various concentration in 12 orifice plates, is further cultured for 48h, collects cell afterwards, according to the operation of DNA extraction kit
Illustrate to extract DNA in sample.Above-mentioned 20 μ L of the DNA sample of extraction liquid are taken respectively, add in the sample-loading buffer of 4 μ L, the mark of 5 μ L
Remember object, analyzed into row agarose gel electrophoresis, voltage is set as 50V, electrophoresis 1h, gel imaging system camera shooting analysis result, hair
The a length of 530nm of ejected wave.
As a result see Fig. 7, administration group DNA ladder shape band (DNA Ladder) occurs, and as shagspine peashrub bark stem and leaf extracts
The increase of object concentration, band is more and more clear, and the above results clearly show that shagspine peashrub bark stem and leaf extract can induce HepG2 cells
Apoptosis occurs, makes the fracture of the DNA double chain Development pattern of its cell.
The influence of experimental example five, shagspine peashrub bark stem and leaf extract and maackiain to protein expression in HepG2 apoptotic cells
During Apoptosis, with a series of change of albumen zymetologys.Bcl-2 families are joined with the variation of Caspase family actives
Startup and implementation procedure with mitochondrial apoptosis access.Bcl-2, Bax, Caspase-3, Caspase-9 are crucial participation point
Son, it, which is detected, can react whether drug induces liver cancer cells to pass through mitochondria pathway apoptosis.
Cell culture and medication are shown in experimental example two.It takes the logarithm the attached cell in growth period, adds in certain density sample
After product effect 48h, cell liquid is collected, PBS washings add in cell pyrolysis liquid cracking, Ultrasonic Pulverization, and boiling water bath 10min is used
BCA determination of protein concentration kit measurement protein concentrations.Concentration glue and separation gel are prepared, using 12%SDS-PAGE gel electrophoresises
Total protein is detached, according to western blotting method by protein delivery to pvdf membrane in glue, was closed with 5% skimmed milk power solution
After night, it is separately added into the antibody at room temperature such as Bax, Bcl-2, Caspase-3, Caspase-9, β-actin and is incubated 3h, TBST washings three
It is secondary, add in the secondary antibody of horseradish peroxidase-labeled, be incubated at room temperature 2h, TBST is washed 3 times, after add successively according to the method for ECL
Enter luminous substrate, exposure, developing and fixing, band is analyzed using Image softwares.
As a result see Fig. 8, significant change occurs for the expression of some intracellular HepG2 important albumen, with terrible arrow brocade
The increase of chicken extract or maackiain administration concentration, Bax, Caspase-3 expression rise, the expression of Bcl-2
Decline, the ratio of Bcl-2/Bax is substantially reduced.This result shows that, shagspine peashrub bark stem and leaf extract and maackiain can lead to
Crossing influences the correlative protein expression of mitochondrial apoptosis access, plays the effect of anti-liver cancer and anti-.
Resisting liver cancer activity is evaluated in experimental example six, shagspine peashrub bark stem and leaf extract body
1. experimental method
Experimental animal uses Kunming mouse, half male and half female, and weight 18-22g is purchased from Disease Prevention Control Center, Hubei Prov
It buys, credit number：SCXK (E) 2008-0005, H22 Ascitic Tumor Cells are given by pharmaceutical college of South-Center University For Nationalities Yang Xin continent teacher.
Lotus H22 ascites tumor mouse of the well-grown without diabrosis is taken, dislocation is put to death, and the abdomen in milk yellow is drawn in super-clean bench
Chamber knurl liquid does well good cell in micro- Microscopic observation sub-argument, the thin of 1 × 106/mL is diluted to sterile saline
Born of the same parents' suspension.Again (1 is diluted with sterile NaCl parenteral solution:3) into cell suspension, in mouse, forelimb armpit inoculates 200 μ L again.
After for 24 hours, mouse is grouped at random, every group 10, blank control group (injecting normal saline), sample controls group (note are set respectively
Penetrate cis-platinum (CDDP), 5mg/kg), the high, medium and low dosage group of shagspine peashrub bark stem and leaf extract (100,50,25mg/kg).By 20g weight
The amount intraperitoneal injection of maximum administered volume 0.4mL, one time a day, successive administration 10d.
