CN108578464A - A kind of Millettia extract application in preparation of anti-tumor drugs - Google Patents
A kind of Millettia extract application in preparation of anti-tumor drugs Download PDFInfo
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- CN108578464A CN108578464A CN201810538736.2A CN201810538736A CN108578464A CN 108578464 A CN108578464 A CN 108578464A CN 201810538736 A CN201810538736 A CN 201810538736A CN 108578464 A CN108578464 A CN 108578464A
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- extract
- caulis spatholobi
- water
- ethyl acetate
- blood
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Classifications
-
- 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
- A61K36/486—Millettia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention relates to a kind of Millettia extract application in preparations of anti-tumor drugs, wherein the Millettia extract is selected from：Caulis Spatholobi water extract, Caulis Spatholobi ethyl acetate extract, Caulis Spatholobi n-butanol extract.The wherein described Caulis Spatholobi water lift extract, preparation method are as follows：Caulis Spatholobi 70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, and 75 DEG C of steam-to-vacuums dry 7h.Caulis Spatholobi water extract 6kg is obtained, water extract is obtained.
Description
Technical field：
The present invention relates to a kind of application that Chinese medicine is new, more particularly to a kind of Millettia extract is preparing antitumor drug
In application.
Background technology
Caulis Spatholobi is the drying rattan of legume spatholobus suberectus Spatholobus suberectus Dunn.With promoting blood circulation
It enriches blood, the effect of menstruction regulating and pain relieving, relaxing tendons and activating collaterals.For irregular menstruation, dysmenorrhoea, Amenorrhea, arthralgia pain due to rheumatism, paralysis and numbness, the deficiency of blood withers
It is yellow.
Blood platelet is one of the visible component in mammalian, is cracked from the megacaryocyte cytoplasm of marrow maturation
The biologically active fritter cytoplasm split away off, small, acellular core.Blood platelet is in anastalsis and thrombosis
Pathophysiological process in play the role of it is vital.Blood platelet is the haemocyte of minimum, but it is approximately white that its quantity, which is,
100 times of number of cells.About 100~300*10^9 blood platelet participates in being damaged blood in the blood circulation of one adult
The reparation of pipe.Blood platelet is mutually assembled, and the fibrin ferment in grumeleuse forming process can be promoted to generate.
The surface receptor of blood platelet, such as GpIb-IX-V compounds are propped up by bundling vWF ELISA (VWF)
The adhesive attraction for holding blood platelet, so as to be combined with Fibronectin (GP).Especially under the high shear force of blood, blood platelet
Distinctive 3 integrin receptors of α IIb β pass through binding fiber proteinogen, GpIa-IIa collagen receptors, platelet collagen receptor
(GpVI) etc. collagen receptors, carry out induced platelet aggregation.Blood platelet by after abnormal activation, tied by the cytoskeleton of blood platelet
Structure recombinates, and causes the release of α particles, dense body and lysosome.The blood platelet of activation causes GpIIb-IIIa receptor conformations to occur
Variation, causes to be combined with high-affinity part and platelet aggregation [2].The electronegative Phospholipids of platelet surface is exposed,
The generation for leading to blood platelet particle (PMPs), to generate procoagulant activity.
Other than blood coagulation hemostatic function, more and more experimental evidences show that blood platelet can be more prior
Aspect carrys out the relationship of homeostasis disease and health, on the one hand, and blood platelet can promote wound healing, regeneration, on the other hand,
It also closely participates in the pathogenesis process of certain major diseases, such as cancer.
Experiment discloses the " blood of the anti-AA inductions of Caulis Spatholobi heterogeneity with laboratory medicine in August, 2016 the 4th phase of volume 34
The experimental study of platelet aggtegation " but without the relationship for illustrating platelet aggregation and tumour.
Blood platelet and the close phase shield effect of tumour cause the concern and attention of people to be faced before more than 100 years already
The phenomenon that bed doctor just observes malignant tumor patient there are thrombocythemias.1872, Riess reported pernicious swollen for the first time
There are thrombocythemia, platelet abnormal incretions can cause piastrenemia for tumor, it be usually seen as it is a kind of it is passive,
The disease of paraneoplastic.19th century, people have found that tumour and thrombus directly have close relationship again.Research thinks, thrombus
Property phlebitis be malignant tumour occur a pre-warning signal.The patient of venous thromboembolism suffers from the Hazard ratio of malignant tumour
Other patients are seven times more.Blood platelet plays considerable effect wherein.
