CN107260752A - A kind of pharmaceutical composition of collaboration anti-pancreatic cancer - Google Patents
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer Download PDFInfo
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- CN107260752A CN107260752A CN201710379871.2A CN201710379871A CN107260752A CN 107260752 A CN107260752 A CN 107260752A CN 201710379871 A CN201710379871 A CN 201710379871A CN 107260752 A CN107260752 A CN 107260752A
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- gemcitabine
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- pancreatic cancer
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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Abstract
The invention provides a kind of pharmaceutical composition of collaboration anti-pancreatic cancer.The medicine acceptable auxiliary material comprising active component and pharmaceutically, it is characterized in that, described active component includes gemcitabine and triterpenoid, described triterpenoid is 3 α, the acid of 11 20 (29) alkene of α dihydroxy lupin 28 or the diacid of 3 α hydroxyls lupin, 20 (29) alkene 23,28.The synergy that drug combination is produced to pancreatic cancer cell is judged to each side such as pancreatic cancer cell growth state, cell growth inhibition percentage, the influences of Cellular cycle and apoptosis.Because of the drug resistance of gemcitabine, drug combination not only provides more drug safeties, and reduces the Clinical practice dosage of gemcitabine, reduces heavy dose of toxic side effect using produced by gemcitabine, safety clinical treatment index is improved, more possibilities are provided for gemcitabine clinical practice.
Description
Technical field
The invention belongs to combination medicine technical field, more particularly, to a kind of drug regimen of collaboration anti-prostate cancer
Thing.
Background technology
Cancer of pancreas is the high malignant tumor of digestive tract of common fatal rate, and the whole world there are about 220,000 people and dies from this every year
Disease, its morbidity and mortality substantially rise in recent years.In China, the annual morbidity of cancer of pancreas is about 5.1/100,000 people, often
Year newly-increased about 5-6 ten thousand.Its death rate is also raised rapidly, accounts for 7-8 of the malignant tumour cause of the death.Further to improve Ji Xita
Treat the effect of advanced pancreatic cancer, many medicines and gemcitabine therapeutic alliance advanced pancreatic cancer, including cis-platinum, Ka Peita in shore
Shore, Docetaxel etc..Although the effective percentage of combined chemotherapy is increased, tumor-related symptoms also have some improvement,
But said medicine and the united extensive randomized clinical research of gemcitabine all fail to show have more than gemcitabine list medical instrument
Good existence benefits, and the chemotherapy of cancer of pancreas has gone to a plateau, and it is swollen both at home and abroad for how realizing the breakthrough in therapeutic effect
The severe challenge that knurl interface is faced.
Gemcitabine is the first-line drug of the treatment cancer of pancreas of U.S. FDA approval, and it can be obviously improved Pancreas cancer patients
Quality of life simultaneously extends life cycle, is described as the goldstandard for the treatment of of pancreatic cancer.Gemcitabine is also increasingly used as simultaneously
The adjuvant therapy medicaments of patients undergoing hepatectomy.Gemcitabine is a kind of precursor medicine of deoxynucleoside analog class, it is necessary to which cell is inhaled
Metabolism after receipts by relevant enzyme produces its active metabolite to play antitumor activity.The active metabolite of gemcitabine
It is main to cause DNA damage and arresting cell cycle progression by disturbing DNA replication dna, so as to suppress cell propagation and inducing cell
Apoptosis occurs.But, in clinical treatment, many Pancreas cancer patients have inborn or acquiredization to gemcitabine
Resistance is treated, the application effect of gemcitabine clinically is limited.Therefore, it is also pancreas that patient produces drug resistance to chemotherapeutics
Cancer is difficult to the major reason cured.For these reasons, finding more effective treatment of pancreatic cancer has importance and urgent
Property.
The content of the invention
The invention aims to the defect for overcoming prior art, there is provided a kind of drug regimen of collaboration anti-pancreatic cancer
Thing.Two triterpenoids in le dish are carried out drug combination by pharmaceutical composition Tong Guo with gemcitabine respectively, with cancer of pancreas
In Pcan-1 cells as research object, increment of the pharmaceutical composition to pancreatic cancer cell is migrated, and invasion and attack etc. have notable
Inhibitory action, be expected to obtain the drug candidate that safer reliable novel therapeutic treats cancer of pancreas.
Above-mentioned purpose of the present invention is achieved by the following technical programs：
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, the medicine includes active component and pharmaceutically acceptable auxiliary
Material, described active component includes gemcitabine and triterpenoid, and the triterpenoid is 3 α, 11 alpha-dihydroxys-feather fan
Beans -20 (29)-alkene -28- sour (E12-1) or 3 Alpha-hydroxies-lupin -20 (29)-alkene -23,28- diacid (E13-1).
Preferably, the mol ratio of described triterpenoid and gemcitabine is (100~4550): (1~10).
Further preferably, the mol ratio of described triterpenoid and gemcitabine is (300~1500): (1.5~8).
It is further preferable that described triterpenoid and the mol ratio of gemcitabine are 170: 1.
Preferably, the pharmaceutical composition of described cancer of pancreas is oral formulations.
It is further preferable that described oral formulations are capsule, tablet, granule or liquor.