After 10 days, mouse is put to death, dissection strips oxter solid tumor, weighs and record, tumor control rate is according to the following formula
It calculates：Tumor control rate IR (%)=(blank group average knurl weight-administration group average knurl weight)/blank group knurl weight.
The mouse put to death to the 10th day, dissection, strips thymus gland, spleen and liver, each internal organs are weighed.Immune organ refers to
Number calculates as follows：Immune Organs Index=immune organ weight (mg)/mice weights (g).
To the mouse of execution, take and win the mouse blood that the method that eyeball takes blood collects administration group, through EDTA anti-freezings, inspection
Survey leukocyte count (WBC), red blood cell number (RBC), hemoglobin (HGB), platelet count (PLT)；Blood through anticoagulant heparin again from
The heart takes supernatant to measure hepatic and renal function index, including transaminase (AST, ALT), urea nitrogen (BUN), uric acid (UA) and creatinine
(CRE)。
To the mouse of execution, the tumor mass of the tumor-bearing mice of administration group is stripped, every group of tumor mass clip phase homogenous quantities are placed in homogenate
It is placed in device and is homogenized on ice, add in 100mM NaCl, 50mM HERPS, 0.1%CHAPS, 1mM DTT and 0.1mM EDTA.Institute
Suspension high speed centrifugation 10min at 4 DEG C must be homogenized.According to described in experimental example six Western Blotting methods detection with
The albumen of apoptosis.
2. experimental result
As a result it is represented using mean+SD (Mean ± SEM), all statistical analyses use GraphPad
Prism 5.0 is analyzed,*P<0.05 shows with significant difference,**P<0.01 shows with pole significant difference.
2.1 shagspine peashrub bark stem and leaf extracts are to the tumor inhibition effect of H22 tumor-bearing mices
As a result see Fig. 9, each dosage group of shagspine peashrub bark stem and leaf extract is respectively provided with apparent suppression to H22 tumor-bearing mice tumour growths
Making use (*P<0.05), and there is certain dose-effect relationship, drug concentration shows apparent correlation with tumor control rate, and
Shagspine peashrub bark stem and leaf extract high dose group shows best activity.
Influence of the 2.2 shagspine peashrub bark stem and leaf extracts to the immune organ and index of immunity of mouse
The result is shown in Figure 10, shagspine peashrub bark stem and leaf extract is to the immune organs of H22 tumor-bearing mices, such as thymus gland and the internal organs of spleen
Exponential effect is little, and larger difference (P is relatively had no with blank group>0.05)；And the organ index of cis-platinum group compares blank group then
Be remarkably decreased (**P<0.01).The result shows shagspine peashrub bark stem and leaf extract is little to the damage of mouse immune ability.
Influence of the 2.3 shagspine peashrub bark stem and leaf extracts to the physiochemical indice of H22 tumor-bearing mices
It the results are shown in Table 1, positive drug cis-platinum group is substantially reduced H22 tumor-bearing mice blood middle leukocytes numbers, and red blood cell number, blood
Lactoferrin, platelet count, which have, slightly to be improved；And relative to cis-platinum group, each dosage group of shagspine peashrub bark stem and leaf extract is significantly increased
Leukocyte count in mouse blood, there is no cis-platinum group occur function of immune system it is low the problem of.
Influence of the 1 shagspine peashrub bark stem and leaf extract of table to the physiochemical indice of H22 tumor-bearing mices
Influence of the 2.4 shagspine peashrub bark stem and leaf extracts to the hepatic and renal function of H22 tumor-bearing mices
Urea nitrogen (BUN), uric acid (UA), urine creatinine (CRE) are to detect the important indicator of renal function, glutamic-oxalacetic transaminease
(AST), glutamic-pyruvic transaminase (ALT) is the important indicator for detecting liver function.The 5 of the liver function of evaluation more than inventors tested a larging, renal function
A important clinical indices, the results are shown in Table 2, the kidney function indicator of the H22 tumor-bearing mices through shagspine peashrub bark stem and leaf extract for treating
Compared with certain improvement that has of blank group, and cis-platinum has the hepatic and renal function of mouse apparent reduction, this is the result shows that terrible arrow
Caragana extract is different from cis-platinum has larger toxicity to the liver kidney organ of mouse, has one to the hepatic and renal function of mouse instead
Fixed improvement result.