Under the prompt of above-mentioned pathological phenomenon, modern oncology studies are gradually, it is realized that progress and blood platelet occur for tumour
Pathologic unusual fluctuation between be not parallel generation the event that occurs together, between the two with height causalnexus.
There is tumour cell induced platelet to occur, numerically with the pathologic potential of changes of function, then to promote thrombus
It is formed.This variation has good suggesting effect for clinical diagnosis malignant tumour and thrombus.Multinomial research report after this
Show in a variety of entity tumors such as lung cancer, the cancer of the esophagus, gastric cancer, breast cancer, liver cancer, colorectal cancer, cancer of pancreas with Clinic Case
In there is thrombocythemia phenomenon, and the tumour patient prognosis with piastrenemia is poor.
It is above-mentioned it turns out that, thrombocythemia can lead to the generation of the hypercoagulative state of blood and thrombus in tumor patient body,
Prompting blood platelet and tumor cell proliferation, hematogenous metastasis and inside tumor angiogenesis has close association, and in tumour
It plays an important role in terms of progression of disease, transfer, wherein TCIPA is the most directly embodiment of blood platelet and tumour interaction
With most crucial regulation process.
Tumour cell can be activated by induced platelet, make platelet activation and be gathered near tumor cells to be formed
Blood platelet --- tumour cell compound, this process are known as TCIPA.This phenomenon was most found earlier than 1865, and gradually
The explaination and verification of system and science are obtained.On the one hand, tumour cell can be by actively enhancing ADP, Thromobin etc.
Promote the expression of blood coagulation molecule, induced platelet number increases and functional activation, forms and maintain the hypercoagulative state of blood samples of patients.
On the other hand, the blood platelet of abnormal activation, can be by effects such as physical bond to tumour cell and biological shieldings, reversely
Tumour cell is acted on, it is protected to be protected from the damage of physical shear power and the killing of immune system removing in the circulatory system,
Promote adherency and field planting of the tumour cell on target organ.
Using TCIPA as target spot, pharmaceutical intervention is carried out using aspirin, there is higher anti-transfer effect.However Ah Si
The successful application of woods does not represent its generally applicable or sustainable application.In the behind effectively applied, but also have
Using upper restricted:If Aspirin Resistance generally occurs in medication patient, highest incidence is up to 60%.
And with part medication patient, gastric ulcer can be accompanied by, gastrorrhagia, tinnitus, hemorrhagic stroke, used in teenager
After aspirin, there is the risk that Reye syndrome occurs, liver renal failure, cerebral injury can be led to quickly, or even dead.Many pairs
Effect generates the above several points and greatly limits application of this therapeutic strategy in clinic, therefore has also pushed other Fei Asi
The research and development and appearance of the drug of the anti-TCIPA of woods class.
Be in anti-TCIPA the core event of anti-tumor metastasis theoretical direction under, can effectively prevent in aspirin
In the case of metastases, in the case of aspirin but also has many insufficient, using traditional Chinese medicine relevant knowledge, from traditional Chinese medicine
Theory is set out, find it is new using anti-TCIPA be the drug of target spot become our matter of utmost importance.
Invention content：
The present invention studies Chinese medicine Caulis Spatholobi, has obtained following technical solution.
The present invention provides a kind of Millettia extract application in preparation of anti-tumor drugs, wherein the Caulis Spatholobi carries
Object is taken to be selected from：Caulis Spatholobi water extract, Caulis Spatholobi ethyl acetate extract, Caulis Spatholobi n-butanol extract.
Application of the present invention, wherein the Caulis Spatholobi water lift extract, preparation method are as follows：Caulis Spatholobi 70kg, often
Secondary 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, and 75 DEG C of steam-to-vacuums dry 7h.It obtains
Caulis Spatholobi water extract 6kg, obtains water extract.
Application of the present invention, wherein the Caulis Spatholobi ethyl acetate extract, preparation method are as follows：Caulis Spatholobi
70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, 75 DEG C of steam-to-vacuum dryings
7h.Caulis Spatholobi water extract 6kg is obtained, obtains water extract, water extract is re-dissolved in 7.2L distilled water, uses ethyl acetate
Extraction, each dosage 10.8L, coextraction 8 times；Combined ethyl acetate layer extract liquor, steams solvent, is dried to obtain ethyl acetate
Extract.
Application of the present invention, wherein the Caulis Spatholobi n-butanol extract, preparation method are as follows：Caulis Spatholobi 70kg,
Every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, and 75 DEG C of steam-to-vacuums dry 7h.It obtains
Caulis Spatholobi water extract 6kg is obtained, obtains water extract, water extract is re-dissolved in 7.2L distilled water, each with extracting n-butyl alcohol
Dosage 14.4L, coextraction 9 times merge n-butanol layer extract liquor, steam solvent, be dried to obtain n-butanol extract.