Application of the upper described pharmaceutical composition in inhibiting tumor cell medicine is prepared.
Preferably, the cancer cell is pancreatic cancer cell.
The pharmaceutical composition of the present invention plays anti-pancreas by described triterpenoid and gemcitabine as active component
The synergy of gland cancer, inventor is screened by lot of experiments to the amount ratio of these three components, and result is described three
Terpene compound and the mol ratio of gemcitabine are in (100~4550): when in the range of (1~10), the increment effect of its anti-pancreatic cancer
Fruit is preferably：In order to reduce the toxic side effect and drug resistance of gemcitabine, The present invention reduces the consumption of the medicine, therefore at this
Invent in most preferably external embodiment, 3 α, 11 alpha-dihydroxys-lupin -20 (29)-alkene -28- sour (E12-1) and Ji Xita
The mol ratio of shore is 170: 1.
In an embodiment of the present invention, the triterpenoid and Ji Xita contained in the pharmaceutical composition of the anti-pancreatic cancer
Shore can be absorbed with faster speed by enteron aisle and play bioactivity, therefore the pharmaceutical composition of the present invention is preferably mouth
Formulation.Pharmaceutical units preparation can be that the pharmaceutically acceptable any conventional that conventional preparation process is made orally is made
Agent, such as capsule, tablet, granule or liquor, to enable above-mentioned preparation type to realize, need to add pharmacy during preparation
Upper acceptable auxiliary material, the pharmaceutically acceptable auxiliary material be filler, disintegrant, lubricant, suspending agent, adhesive or
Sweetener.It is one kind in starch, lactose, microcrystalline cellulose or sucrose to use filler in an embodiment of the present invention, also may be used
To use any mixture of above-mentioned filler；The disintegrant used is in starch, sodium carboxymethyl starch or pregelatinized starch
It is a kind of, it is possible to use other known disintegrant such as low-substituted hydroxypropyl cellulose or Ac-Di-Sol, it can also make
With any mixture of above-mentioned disintegrant, the lubricant used is magnesium stearate or silica, it is possible to use other known
In lubricant such as lauryl sodium sulfate, any mixture of above-mentioned lubricant can also be used；The suspending agent is poly- second
One or more of alkene pyrrolidone, sucrose or hydroxypropyl methyl cellulose；Described adhesive is hydroxypropyl methyl cellulose, formed sediment
One or more of slurry or polyvinylpyrrolidone；The sweetener be saccharin sodium, enoxolone, sucrose, aspartame or
One or more of honey element.
The present invention is using the Pcan-1 cells in cancer of pancreas as research object, by by triterpenoid E12-1 and E13-1
Drug combination is carried out with gemcitabine respectively, Anticancer Activity in vitro test is carried out to Pcan-1 cells.As a result show three in , le dishes
Terpene compound E12-1 and E13-1 growth conditions being used in combination to pancreatic cancer cell respectively with gemcitabine, cell growth suppression
The influence of percentage processed, Cellular cycle and apoptosis.The present invention because gemcitabine be treat cancer of pancreas standard front-line chemotherapeutic agents, but
Easily occurs resistance phenomenon, Apoptosis inhibitor is one of major reason that gemcitabine produces resistance.Research finds the related egg of Bcl families
White to be played a key effect in regulation and control apoptosis of pancreatic cancer cell, tumour cell can be strengthened to chemotherapy drug susceptibility by suppressing Bcl-2.
Thus, invention is with the human pancreatic cancer cell of in vitro culture from inquiring into gemcitabine in integral level He triterpene compound in le dish
Inhibitory action and mechanism, it is intended to combine 3 α, 11 alpha-dihydroxys-lupin -20 (29)-alkene -28- acid for gemcitabine
And 3 Alpha-hydroxies-lupin -20 (29) (E12-1)-alkene -23,28- diacid (E13-1) clinical treatments cancer of pancreas provides theoretical and real
Test foundation.
The separating extractive of triterpenoid E12-1 and E13-1 Shi le dishes of the present invention.Le dish, scientific name Wei Bai le
(Acanthopanax trifoliatus (Linn.) Merr.), also known as E Zhang le, Trifoliate Acanthopanax Root, Radix Acanthopanacis Trifoliati, bitter thorn, thorn three
Plus, it is that one kind of Araliaceae (Araliaceae) Acanthopanax (Acanthopanax) climbs up by holding on to shape shrub.Le dish is that integration of drinking and medicinal herbs is planted
Thing, China's data is early on the books to it, and Li Shizhen (1518-1593 A.D.) is said：" slender acanthopanax controls rheumatism paralysis storehouse, strengthens the bone, the good depth of its work("；The Ming Dynasty《Food sheet
Grass》Described in " slender acanthopanax, leaf makees cooked food, goes skin rheumatism "；《Guangdong medicinal plant short course》Record " its root, cauline leaf can be used as medicine,
Root, dispelling wind and eliminating dampness, dispersing blood stasis analgesic；Leaf, dispelling wind, detumescence, it is antipruritic, relax the muscles and stimulate the blood circulation, subdhing swelling and detoxicating ".Le dish complete stool is equal
Can be used as medicine, it is bitter, pungent, cool, lived with clearing heat and detoxicating, expelling wind and removing dampness, relaxing muscles and tendons, it is relieving cough and asthma the effect of.There are reported in literature , le dishes
In contain in Bai le containing the medicinal ingredient such as flavones, saponin(e, cumarin, polysaccharide, cardiac glycoside.
