CN105726726A - Combined application of extract of traditional Chinese medicaments coptischinensis and evodia rutaecarpa and 5-fluorouracil to preparation of medicament for treating intestinal cancer - Google Patents
Combined application of extract of traditional Chinese medicaments coptischinensis and evodia rutaecarpa and 5-fluorouracil to preparation of medicament for treating intestinal cancer Download PDFInfo
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- CN105726726A CN105726726A CN201610122172.5A CN201610122172A CN105726726A CN 105726726 A CN105726726 A CN 105726726A CN 201610122172 A CN201610122172 A CN 201610122172A CN 105726726 A CN105726726 A CN 105726726A
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- zuojin
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- 239000003814 drug Substances 0.000 title claims abstract description 49
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- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
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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)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
- A61K36/718—Coptis (goldthread)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- 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)
- A61K36/75—Rutaceae (Rue family)
- A61K36/754—Evodia
-
- 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
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides combined use of extract (Zuojin Prescription) of traditional Chinese medicaments coptischinensis and evodia rutaecarpa and 5-fluorouracil to preparation of a medicament for treating intestinal cancer. The Zuojin Prescription is combined with the 5-fluorouracil to remarkably increase the survival rate of cells, greatly reduce the toxic or side effects of the 5-fluorouracil, promote apoptosis of intestinal cancer cells HT-29 by promoting high expression of Bax proteins in the cells and low expression of Bcl-2 and Survivin proteins, and achieve curative effects better than those achieved by independent use of the Zuojin Prescription or the 5-fluorouracil on intestinal cancer.
Description
Technical field
The present invention relates to the new application of Chinese medicine compound Zuojin prescription and chemotherapy drugs in combination treatment.
Background technology
Colorectal cancer is one of malignant tumor common in digestive system, including colon cancer and rectal cancer, and the whole world every year
There are about 1,200,000 patients to be diagnosed as colorectal cancer, and have more than 600,000 patients and directly or indirectly die from knot directly
Intestinal cancer, its M & M lays respectively at the 3rd and the 4th.The sickness rate of male's colorectal cancer is higher than
Women, colorectal cancer incidence rate age bracket below 40 years old is relatively low, but can increase along with the increase at age.Greatly
Intestinal cancer be China most common be also one of malignant tumor the most of greatest concern.From the nineties, developed country
Colorectal cancer incidence rate start to decline year by year, but the developing Asian countries such as China rise the most year by year, man
In property patient, this epidemiologic feature becomes apparent from, and the healthy of the mankind in serious threat.
Operation is considered as the Main Means treating colon cancer at present, but due to colon cancer morbidity disguise and examine
The limitation for the treatment of means, most colon cancer have been enter into colon cancer morbidity middle and advanced stage, are unsuitable for operative treatment when finding.
Typically take intestinal cancer based on chemotherapy clinically at present, radiotherapy, biological immune treatment etc. are auxiliary treatment means.
But chemotherapy has huge untoward reaction, causes acute liver damage and kidney injury etc., greatly limit chemotherapeutics and use
Amount, hinders the performance of efficacy of drugs, and radiotherapy in the treatment is easily caused radioactivity brain, spinal cord injury, radioactivity brain in addition
The complication such as nerve injury, the damage of radioactivity salivary gland and radioactive rear induction cancer.
Therefore, Chinese medicine combines chemotherapeutic treatment middle and advanced stage intestinal cancer and increasingly shows advantage.Chinese medicine is to colorectal cancer
Therapy study has been directed to the fields such as tumorigenesis, death, aging, plays in treatment of colorectal cancer
Positive effect.A large amount of medical practices prove: the auxiliary treatment after Chinese medicine coordinates colorectal neoplasm operation excision (is changed
, targeting, gene, radiation, endocrine, biotherapy etc.), reduction is recurred, alleviates toxicity, prolonged
Long patient life cycle, improve patients ' life quality and have obvious advantage. additionally, the advantage of lower cost of Chinese medicine,
Practicality is wide.But Traditional Chinese Medicine Anti intestinal cancer preparation is dull, and uncertain therapeutic efficacy is cut, and there is no at present and has extensive therapeutical effect
Clinical preparation come out, most of Chinese medicine preparation for auxiliary medicine for treatment, its clinical practice lack specification, and should
Undesirable by effect, seriously constrain extensively applying and obtaining recognizing of international community of anti-intestinal cancer Chinese medicine and compound recipe thereof
Can.