Application of the present invention, wherein the inhibition metastases are lured by the anti-extract tumour cell of Caulis Spatholobi
What the platelet aggregation led was realized.
Application of the present invention, wherein the inhibition metastases are to inhibit blood platelet to live by Millettia extract
What change aggregation and blood coagulation resisting function were realized.
Application of the present invention, wherein the tumour is selected from：Colon cancer, breast cancer, lung cancer, cancer of pancreas or liver cancer.
Preferably, the tumour is colon cancer.
The present invention further provides a kind of Caulis Spatholobi ethyl acetate extract, preparation method is as follows：Caulis Spatholobi 70kg,
Every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, and 75 DEG C of steam-to-vacuums dry 7h.It obtains
Caulis Spatholobi water extract 6kg is obtained, water extract is obtained, water extract is re-dissolved in 7.2L distilled water, is extracted with ethyl acetate,
Each dosage 10.8L, coextraction 8 times；Combined ethyl acetate layer extract liquor, steams solvent, is dried to obtain ethyl acetate extraction
Object.
The invention also includes the pharmaceutical compositions of the Caulis Spatholobi ethyl acetate extract.
The present invention most preferably Caulis Spatholobi ethyl acetate extract.
Pharmaceutical composition of the present invention containing Millettia extract includes the preparation shape that any type is suitble to take
Formula, such as tablet, capsule, oral solution, granule, pill, pill, pulvis etc..
Described pharmaceutical composition can be prepared by existing conventional formulation preparation method.
When in use, dosage adjusts according to the patient's condition, and the method that can be used 1-3 times on the 1st is taken.Below by way of reality
Test the advantageous effect that data further illustrate the present invention：
In testing below, the drug used is named as SET for Millettia extract, and extraction preparation method is referred to
Any one technical solution in the prior art, can also commercially obtain or preparation side using the present invention
It is prepared by method.
If it is buying, the following product that can be commercially available：
The Millettia extract that the Xi'an bio tech ltd Yuan Sen produces
Extract part：Rattan Extraction solvent：Aqueous shape：Brownish-yellow powder fineness：100%, which crosses 80 mesh sieve specification, is：5：1
Or 10：1
Experiment is carried out according to this experiment actual conditions below.
It tests 1, SET and inhibits platelet aggregation and the drug efficacy study of blood coagulation
Research process is see Fig. 1
Experiment, 2, SET anti-TCIPA drug efficacy studies in vitro
Research process is see Fig. 2
Test anti-tumor metastasis drug effect research in 3, SET bodies
Research process is see Fig. 3
Result of study：
It tests 1, SET and inhibits platelet aggregation and the drug efficacy study of blood coagulation
SET can significantly extend removal calcium ion blood plasma recalcification time, and anticoagulating active dose dependent increases 40 μ g/mL
It is small that there is SET under concentration preferable inhibition 20,40,80 μ g/mL of platelet aggregation activity can significantly inhibit ADP induction blood
Plate is assembled, and inhibitory activity dose dependent increases.
Test 2, SET anti-TCIPA drug efficacy studies in vitro
SET can effectively inhibit the platelet adhesion reaction that 4T1 cells induce, and significantly reduce the aggregation rate of blood platelet.SET energy
The inhibition TCIPA processes of enough specificity.
Test anti-tumor metastasis drug effect research in 3, SET bodies
In experiment in vivo, SET can significantly inhibit the Lung metastases model that tail vein injection mode is built, and reduce Lung metastases
Stove intensity.It is daily to be administered continuously, lung tissue's structure can be improved, consolidation degree is relieved, and can finally extend Lung metastases
Survival time of mice reduces the death rate.
SET inhibits platelet aggregation and the drug efficacy study result of blood coagulation see Fig. 4-6, and SET in vitro grind by anti-TCIPA drug effects
Result is studied carefully see Fig. 7-8, and anti-tumor metastasis drug effect result of study is see Fig. 9-13 in SET bodies.
Description of the drawings：
Fig. 1,1, SET of experiment inhibit platelet aggregation and the drug efficacy study of blood coagulation
Research process is see Fig. 1
It in this experiment, is tested by external platelet aggregation-against, builds ADP induced platelet aggregation models, mould in vitro
The inducing action for intending tumour cell in environment in blood, has carried out tentatively the platelet aggregation-against optimum medicine efficacy dosage of SET
It determines.Meanwhile being tested by multiple calcium, influences of the SET to the clotting time is demonstrated, Caulis Spatholobi extraction is more intuitively reflected
Object blood coagulation resisting function.Final optimum medicine concentration and the action time that SET platelet aggregation-againsts are determined.