Inventor has carried out many basic research , by long-term research , Dui le dishes and has isolated various active Cong le dish
Stronger compound, studies its oxidation resistance, active anticancer, anti-inflammatory activity etc..This research is separated to two Cong le dish
The α of triterpene compound 3,11 alpha-dihydroxys-lupin -20 (29)-alkene -28- acid and 3 Alpha-hydroxies-lupin -20 (29)-alkene -
23,28- diacid (as shown in Figure 1), and the test of Basal activity has been carried out to it, as a result show, such compound has anti-
Tumour, anti-inflammatory suppresses the effect such as alpha-glucosidase (patent CN104922173A).
Compared with prior art, the invention has the advantages that：
1. the present invention passes through two activity that Jiang be isolated Cong le dish stronger triterpene compound E12-1 and E13-1
Drug combination is carried out with the line clinical medicine gemcitabine of anti-pancreatic cancer one respectively, to pancreatic cancer cell growth state, cell growth
Suppress each side such as percentage, the influence of Cellular cycle and apoptosis and judge the collaboration work that drug combination is produced to pancreatic cancer cell
With.The survival rate of E12-1 in the present invention, E13-1 and gemcitabine cell is respectively 80.3%, 75.5% and 77.9%.And work as
E12-1 and E13-1 respectively with after gemcitabine drug combination, the survival rates of Pcan-1 cells is only 49.7% He respectively
44.2%.Cells survival rate is by have dropped 31%-36% or so after medication combined.Thus illustrate to share in the comparable sodium second line of a couplet
Medicine can more effectively suppress the growth of Pcan-1 cells, there occurs the effect of Synergistic.
2. triterpenoid E12-1 in the present invention, E13-1 are combined apoptosis-induced related gene B cl- with gemcitabine respectively
2nd, the change of the expressing quantity such as IL-6, NF- κ B and P-State3, it is found that triterpenoid E12-1 and E13-1 can strengthen Ji Xi
He kills pancreatic cancer cell in shore.
3. triterpenoid E12-1 of the present invention, E13-1 respectively with pharmaceutical composition associated with gemcitabine, not only provide
More drug safeties, and the Clinical practice dosage of gemcitabine is reduced, reduce heavy dose of using produced by gemcitabine
Toxic side effect, improve safety clinical treatment index, reduce gemcitabine drug resistance.Provided for gemcitabine clinical practice
More possibilities.
4. triterpenoid E12-1 of the present invention, E13-1 respectively with pharmaceutical composition associated with gemcitabine and traditional list
Medicine treatment is compared, and apoptosis rate is increased substantially, and respectively 44.3% and 48.2%.Illustrate that drug combination promotes Apoptosis
The effect that is administered than single medicine of effect improve, Apoptosis is added 21%-26%.Further illustrate, the effect of drug combination
Fruit is better than single medicine administration.A variety of signal of interest paths can be targetted simultaneously, overcome tumor drug resistance, reduction toxic side effect etc..
Brief description of the drawings
Fig. 1 is 3 α, and 11 alpha-dihydroxys-lupin -20 (29)-alkene -28- is sour (E12-1) (a), 3 Alpha-hydroxies-lupin -20
(29) chemical structural formula of-alkene -23,28- diacid (E13-1) (b) and gemcitabine (c).
Fig. 2 is the influence of triterpenoid E12-1, E13-1 and gemcitabine to Pcan-1 cell growths.
Fig. 3 is that triterpenoid E12-1, E13-1 and gemcitabine dye representational microphoto by PI.
Fig. 4 is triterpenoid E12-1, E13-1 and gemcitabine to apoptotic morphologic after the processing of Pcan-1 cells
Ratio.
Fig. 5 is 0h after triterpenoid E12-1 and gemcitabine are handled Pcan-1 cells, 5h and 24h cell cuts are cured
Close cellular morphology microphoto.
Fig. 6 is 0h after triterpenoid E13-1 and gemcitabine are handled Pcan-1 cells, 5h and 24h cell cuts are cured
Close cellular morphology microphoto.
Fig. 7 is triterpenoid E12-1 and E13-1 respectively with gemcitabine to triterpenoid after the processing of Pcan-1 cells
With gemcitabine to migration results after the processing of Pcan-1 cells.
Fig. 8 be triterpenoid E12-1 and E13-1 respectively with gemcitabine to associated signal paths in Pcan-1 cells and
The influence of expression of apoptosis protein.
Embodiment
Present disclosure is further illustrated with reference to specific embodiment, but be should not be construed as limiting the invention.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.Except non-specifically
Illustrate, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.