Zuojin prescription (ZoujinPerscription) comes from " danxi's experiential therapy ", by monarch drug Rhizoma Coptidis (Coptischinensis Fr)
6:1 part by weight composition is pressed, by each medicine with adjuvant Fructus Evodiae (Evodiarutaecarp (Juss.) benth.)
Allusion quotation is recorded, and its property of medicine is the coldest, cures mainly pathogenic fire derived from stagnation of liver-QI, stomach-QI being unable to descend normally, hypochondriac pain gastral cavity painful abdominal mass, vomiting acid regurgitation, noisy seemingly
Hungry, wiry and frequent pulse and the bitter taste tongue relatively stomach-fire such as red are more notably.Modern medical knowledge research shows that the protected stomach of Zuojin prescription glues
Film, gastric acid secretion inhibiting, antiulcer, affect the effect of gastrointestinal motility.Clinic is usually used in acute or chronic gastritis at present
And peptic ulcer.Recently, research proves that Zuojin prescription can induce the apoptosis of In vitro culture colon-cancer cell HT-29,
It reduces the expression of Bcl-2 and Survivin albumen by raising the expression of the Bax albumen in tumor tissues, aobvious
Write the growth inhibiting people's intestinal cancer Nude Mice.
Chemicotherapy based on preoperative fluorouracil (5-FU) is the standard care of Locally Advanced rectal cancer at present.
5-FU is radiotherapeutic sensitizer, but the best to the therapeutic effect having metastasis patient, can band serve such as anus and
The problem of sexual function damage, and can not make extend further life cycle.So, owing to 5-FU has biological utilisation
The shortcomings such as spending low, little to tumor cells selectivity, toxic and side effects is big, and therapeutic dose is close with toxic dose, greatly
Limit greatly its clinical practice.
Chinese patent application 101785807A discloses the Chinese Drug Rhizomes of Coptis and the extraction of Fructus Evodiae that mass ratio is 6:1
Thing application in preparation treatment gastric cancer medicament, and Chinese medicine compound Zuojin prescription is in preparation treatment gastric cancer medicament
Application.
Summary of the invention
It is an object of the invention to provide Chinese Drug Rhizomes of Coptis to combine with 5-FU (5-fluorouracil) with the extract of Fructus Evodiae
Purposes in preparation treatment bowelcancer medicine.
According to an aspect of the present invention, Chinese Drug Rhizomes of Coptis is combined with 5-FU (5-fluorouracil) with the extract of Fructus Evodiae
For treating intestinal cancer, preferably by extract and the Chemo-Therapy of Chinese Drug Rhizomes of Coptis that mass ratio is 6:1 and Fructus Evodiae
Treat agent 5-fluorouracil use in conjunction, more preferably carry described in the preparation method preparation of Chinese medicine compound Zuojin prescription
Taking thing, therefore compound Zuojin prescription and the Zuojin Wan prepared according to this compound recipe are all contained in the scope of the invention, this
Outward, it will be appreciated by persons skilled in the art that prescription based on Zuojin prescription, add other auxiliary medical material systems
Standby compound medicine is also contained in the present invention.In the treatment of intestinal cancer, described combine refer to be administered simultaneously or
Sequential administration, i.e. prior to or while giving patient's extract or give 5-FU afterwards.
The present invention is proved by cells in vitro test, the extract of Chinese Drug Rhizomes of Coptis and Fructus Evodiae and 5-FU drug combination
More independent than extract, or the independent medication of 5-FU is more preferable to the therapeutic effect of intestinal cancer.Chinese Drug Rhizomes of Coptis and Fructus Evodiae
Extract and 5-FU drug combination, significantly improve the survival of FHC people's Normal Colon mucous membrane of rectum epithelial cell
Rate, significantly reduces the cytotoxic effect of 5-FU, by promoting the high expressed of Bax albumen in HT-29 cell
With the low expression of Bcl-2, Survivin albumen, promote the apoptosis of people's colon-cancer cell.The achievement in research pair of the present invention
Reduce the toxic and side effects of clinical anti-intestinal cancer chemotherapeutics, improve therapeutic effect, be significant.