Fig. 2, experiment, 2, SET anti-TCIPA drug efficacy studies in vitro
Research process is see Fig. 2
In this part is tested, fluorescent marker is carried out to blood platelet using CFDA-SE, it is aobvious by laser co-focusing first
Micro mirror intuitively qualitatively judges adhering to each other and assembling for tumour and blood platelet, then passes through flow cytometer pair
The quantity for gathering tumor cell surface blood platelet carries out Quantitative measurement, while commenting the drug effect of SET antiplatelets adherency
Estimate.
Anti-tumor metastasis drug effect research in Fig. 3, experiment 3, SET bodies
Research process is see Fig. 3
The tumor cell injection for stablizing expression fluorescence is entered mouse blood cycle by this experiment by way of tail vein injection
In system, the growth course of Nasopharyngeal neoplasms stove is observed in real time by small animal living body imaging system, and most finish-unification fluorescence is strong
Degree assessment transfer stove size intuitively judges inside tumor morphological change by immunohistochemistry HE dyeing.
Fig. 4-6, SET inhibit the drug efficacy study result of platelet aggregation and blood coagulation
Fig. 7-8, SET anti-TCIPA drug efficacy studies result in vitro
Anti-tumor metastasis drug effect result of study in Fig. 9-13, SET bodies
Figure 14 Caulis Spatholobi series extracts influence blood platelet LDH releases
Figure 15 Caulis Spatholobi series extracts are to colon cancer cell MC38 proliferative effects
Figure 16 Caulis Spatholobi series extracts are to colon cancer cell HCT116 proliferative effects
The drug effect of the platelet aggregation of the anti-ADP inductions of Figure 17 Caulis Spatholobi series extracts
Figure 18 20h each group cellular morphologies change
Figure 19 Millettia extracts influence MC38 transfer abilities
Figure 20 Millettia extracts influence MC38 transfer abilities
Figure 19 and Figure 20 results are shown：In blank group, cellular portions migrate, and have certain transfer ability.With blank
Group compares, and after giving blood platelet and co-culturing, cell migration ratio increases model group；In aspirin group, n-butanol group and water
In layer group, cell migration number substantially reduces (P ＜ 0.05) compared with model group.And compared with model group, ethyl acetate extract can
Increasing (P ＜ 0.01) by the cell migration number caused by blood platelet is significantly reduced, and significant effect is better than positive group and other
Each group (P ＜ 0.01).It is above-mentioned the experiment results show that blood platelet can induce the transfer ability of enhancing MC38 cells, and Caulis Spatholobi
The addition of ethyl acetate extract can effectively weaken the transfer ability of MC38 cells.
Figure 21 Millettia extracts influence HCT116 cell E-cadherin protein expressions
Figure 21 results are shown：In blank group, the E-cadherin expressions of HCT116 cells are normal.Compared with blank group,
Model group can cause the E-cadherin protein expression levels of HCT116 cells to significantly reduce after giving blood platelet and co-culturing
(P ＜ 0.01)；And when Caulis Spatholobi series extract is added, it can significantly increase the expression (P of E-cadherin albumen
＜ 0.01), and it is better than positive drug aspirin.Above-mentioned experimental result explanation：Blood platelet can induce the E- of HCT116 cells
The reduction of cadherin protein expressions, and three kinds of Millettia extracts are capable of the expression of apparent increase E-cadherin, and second
The effect of acetoacetic ester group is the most notable.
Specific implementation mode：
The experimentation and experimental result further illustrated the present invention by the following examples.
Embodiment 1
Cell culture
Cell growth degree of converging carries out cell passage when reaching 70-80%.Old culture medium in original culture bottle is carefully sucked out
It is spare, it avoids pipette from touching cell, cell 2 times in culture bottle is washed using PBS buffer solution.0.05% trypsase is added
(containing EDTA) 1mL, makes cell fully infiltrate, and digestion reaction is carried out at 37 DEG C, and digestion time is controlled under inverted microscope.