Laboratory apparatus employed in embodiment below has the CO of Thermo Scientific Series 80002Culture
Case (Thermo companies of the U.S.)；Class IIA/B3Level stream clean work station (Thermo companies of the U.S.)；MDF-382E is ultralow
Temperature refrigerator (Japanese Sanyo companies)；CPKR supercentrifuges (Beckman Coulter companies of the U.S.)；Allegra X-22R are cold
Freeze centrifuge (Beckman Coulter companies of the U.S.)；The type liquid nitrogen containers of Locator 4 (Thermo companies of the U.S.)；Hotpack types
High-temperature disinfection cabinet (Thermo companies of the U.S.)；Liquid-transfering gun (German Eppendorf companies)；Nikon Optiphot light microscopes
(Japanese Nikon companies)；Nikon Eclipse TE200 fluorescence microscopes (Japanese Nikon companies)；Nikon EFD-3 are micro-
Mirror (Japanese Nikon companies)；Infinite M200PRO multi-function microplate readers (Austrian TECAN companies)；Sirius single hoses
Chemiluminescence detector (German Berthold Detection Systems companies)；ChemiDocTMXRS Imager imagings system
Unite (Bio-Rad companies of the U.S.).
The experiment reagent used has DMEM culture mediums (Gibco companies of the U.S.)：DMEM dry powder (1L with) is dissolved in about
In 800mL distilled water, stirring adds penicillin and streptomysin mixed liquor (final concentration to being completely dissolved：Penicillin 100units/
ML, the μ g/mL of streptomysin 100), 2g sodium carbonate is added, 1000mL is settled to.Then 10% hyclone is added, after filtration sterilization
It is dispensed into sterile serum bottle, 4 DEG C save backup.(U.S. Serum Source International are public for hyclone (FBS)
Department)；Trypsin-EDTA solutions (1 ×) (Sigma-Aldrich)；0.4% trypan blue solution (U.S. Cambrex
Bio Science companies)；Protein determination kit (Bio-Rad companies of the U.S.)；Luciferase Assay Reagent box (the U.S.
Promega companies)；SuperSignal West Femto maximum sensitivities chemical luminous substrate (Thermo companies of the U.S.)；
MTT, PI, DMSO (Sigma-Aldrich)；Primary antibody：β-actin, p-STAT3, IL-6, Bcl-2, the Cox-2 (U.S.
Cell Signaling companies)；The anti-igg secondary antibody (Santa Cruz companies of the U.S.) of HRP marks；Other reagents are the U.S.
Sigma companies analyze pure ratings above.
Embodiment 1
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-1200 parts of compound, 1 part of gemcitabine, 600 parts of microcrystalline cellulose, after fully mixing, filling No. 1 capsule 100.
Embodiment 2
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-11000 parts of compound, 10 parts of gemcitabine, 300 parts of microcrystalline cellulose, after fully mixing, filling No. 1 capsule 100.
Embodiment 3
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-1600 parts of compound, 1 part of gemcitabine, 650 parts of lactose after being well mixed, adds 10% shallow lake that 300 parts of starch are made
Slurry, carries out spraying granulation.130 parts of 100 parts of magnesium stearate and PVP are added, after being well mixed, tabletting is made 500.
Embodiment 4
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-1100 parts of compound, 1 part of gemcitabine, 350 parts of lactose after being well mixed, adds 10% shallow lake that 800 parts of starch are made
Slurry, carries out spraying granulation.80 parts of 60 parts of magnesium stearate and PVP are added, after being well mixed, tabletting is made 500.
Embodiment 5
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-1300 parts of compound, 1 part of gemcitabine, 300 parts of starch, 250 parts of carboxymethyl cellulose is dried in vacuo, and is well mixed, mistake
100 mesh sieves, are made granule.
Embodiment 6
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-1340 parts of compound, 2 parts of gemcitabine, 350 parts of starch, 250 parts of carboxymethyl cellulose is dried in vacuo, and is well mixed, mistake
80 mesh sieves, are made granule.
Embodiment 7
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-14550 parts of compound, 1 part of gemcitabine, 700 parts of silica, 280 parts of starch slurry, 460 parts of sucrose is mixed, until
Oral liquid is made in even liquid, filtration sterilization, packing.
Embodiment 8
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-1455 parts of compound, 1 part of gemcitabine, 450 parts of silica, 90 parts of starch slurry, 500 parts of sucrose is mixed, until uniform
Liquid, filtration sterilization, packing, oral liquid is made.
Embodiment 9
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-1300 parts of compound, 1.5 parts of gemcitabine, 500 parts of sodium carboxymethyl starch, 750 parts of pregelatinized starch is mixed, and crosses 120 mesh
Sieve, pelletizes, and dries, whole grain, adds 300 parts of magnesium stearate mixing, and tabletting is made 200.
Embodiment 10
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E12-1300 parts of compound, 8 parts of gemcitabine, 300 parts of sodium carboxymethyl starch, 500 parts of pregelatinized starch is mixed, and crosses 60 mesh sieves,
Granulation, is dried, whole grain, adds 200 parts of magnesium stearate mixing, and tabletting is made 200.
Embodiment 11
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-11500 parts of compound, 1.5 parts of gemcitabine, 250 parts of pregelatinized starch, 650 parts of lactose is well mixed, and is pelletized, and is dried,
320 parts of silica is added as lubricant, is mixed again, 120 mesh sieves are crossed, 50 capsules are made in filling.