The influence of the hepatic and renal function of 2 MH22 tumor-bearing mices of table
Influence of the 2.5 shagspine peashrub bark stem and leaf extracts to the apoptosis of tumor cells of H22 tumor-bearing mices
Experimental implementation as described above, we take out tumor mass, carry out HOECHST dyeing, the result is shown in Figure 11, with cis-platinum
Group is similar, the tumor tissue sections of 2 shagspine peashrub bark stem and leaf extract for treating groups (middle and high dosage group) through fluorescence microscope,
Occurs the typical cells apoptosis phenomenon of karyopycnosis and nuclear fragmentation in various degree.
2.6 shagspine peashrub bark stem and leaf extracts are to the influence with apoptosis-related protein in the tumour cell of H22 tumor-bearing mices
The result is shown in Figure 12, it is similar to the influence of the relevant albumen of apoptosis into the cell to HepG2 to shagspine peashrub bark stem and leaf extract,
In shagspine peashrub bark stem and leaf extract administration group, significant change occurs for the expression of some intracellular important albumen in H22 tumor mass,
With the increase of administration concentration, Bax, Caspase-3 expression rise, and the expression of Bcl-2 declines, Bcl-2/Bax's
Ratio is substantially reduced.This result shows that, shagspine peashrub bark stem and leaf extract is in vivo again by influencing mitochondrial apoptosis access
Correlative protein expression plays the effect of anti-liver cancer and anti-.
To sum up, shagspine peashrub bark stem and leaf extract whether study in vitro or in vivo by resisting liver cancer activity, and display is good
Effect.In vitro in resisting liver cancer activity activity research, shagspine peashrub bark stem and leaf extract can effectively induce two kinds of hepatoma cell strains
Apoptosis occurs, blocks the cell cycle in the G2/M phases.Find that shagspine peashrub bark stem and leaf extract can increase in the Mechanism Study to its apoptosis
Add the expression of Bax albumen, slightly reduce the level of Bcl-2, it is another to raise so as to reduce the ratio of Bcl-2/Bax
The level of Caspase-3, and lead to inducing cell apoptosis.Internal anti-liver cancer and anti-zoopery shows, in shagspine peashrub bark stem and leaf extract,
High dose can significantly inhibit the growth of H22 tumor-bearing mice tumours, and immune organ, liver kidney, the hemopoietic system to animal are not shown
Show very strong toxic side effect, to its internal resisting liver cancer activity the study found that shagspine peashrub bark stem and leaf extract can also increase Bax albumen
Expression, slightly reduce the level of Bcl-2, so as to reduce the ratio of Bcl-2/Bax, the another level for raising Caspase-3,
Active anticancer is played by inducing H22 Apoptosis.The result of inside and outside anti-liver cancer and anti-experiment can be mutually authenticated, it was demonstrated that terrible arrow
Caragana extract is a potential medicines resistant to liver cancer for being worth exploitation.
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 (5)
- A kind of 1. purposes of pharmaceutical composition in the drug for preparing liver cancer apoptosis reducing, which is characterized in that the drug Composition includes maackiain and pharmaceutically acceptable carrier or auxiliary material.
- 2. purposes according to claim 1, which is characterized in that described pharmaceutical composition is believed by influencing mitochondrial apoptosis Number Signal Transduction Pathways induce the cancer cell-apoptosis.
- 3. purposes according to claim 2, which is characterized in that described pharmaceutical composition is the table by raising Bax albumen The mitochondrial apoptosis signal transduction pathway is influenced up to level.
- 4. purposes according to claim 2, which is characterized in that described pharmaceutical composition is to activate Caspase- by up-regulation 3 expression influences the mitochondrial apoptosis signal transduction pathway.
- 5. 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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