When cell compartment increases, and index of refraction increases, old culture medium is added and terminates digestion reaction.Culture bottle is drawn using elbow straw
Middle cell suspension, slight, piping and druming culture bottle wall attachment repeatedly cell, is that cell completely disengages.Cell suspension is transferred to nothing
In bacterium centrifuge tube, 1000rpm centrifuges 5min.It discards supernatant, is added in new late period culture medium to centrifuge tube after centrifugation, is resuspended
Cell is simultaneously blown and beaten uniformly, and after being passed in required ratio or carried out other related experiments, Tissue Culture Flask is placed in containing 5%
It is cultivated in 37 DEG C of the cell incubator of CO2, relative humidity 95%.Blood platelet extracts
Mouse orbit takes the total 5mL of blood, all mouse not to take any anticoagulant in two weeks and have to platelet function
The drug of influence.It is placed in 3.8% sodium citrate anticoagulant tube, turned upside down mixing.Room temperature preservation, blood sample was at two hours
Within carry out in next step experiment detection.
Prepare PRP
Sodium citrate anticoagulation 280g is centrifuged into 10min, supernatant blood plasma and the buffy coat of middle layer are carefully moved to
In new EP pipes.280g repeated centrifugations 10 minutes draw supernatant blood plasma, obtain platelet rich plasma (PRP).
It is screened by the pharmaceutical activity of index of platelet activation
Platelet aggregation instrument, computer, computer monitor are opened successively.Silicification reaction cup with bar magnet is inserted into pre-temperature
Slot, pre-temperature 10 minutes to 37 DEG C.It takes after solution 1mL is as reaction cup in siliconized tubes in above-mentioned steps, then pre-temperature 5 minutes, inserts
Enter electrode, the other end is connected to aggregation instrument.The mixing speed that bar magnet is arranged is 1200rpm.By computation,
Run Test buttons are selected in aggregoment menus, input experimental subjects group names.OK is clicked after selection electric-resistivity method,
Operation curve can be observed on the screen.Curve baseline is set at 0% using Impendence Zero Knob, is pressed
Calibrate buttons, adjustment Gain buttons are that aggregation curve is located at 50% position, select ADP for derivant, run 6 minutes, point
It hits Stop test and stops experiment.It determines Set start time and stop time, calculates resistance value automatically.Take out electricity
It is wiped only using dustless blotting paper using tap water and normal saline flushing pole.Instrument temperature is once closed after experiment to open
Pass, power supply and computer.
Tail vein injection model construction
Primary tumor across Nasopharyngeal neoplasms grows and enters vascular process, and the target spot of drug effect is focused on tumour cell
Survival processes in blood exclude influence of the primary tumor to Lung metastases, for SET Composition analyzed provide it is more specific
Effect section.
Embodiment 2
Method
2.1 cell lines and cell culture
Mouse colonic cell mc38 and human colon cancer cell HCT116 purchases are in Beijing consonance cell bank.Cell culture
Condition:1640 culture mediums contain 10% calf serum and 1% dual anti-(penicillin 100UmL-1,100 μ gmL-1 of streptomysin),
It is placed in 37 DEG C, secondary culture in the insulating box of 5%CO2.
The preparation of 2.2 reagents
Millettia extract is extracted by Department Of Medicine, Peking University teacher Yang Xiuwei laboratory：Caulis Spatholobi 70kg, 5 times of amounts are steamed
Distilled water refluxing extraction 2 times, each 2h is concentrated under reduced pressure, steam-to-vacuum drying, 75 DEG C, dry 7h.Obtain Caulis Spatholobi water extract
6kg is re-dissolved in 7.2L distilled water, is extracted every time with 10.8L ethyl acetate, 8 times；Then use 14.4L extracting n-butyl alcohols, 9
It is secondary.It is final to obtain, acetic acid ethyl ester extract 300g, n-butyl alcohol extract 581g and water layer extract.
Medicine preparation：Acetic acid ethyl ester extract, n-butyl alcohol extract and each 35mg of water layer extract powder are weighed respectively,
175 μ L DMSO respectively are dissolved in, fully dissolving, mixing, until without precipitation.30 μ L are respectively asked for again in mortar, are added while stirring
Enter 1470 μ L physiological saline, until solution clear, no precipitation and crystal are precipitated, and final concentration is 4mg/mL.Using new sterile
After the packing of EP pipes, -20 DEG C of Cord bloods.
It is prepared by 2.3 animal's whole blood sample preparations and platelet rich plasma PRP
Mouse orbit takes blood, is placed in the anticoagulant tube of sodium citrate containing 20U/mL, slight mixing, as whole blood sample.By Chinese holly
Rafter acid sodium anti-freezing blood vessel 280g centrifuges 10min, and supernatant blood plasma and the buffy coat of middle layer are carefully moved in new EP pipes.