Embodiment 12
A kind of pharmaceutical composition of collaboration anti-pancreatic cancer, is measured comprising the triterpene isolated Cong le dish alcohol extract by mol
E13-1750 parts of compound, 4 parts of gemcitabine, 250 parts of pregelatinized starch, 130 parts of lactose is well mixed, and is pelletized, and is dried, plus
Enter 250 parts of silica as lubricant, mix again, cross 60 mesh sieves, 50 capsules are made in filling.
The pharmaceutically acceptable auxiliary material added in above-described embodiment 1-12 can be effectively moistureproof, at the same be also beneficial to medicine into
Type.
Embodiment 13MTT colorimetric methods
MTT is a kind of to receive H+ weld.In living cells mitochondria respiratory chain, succinate dehydrogenase and cell
Pigment C can make MTT tetrazole ring opening, the first a ceremonial jade-ladle, used in libation crystallization of generation bluish violet, and this dehydrogenase is free of in dead cell.First a ceremonial jade-ladle, used in libation
The growing amount of crystallization is directly proportional to viable count, can evaluate the shadow of medicine cell proliferation by detecting the amount of first a ceremonial jade-ladle, used in libation crystallization
Ring.Pcan-1 cells are with inoculum density 1 × 105Individual/mL concentration is inoculated in 96 orifice plates (100 μ L/ holes), while setting blank to adjust
Zero hole (culture medium bed board hole) and control wells dimethyl sulfoxide (DMSO) (DMSO) processing hole, 5%CO2, 37 DEG C of overnight incubations, addition concentration
The compound and gemcitabine of gradient are handled, and each sample sets 4 multiple holes, 5%CO2, 37 DEG C be incubated 72h after carefully inhale
Go out nutrient solution, the MTT solution (5mg/mL, i.e. 0.5%MTT) that 100 μ L culture mediums are prepared is added per hole；Continue to cultivate after 1h, it is small
The heart sucks nutrient solution in hole.100 μ L DMSO are added per hole, low-speed oscillation 10min on shaking table is put, crystal is fully dissolved；With
The light absorption value in each hole, the inhibiting rate of medicine cell growth is calculated with formula (1), as a result such as the institute of table 1 at ELIASA measurement 570nm
Show.
Inhibitory rate of cell growth (%)=(1-ASample sets/AControl groupThe formula of) × 100% (1)
The increment inhibitory action (mtt assay) of the triterpenoid of table 1 and gemcitabine to Pcan-1 cells
| E12-1 | E13-1 | Gemcitabine Gemcitabine | |
| IC50 | 66.13±5.65μM | 98.38±6.40μM | 2.78±3.39μM |
The α of triterpene compound 3,11 alpha-dihydroxys-lupin -20 (29)-alkene -28- acid (E12- are determined using mtt assay
1) inhibitory activity bred with 3 Alpha-hydroxies-lupin -20 (29)-alkene -23,28- diacid (E13-1) to Pcan-1 cells.As a result
Show, all compounds have the activity of stronger suppression pancreatic cancer cell growth, IC50Respectively：66.13,98.38 Hes
2.78μM。
The trypan exclusion stain of embodiment 14
Trypan Blue Cleaning Principle：Normal living cells, after birth structural integrity can repel trypan blue, and being allowed to can not
Enough enter intracellular；And loss of activity or the incomplete cell of cell membrane, the permeability increase of after birth, indigo plant can be dyed by trypan blue
Color.It has been generally acknowledged that cell membrane integrity is lost, you can think that cell is dead, this is opposite with dimethyl diaminophenazine chloride effect.Therefore, by
Trypan Blue can simply and quickly distinguish living cells and dead cell very much.Pcan-1 cells are with inoculum density 0.2 × 105
Individual/mL concentration is inoculated in diameter 35mm culture dishes (2mL/ culture dishes) (6 orifice plates, 2mL/ holes), 5%CO2, 37 DEG C were cultivated
Night.Addition is dissolved in the compound and gemcitabine of the high, medium and low concentration in dimethyl sulfoxide (DMSO) (DMSO), and is entered
Row administering drug combinations, are carried out after processing 72h to Pcan-1 cells, and compounds on cell growth is determined using the method for Trypan Blue
With dead influence.Specific method is：The cell of adherent growth is digested using pancreatin, by 80 μ L cell suspensions and 20 μ L 0.4%
Trypan blue solution uniformly mix, stand 2min after, use micro- sem observation and counting.Under the visual field, clear transparent cell is calculated as
Living cells, trypan blue dye is calculated as dead cell for blue cell.Experiment dimethyl sulfoxide concentration is no more than 0.1% every time.As a result such as
Shown in table 2.
The influence of the triterpenoid of table 2 and gemcitabine to Pcan-1 cell survival rates
Compound E12-1, E13-1 and fatal rate of the gemcitabine to Pcan-1 cells are tested with trypan exclusion stain,
Concentration needed for selection drug combination is according to its IC50, the concentration value of medicine, determines E12- when selection cell lethality is about 20%
Concentration needed for 1, E13-1 is 10uM, and 35uM, gemcitabine concentration is 0.2uM.Determine after administration concentration, medicine is carried out to it
Combination.Fig. 2 is the influence of triterpenoid and gemcitabine to Pcan-1 cell growths.As can be seen from Figure 2, drug combination pair
The fatal rate of Pcan-1 cells is better than the fatal rate effect that single medicine is administered.During single medicine, E12-1, E13-1, gemcitabine cell
Survival rate is 80.3%, 75.5% and 77.9% respectively.And after drug combination, the survival rate of Pcan-1 cells is only respectively
49.7% and 44.2%.Cells survival rate is by have dropped 31%-36% or so after medication combined.Thus prove, equal dense
The lower drug combination of degree can more effectively suppress the growth of Pcan-1 cells, there occurs the effect of Synergistic.
The Apoptosis of embodiment 15 is detected
The Pcan-1 cell suspending liquids after compound processing 72h are combined during trypan blue exclusion is tested, is centrifuged and applied with cell
Cell smear is made in piece device, through acetone/methanol (1:1) solution is fixed after 10min, with propidium iodide (PI) PBS solution (10 μ g/
ML) dye, it is observed using fluorescence microscope and morphological analysis, DNA takes on a red color fluorescence.Apoptosis is by classics
Morphological feature is determined, includes the formation of karyopycnosis, cellular contraction, and apoptotic body.Nucleus is in fine and close dense dye during observation, or is in
The fine and close dense dye of chunky shape.Each sample is in the several visuals field of random observation under microscope, and no less than 200 cells, count every altogether
The percentage of the apoptotic cell included in the individual visual field (× 200).
Experiment is contrasted by single medicine and drug combination, and Pcan-1 cells are carried out after 72h processing, through fixing and passing through
After PI dyeing, it is observed using fluorescence microscope.It is representational that Fig. 3 is that triterpenoid and gemcitabine are dyed by PI
Microphoto.Wherein, (A) be using DMSO as positive control, the microphoto acted on after being dyed after cell by PI, (B), (C),
(D), (E) and (F) be respectively E12-1, E13-1 and gemcitabine list medicine administration and drug combination after, after cell is dyed by PI
Microphoto.As can be seen from Figure 3, apoptotic cell nucleus takes on a red color (grey parts in Fig. 3) fine and close dense dye, or the chunky shape that takes on a red color
Mutation occurs for fine and close dense dye, nuclear fractions.The more single medicine of Apoptosis situation of combination medicine is more serious.Fig. 4 is triterpene
Compound and gemcitabine are to apoptotic morphologic ratio after the processing of Pcan-1 cells.As can be seen from Figure 4, during single medicine, E12-1,
The apoptosis rate of E13-1 and gemcitabine cell is 25.4%, 22.4% and 24.3% respectively.When medication combined, apoptosis rate
Increase substantially, E12-1, E13-1 is combined with gemcitabine respectively, is as a result respectively increased to 44.3% and 48.2%.As a result say
Bright, E12-1 and E13-1 promote the effect of Apoptosis to be improved than the effect that single medicine is administered with gemcitabine drug combination respectively,
Apoptosis is set to add 21%-26%.Further illustrate, the effect of E12-1 and E13-1 respectively with gemcitabine drug combination
It is better than single medicine administration.
The Cell migration assay of embodiment 16
Pcan-1 cells are with inoculum density 4 × 106Individual/mL concentration is inoculated in diameter 35mm culture dishes (2mL/ culture dishes)
(6 orifice plates, 2mL/ holes), 5%CO2, 37 DEG C of culture 36-48h.When the cell fusion degree of observation culture dish reaches 90%-100%,
Rule with micro pipette tips or other hard things in the middle section of cell growth, remove the cell of middle body, addition is dissolved in
Compound and gemcitabine in DMSO, and administering drug combinations are carried out, after handling Pcan-1 cells, observation
When 0h, 5h and 24h, the healing state of cell.Specific method is：With the middle section of micro pipette tips cell growth in culture dish
Line, it is ensured that line position even thickness, twice of cell is washed with PBS, the cell and fraction floats cell of middle body is removed, changes
Continuation culture is carried out to cell into serum free medium, added according to the drug concentration set in culture medium.Added in medicine
Afterwards, when 0h, 5h and 24h, observe and take pictures, record cellular morphology and its migration situation.
Using cell scratch experiment, E12-1 and E13-1 can be determined respectively with gemcitabine medicine to Pcan-1 cells
Healing ability and the form of cell movement, experiment are contrasted by single medicine and drug combination, melt Pcan-1 cell growths
Close after 90%-100%, scratching is carried out to it.Fig. 5 and 6 be respectively triterpenoid E12-1 and E13-1 respectively with Ji
To 0h, 5h and 24h cell cut healing cell form microphotos after the processing of Pcan-1 cells after his shore joint of west.By Fig. 5 and
Fig. 6 passes through the Follow-up observations of different time sections, the form of cell and its healing, healing ability of the analysis medicine to cell.