280g repeated centrifugations 10 minutes draw supernatant blood plasma, obtain platelet rich plasma (PRP).
2.4 lactic dehydrogenases (LDH) detect
Using LDH kits detection (Nanjing build up Science and Technology Ltd. production), sample treatment and enzyme assay according to
Kit specification is operated.OD values are measured under the conditions of 450nm using microplate reader after reaction.
2.5 MTT colorimetric methods
MC38, HCT116 tumour cell in exponential phase is made single cell suspension, adjust cell concentration be 2 ×
107L-1 is inoculated in 96 orifice plates, per 50 μ L of hole, after being cultivated for 24 hours in 37 DEG C, the constant incubator of 5%CO2, is added per hole
50 μ L drug containings or without medicine culture solution, if blank control group, each concentration medication group, positive drug control group, every group sets 3 multiple holes.
Continue after cultivating 72h, MTT (5gL-1) 10 μ L are added per hole, equal conditions continue to cultivate 4h.Culture solution is abandoned, is added per hole
150 μ L DMSO, fully shaking mixing make crystallization dissolve, and absorbance (A values) at 470nm is surveyed on enzyme-linked instrument, and calculate thin
Born of the same parents' proliferation inhibition rate (IR).Cell proliferation inhibition rate (%)=(1- experimental groups OD values/control group OD values) × 100%.Experiment
It repeats 3 times.
2.6 electric-resistivity methods detect the whole-blood platelet aggregation experiment of anti-ADP inductions
Blank group, modeling group, water is respectively set and puies forward group, ethyl acetate extract group, n-butanol extract group and positive drug
Group.Medicine group concentration is 100 μ g/mL, and positive drug Aspirin concentrations are 300 μM.In addition to blank group, every group of 10 μ of addition
The ADP derivants of L10mM concentration.It is as follows：Divide equally into each group siliconized tubes by often pipe 0.5mL blood, and respectively
Isometric test medicine is added.Siliconized tubes with bar magnet are inserted into pre-temperature 5 minutes to 37 DEG C in pre-temperature slot.Later by silicon
Change test tube to be transferred in reaction cup, be inserted into electrode, the other end is connected to aggregoment platelet aggregation instruments.Stirring for bar magnet is set
It is 1200rpm to mix speed, is run after selecting electric-resistivity method, operation curve can be observed on the screen.Select ADP for derivant,
Operation stops experiment after five minutes, calculates resistance value automatically.
2.7 morphological observation
Mouse mc38 cells are laid on by every hole 1x106 in six orifice plates, per hole culture medium containing 1mL1640.
Blank group, modeling group, water is respectively set and puies forward group, ethyl acetate extract group, n-butanol extract group
With positive drug group.In addition to blank group, 200 μ LPRP are added in bed board in remaining each group afterwards for 24 hours.Medicine group concentration is 100 μ g/
ML, positive drug Aspirin concentrations are 300 μM.It is common to be incubated after 20h comparison of taking pictures.
2.8 Transwell methods detect MC38 transfer abilities
Exponential phase MC38 cells are digested, its a concentration of 106/ml is adjusted, each small indoor addition 0.1ml (1 × 105
A cell).Cell is respectively divided into blank control group according to processing mode, (upper chamber is added blood platelet and trains altogether model control group
Support) and each administration group.1640 culture medium, the 600 μ L containing 10%FBS are added in lower room.Upper chamber is taken after 18h, is gently wiped away with cotton swab
Cell on cell filter membrane.The inhibiting rate of cell migration is calculated simultaneously：The inhibiting rate of cell migration=(control group migrating cell
Number-blank group group migrating cell number)/control group migrating cell number × 100%.
2.9 Western blot methods detect MC38, HCT116 cell CDH1 protein expression level logarithmic growth phases
MC38, HCT116 cell are inoculated in 5 × 104/hole cell density in 12 orifice plates, after culture for 24 hours, each group in addition to blank group
It is separately added into PRP and Millettia extract processing, is discarded supernatant after 4h, 20h, cell is cleaned 3 times with precooling PBS, each hole
RIPA is added and cracks thin 30min on ice, keeps cell cracking complete, is transferred in centrifuge tube, 13000 × g centrifuges 5min, draws
Supernatant, BCA kit quantifications and is uniformly diluted to same concentration.Per 12 μ L, SDS-PAGE protein isolates of hole loading, wet turn
Method is transferred on PVDF films, and 5%BSA closes 1h, primary antibody is added, 4 DEG C overnight, and TBST washes film 3 times, and secondary antibody is added, and room temperature is incubated
2h is educated, TBST washes film 3 times, shines.Experiment is repeated 3 times, and counts gray value.
3 statistical methods and processing
Experimental data carries out data statistic analysis using 23.0 softwares of IBM SPSS, is divided using single factor test variance method
Analysis, is as a result indicated with x ± s, with P<0.05 has statistical significance for difference, is that difference is united with pole conspicuousness with P ＜ 0.01
Meter learns meaning.
4 experimental results
4.1 Caulis Spatholobi series extract administration concentrations select
This experiment uses tumour-blood platelet co-culture system, to exclude drug for the straight of tumour cell or blood platelet
Effect is connect, we use LDH experiments and MTT experiment to have rated influence of the drug for blood platelet and tumour cell respectively first,
Ensure drug for the existence of blood platelet or tumour and function without larger impact.
Figure 14 results are shown：Blood platelet Caulis Spatholobi series extract five kinds of drug concentrations (25 μ g/mL, 50 μ g/mL,
100 μ g/mL, 200 μ g/mL, 400 μ g/mL) under effect, LDH activity is compared with negative control group there are no significant difference.Experimental result
Explanation：Caulis Spatholobi series extract is on blood platelet LDH releases without influence.
Figure 15 results are shown：When three kinds of extracts low concentration (<100 μ g/mL), under the action of the short time (24H), it is right
The inhibiting rate of MC38 cells is below 20%.And when action time is more than 24H, and drug concentration is more than 200 μ g/mL, it is each to extract
Object all has a strong inhibitory effect MC38 cells.
Figure 16 results are shown：Ethyl acetate extract and n-butanol extract are thin to HCT116 under 5 kinds of concentration doses
The proliferation inhibiting effect of born of the same parents is weaker.It is thin to HCT116 under only 800 μ g/mL concentration when water layer group action time is 24H
Born of the same parents have higher inhibiting rate；When water layer group action time is 48H, each concentration all has obviously HCT116 cell Proliferations
Inhibiting effect.
In conjunction with above-mentioned experimental result, we select 100 μ g/mL as the suitable administration of follow-up each medicine group experiment in vitro
Concentration.
4.2 electric-resistivity methods detect the platelet aggregation test of the anti-ADP inductions of each extract of Caulis Spatholobi
The most direct pathologic phenomenons of TCIPA are the abnormal activations of platelet function.And then we select anti-ADP to induce
Platelet aggregation test fast and effectively evaluate and screen Caulis Spatholobi series extract platelet aggregation-against in vitro
Drug activity.
The results show that compared with model group, positive drug aspirin, ethyl acetate extract and n-butanol extract Figure 17
Object can significantly inhibit the platelet aggregation (P of ADP inductions<0.01), and the drug effect of Caulis Spatholobi ethyl acetate extract by
In aspirin group and n-butanol group.In addition, there was no significant difference compared with model group for water layer group.The experiment results show that acetic acid
Ethyl ester extract and n-butanol extract may containing the active constituent of platelet aggregation-against, and water layer group active constituent compared with
It is low.
4.3 Millettia extracts are in tumour cell on morphologic influence and detection
After primarily determining pharmaceutical activity position, we evaluate Caulis Spatholobi series extract pair from morphological indexes first
The influence of MC38 cells.
It can be seen that from Figure 18 (lower right corner is partial enlarged view)：In blank group, cell is oval, and connection is tight
It is close.Compared with blank group, model group is after giving blood platelet and co-culturing, and cell is in spindle shape, and length-width ratio increases and pseudopodium increases
More, Cell tracking is loose, and cell is easy to fall off.In aspirin group, cell length-width ratio decreases, and pseudopodium is reduced, cell
It is less easy to fall off.And in Caulis Spatholobi ethyl acetate extract group, cell length-width ratio restores normal, and pseudopodium disappears.N-butanol
Group changes unobvious compared with water puies forward group compared with model group.Above-mentioned experimental phenomena prompt, blood platelet occur induction of mc38 cells
Apparent interstitial sample becomes；And the addition of Caulis Spatholobi ethyl acetate extract, its interstitial sample can be reversed to become.
4.4 Millettia extracts influence MC38 transfer abilities
Once EMT variations occur, the transfer ability of tumour cell will greatly improve tumour cell, contribute to tumour thin
Born of the same parents, which invade, enters such as blood vessel, to carry out being far apart organ metastasis by blood circulation.Therefore, we are based on Morphological Experimental
Variation, has detected influence of the Caulis Spatholobi series extract to the transfer ability of tumour cell.