As a result show, with the increase of cell growth time, the cell being scratched gradually recovers original form, and some are irreclaimable
Cell is constantly die, and the healing of cell constantly strengthens, and scratch area is tapered into.But the cell of drug combination group, more
Conjunction ability is substantially weaker than the healing ability that single medicine is administered.Fig. 7 be triterpenoid E12-1 and E13-1 respectively with gemcitabine pair
Triterpenoid and gemcitabine are to migration results after the processing of Pcan-1 cells after the processing of Pcan-1 cells.As can be seen from Figure 7, E12-
1 and E13-1 is significantly lower than single medicine administration after medication combined with gemcitabine to the healing ability of Pcan-1 cells respectively, and it heals
The more single medicine administration of ability reduces 20%-35% or so.I.e. the effect of drug combination more can make cell can not normal growth.
The Western blot of embodiment 17
Immunoblotting (Western blot), is to shift the cell or tissue gross protein after electrophoretic separation from gel
Onto solid support NC films or pvdf membrane, then with a kind of protein detection skill of detection of specific antibody specific antigen
Art, has been widely used in gene in multiple sides such as the expression study of protein level, antibody activity detection and disease early diagnosis
Face.
1. cell holoprotein is extracted：Pcan-1 cells are with 1 × 105Individual/mL density is seeded in 100mm culture dishes, and 37
DEG C culture 24h.Then the compound and gemcitabine being dissolved in DMSO are added, and is carried out administering drug combinations processing
24h, DMSO are control treatment group.Then inhale and abandon all solution, after the PBS cell of ice bath, then add 200 μ
LRIPA lysis buffers, complete cell lysis is scraped down from culture dish attached cell using cell scraper, collects thin
Born of the same parents' suspension, being blown and beaten repeatedly using syringe needle makes cell crack completely, in 4 DEG C of centrifugations, takes supernatant stand-by.
2. determination of protein concentration：20 μ L reaction solutions S are added in 1mL reaction solutions A and prepare reaction solution A '；Ox is prepared with PBS
Seralbumin BSA 16mg/mL, and it is molten to be diluted with water to 0.1,0.2,0.4,0.8,1.6mg/mL series BSA standards successively
Liquid；5 μ L protein samples (if protein concentration is too high, suitably dilute) and BSA standard liquids are respectively taken in 96 orifice plates, successively
25 μ L reaction solutions A ', 200 μ L reaction solution B are added, are mixed, rocked at room temperature reaction 15min；Absorbance is read at wavelength 650nm
Value, draws standard curve.Testing protein sample concentration is calculated according to standard curve.
The μ L of supernatant 5 are taken to determine luminous with Sirius single hoses chemiluminescence detector immediately after being mixed with 25 μ L substrate
Intensity.Each sample sets 3 multiple holes, determines 5 times, averages per hole.Uciferase activity luminous intensity and albumen are dense
The ratio of degree represents, and is scaled the percentage of blank control group (DMSO processing) and represents.
3.SDS-PAGE electrophoresis：The above-mentioned μ g of protein sample 30 are taken, 2 × protein electrophoresis sample buffer, boiling water bath is added
5min, is then centrifuged for 30s, puts standby on ice；By specification installs electrophoretic apparatus；Take out Minin-protean TGT pre-prepared colloids
Comb, cleans well, is put into electrophoresis tank, adds Tris- glycine running buffers；Sample is carefully added into loading hole
And standard protein molecular weight Marker；Switch on power, 100V, 110min；Electrophoresis to sample-loading buffer dyestuff reaches gel front
At about 1cm, stop electrophoresis.
4. western blot test：
(1) transferring film：After electrophoresis terminates, appropriately sized pvdf membrane and 2 filter paper is intercepted, pvdf membrane is first soaked in methyl alcohol
Moisten 10s, 2min is then infiltrated in deionized water, then be together dipped in filter paper in transfering buffering liquid, balance 10-15min, simultaneously
Gel is dipped in appropriate transfering buffering liquid, installs clip according to device specification, in order：Sponge-filter paper-glue-film-filter
Paper-sponge, after every layer is put well, bubble of being rushed with test tube forms sandwich structure.Transfer groove is placed in ice bath, Sanming City is put into
Structure, plus transfering buffering liquid are controlled, electrode, constant current 90V transferring films 90min is plugged.
(2) close：The film for turning to have albumen is dipped in 5%BSA confining liquids and closes 1h.With containing 0.05%Tween-20's
Tris buffer salt solutions (TBST) wash film 3 times, each 5mim.
(3) hybridize：The film closed is taken out, is subsequently placed in corresponding primary antibody solution, 4 DEG C overnight.Actin is used as sample-adding
Reference protein.Film is washed with TBST 4 times, each 15mim.The general anti-igg secondary antibody (1 of HRP marks is placed in again：5000) dilute) it is molten
In liquid, room temperature lucifuge shaking l h.Film is washed with TBST 4 times, each 15min.The SuperSignal West Femto reagents used
Box carries out chemiluminescence detection, and Quantity One systems are scanned.With the softwares of Image Studio Lite Ver 4.0
Analyzed.