Figure 19 and Figure 20 results are shown：In blank group, cellular portions migrate, and have certain transfer ability.With
Blank group compares, and after giving blood platelet and co-culturing, cell migration ratio increases model group；In aspirin group, n-butanol group
In water layer group, cell migration number substantially reduces (P ＜ 0.05) compared with model group.And compared with model group, ethyl acetate extract
Increasing (P ＜ 0.01) by the cell migration number caused by blood platelet can be significantly reduced, and significant effect is better than positive group
With other each groups (P ＜ 0.01).It is above-mentioned the experiment results show that blood platelet can induce enhancing MC38 cells transfer ability, and
The addition of Caulis Spatholobi ethyl acetate extract can effectively weaken the transfer ability of MC38 cells.
4.5 Millettia extracts influence HCT116 cell E-cadherin protein expressions
On a molecular scale, we have detected molecule E-cadherin most crucial in EMT variations.Figure 21 results are shown：
In blank group, the E-cadherin expressions of HCT116 cells are normal.Compared with blank group, model group is to give blood small
Plate can cause the E-cadherin protein expression levels of HCT116 cells to significantly reduce (P ＜ 0.01) after co-culturing；And works as and add
When entering Caulis Spatholobi series extract, the expression (P ＜ 0.01) of E-cadherin albumen can be significantly increased, and better than sun
Property medicine aspirin.Above-mentioned experimental result explanation：Blood platelet can induce the E-cadherin protein expressions of HCT116 cells
It reduces, and three kinds of Millettia extracts are capable of the expression of apparent increase E-cadherin, and the effect of ethyl acetate group is the most
Significantly.
Claims (10)
1. a kind of Millettia extract application in preparation of anti-tumor drugs, wherein the Millettia extract is selected from：Chicken blood
Rattan water extract, Caulis Spatholobi ethyl acetate extract, Caulis Spatholobi n-butanol extract.
2. application according to claim 1, which is characterized in that the wherein described Caulis Spatholobi water lift extract, preparation method is such as
Under：
Caulis Spatholobi 70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, 75 DEG C of steam
It is dried in vacuo 7h.Caulis Spatholobi water extract 6kg is obtained, water extract is obtained.
3. application according to claim 1, which is characterized in that the wherein described Caulis Spatholobi ethyl acetate extract, preparation side
Method is as follows：
Caulis Spatholobi 70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, 75 DEG C of steam
It is dried in vacuo 7h.Caulis Spatholobi water extract 6kg is obtained, obtains water extract, water extract is re-dissolved in 7.2L distilled water, uses second
Acetoacetic ester extracts, each dosage 10.8L, coextraction 8 times；Combined ethyl acetate layer extract liquor, steams solvent, is dried to obtain acetic acid
Ethyl ester extract.
4. application according to claim 1, which is characterized in that the wherein described Caulis Spatholobi n-butanol extract, preparation method
It is as follows：
Caulis Spatholobi 70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, 75 DEG C of steam
It is dried in vacuo 7h.Caulis Spatholobi water extract 6kg is obtained, obtains water extract, water extract is re-dissolved in 7.2L distilled water, with just
The each dosage 14.4L of butanol, before immunoassay, coextraction 9 times merge n-butanol layer extract liquor, steam solvent, be dried to obtain n-butanol and carry
Take object.
5. application according to claim 1, wherein the inhibition metastases are thin by the anti-extract tumour of Caulis Spatholobi
What the platelet aggregation of born of the same parents' induction was realized.
6. application according to claim 1, wherein the inhibition metastases are to inhibit blood small by Millettia extract
What plate activation aggregation and blood coagulation resisting function were realized.
7. application according to claim 1, wherein the tumour is selected from：Colon cancer, breast cancer, lung cancer, cancer of pancreas or liver
Cancer.
8. application according to claim 1, wherein the tumour is colon cancer.
9. a kind of Caulis Spatholobi ethyl acetate extract, preparation method are as follows：
Caulis Spatholobi 70kg, every time with 5 times of amount distilled water refluxing extractions, totally 2 times, each 2h is concentrated under reduced pressure after merging, 75 DEG C of steam
It is dried in vacuo 7h.Caulis Spatholobi water extract 6kg is obtained, obtains water extract, water extract is re-dissolved in 7.2L distilled water, uses second
Acetoacetic ester extracts, each dosage 10.8L, coextraction 8 times；Combined ethyl acetate layer extract liquor, steams solvent, is dried to obtain acetic acid
Ethyl ester extract.
10. the pharmaceutical composition containing Caulis Spatholobi ethyl acetate extract described in claim 9.
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