There are some researches show the signal paths of STAT3 and NF- κ B two are interconnected, they can adjust jointly some with it is swollen
Knurl and the related gene expression of inflammation, such as NF- κ B, IL-6, P-State3 and Bcl-2.Therefore, the present invention passes through Western
Blot methods are to 3 α, and 11 alpha-dihydroxys-lupin -20 (29)-alkene -28- is sour, 3 Alpha-hydroxies-lupin -20 (29)-alkene -23,
The level of related apoptosis albumen is examined in Pcan-1 cells after 28- diacid and gemcitabine and respectively drug combination effect
Survey.Fig. 8 is triterpenoid E12-1 and E13-1 respectively with gemcitabine to associated signal paths and apoptosis in Pcan-1 cells
The influence of protein expression.As a result show, for Bcl-2 antibody, drug combination effect is relatively better than single medicine administration, especially 3 α, 11
Alpha-dihydroxy-lupin -20 (29)-alkene -28- acid, colour band color is substantially shallower, i.e. the obvious reduction of Bcl-2 expression.IL-6 resists
Body, single medicine expression is higher, and gemcitabine expresses relatively low relative to triterpene compound, and E12-1 and E13-1 respectively with Ji Xi
The more single medicine of IL-6 expression is lower after his shore combination.NF- kappa B antibodies, E12-1 and E13-1 respectively with gemcitabine drug combination effect
Relatively it is better than single medicine administration, the obvious reduction of especially 3 Alpha-hydroxies-lupin -20 (29)-alkene -23,28- diacid, i.e. NF- κ B expression.
P-state3 antibody, clearly, drug combination effect is significantly stronger than single medicine administration, illustrates after drug combination for the change of colour band,
The reduction of P-state3 antibody expressions is a lot.3 α, 11 alpha-dihydroxys-lupin -20 (29)-alkene -28- acid, 3 Alpha-hydroxies-lupin -
20 (29)-alkene -23,28- diacid is stronger with gemcitabine drug combination inhibitory action respectively, IL-6 expression reductions, P-tate3 tables
Up to also reducing.Illustrate that the drug combination of E12-1 and E13-1 respectively with gemcitabine suppresses the possible letter of growth of Pcan-1 cells
Number path.Exist between side light NF- κ B and P-state3 and influence each other, there is interaction between this two bars conduction path
Relation, drug combination can influence this two bars conduction path.
The statistical analysis of embodiment 18
Variance analysis is carried out to experimental result, as a result represented with means ± SD form.Between sample treatment group and control group
Data variance analysis is examined using Student's t and carried out.P<0.05 (*) and P<0.001 (* *) represents difference and shown respectively
Write and difference is extremely notable.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, is combined and simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of pharmaceutical composition of collaboration anti-pancreatic cancer, the medicine acceptable auxiliary material comprising active component and pharmaceutically,
Characterized in that, described active component includes gemcitabine and triterpenoid, described triterpenoid is 3 α, 11 α-two
Hydroxyl-lupin -20 (29)-alkene -28- acid or 3 Alpha-hydroxies-lupin -20 (29)-alkene -23,28- diacid.
2. the pharmaceutical composition of anti-pancreatic cancer is cooperateed with according to claim 1, it is characterised in that described triterpenoid
Mol ratio with gemcitabine is (100~4550): (1~10).
3. cooperate with the pharmaceutical composition of cancer of pancreas according to claim 2, it is characterised in that described triterpenoid and
The mol ratio of gemcitabine is (300~1500): (1.5~8).
4. the pharmaceutical composition of anti-pancreatic cancer is cooperateed with according to claim 3, it is characterised in that described triterpenoid
Mol ratio with gemcitabine is 170: 1.
5. according to the pharmaceutical composition of all collaboration anti-pancreatic cancers in claim 1, it is characterised in that described anti-prostate cancer
Pharmaceutical composition be oral formulations.
6. according to the pharmaceutical composition of all collaboration anti-pancreatic cancers in claim 5, it is characterised in that described oral formulations are
Capsule, tablet, granule or liquor.
7. application of any one of the claim 1-6 described pharmaceutical composition in inhibiting tumor cell medicine is prepared.
8. application according to claim 7, it is characterised in that the cancer cell is pancreatic cancer cell.
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| CN102600077A (en) * | 2012-03-29 | 2012-07-25 | 江苏豪森药业股份有限公司 | Gemcitabine or gemcitabine salt nano-emulsion injecta and preparation method thereof |
| CN104922173A (en) * | 2015-05-21 | 2015-09-23 | 五邑大学 | Application of radix acanthopanacis trifoliate extract in inhibiting alpha-glucosidase |
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| CN102600077A (en) * | 2012-03-29 | 2012-07-25 | 江苏豪森药业股份有限公司 | Gemcitabine or gemcitabine salt nano-emulsion injecta and preparation method thereof |
| CN104922173A (en) * | 2015-05-21 | 2015-09-23 | 五邑大学 | Application of radix acanthopanacis trifoliate extract in inhibiting alpha-glucosidase |
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| CN110742886A (en) * | 2019-10-14 | 2020-02-04 | 五邑大学 | Application of jatrorrhizine hydrochloride in reversing drug resistance of pancreatic cancer cells |
| CN110742886B (en) * | 2019-10-14 | 2022-06-07 | 五邑大学 | Application of jatrorrhizine hydrochloride in reversing drug resistance of pancreatic cancer cells |
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