Accompanying drawing explanation
Fig. 1 is the CCK-8 test knot that Zuojin prescription of the present invention acts on FHC people's colorectal mucous membrane epithelial cell
Really, display extract concentrations, action time and the relation of FHC survival rate；
Fig. 2 is the CCK-8 test knot that 5-FU of the present invention acts on FHC people's colorectal mucous membrane epithelial cell
Really, display 5-FU concentration, action time and the relation of FHC survival rate；
Fig. 3 is the flow cytomery result that Zuojin prescription of the present invention acts on colon cancer cell HT-29, display
The relation of extract concentrations, action time and HT-29 apoptosis rate；
Fig. 4 is the flow cytomery result that 5-FU of the present invention acts on colon cancer cell HT-29, display
The relation of 5-FU concentration, action time and HT-29 apoptosis rate；
Fig. 5 is blank, 1.00 μ g/mL Zuojin prescriptions, 50.0 μ g/mL 5-FU and 1.00 μ g/mL Zuojin prescriptions
Combine 50.0 μ g/mL 5-FU and act on the CCK-8 result of the test of FHC cell 24h respectively, show certain concentration
Extract, 5-FU, extract associating 5-FU Yu FHC survival rate relation；
Fig. 6 is blank, 1.00 μ g/mL Zuojin prescriptions, 100.0 μ g/mL 5-FU and the 1.00 left gold of μ g/mL
Fang Lianhe 100.0 μ g/mL 5-FU acts on the CCK-8 result of the test of FHC cell 24h respectively, shows specific
The relation of the extract of concentration, 5-FU, extract associating 5-FU Yu FHC survival rate；
Fig. 7 is blank, 1.00 μ g/mL extracts, 50.0 μ g/mL 5-FU, 1.00 μ g/mL extracts
Combine 50.0 μ g/mL 5-FU and act on the flow cytomery result of HT-29 cell respectively, show certain concentration
Extract, 5-FU, extract associating 5-FU Yu HT-29 apoptosis rate relation；
Fig. 8 is blank, 1.00 μ g/mL extracts, 100.0 μ g/mL 5-FU, and 1.00 μ g/mL extract
Internet of Things is closed 100.0 μ g/mL 5-FU and is acted on the flow cytomery result of HT-29 cell respectively, shows specific
The relation of the extract of concentration, 5-FU, extract associating 5-FU Yu HT-29 apoptosis rate；
Fig. 9 is blank, 1.00 μ g/mL Zuojin prescriptions, 100.0 μ g/mL 5-FU, the 1.00 left gold of μ g/mL
Fang Lianhe 100.0 μ g/mL 5-FU acts on HT-29 cell 24h respectively, in regulation cell BAX, Bcl-2 and
Western Blotting gel electrophoresis figure (n=6) of Survivin protein expression；
Figure 10 is blank, 1.00 μ g/mL extracts, 50.0 μ g/mL 5-FU, 100.00 μ g/mL 5-FU,
1.00 μ g/mL extracts combine 50.0 μ g/mL 5-FU and 1.00 μ g/mL extracts combine 100.0 μ g/mL
5-FU acts on morphocytology microscope observation figure (× 200) of HT-29 cell respectively.
Detailed description of the invention
One, test material
Cell strain: FHC cell (people's Normal Colon mucous membrane of rectum epithelial cell), HT-29 cell (human colon carcinoma
Cell) purchased from Zhongshan University's Animal Experimental cell centre.With containing the FBS's that volume fraction is 10%
DMEM complete medium, in 37 DEG C, in the CO2 incubator of 5% cultivate within 3 days, once pass on, pass on by
1:3 is carried out.Take trophophase cell to test.
Chinese Drug Rhizomes of Coptis and the extract (calling extract or Zuojin prescription in the following text) of Fructus Evodiae: Chinese Drug Rhizomes of Coptis (Coptis
Chinensis Fr) with Fructus Evodiae (Evodiarutaecarp (Juss.) benth.) be commercially available purchase, by 6:1 matter
Amount mixing, makes water extract through decocting, concentration and lyophilization.Crude drug, after water carries, obtains water extract 260
(mg)/crude drug (g).
Zuojin prescription traditional decoction lyophilized powder: provided by this laboratory, takes 30g Rhizoma Coptidis and 5g Fructus Evodiae, adds 8 times
Distilled water immersion 30min, boiling 3 times, each 30min, filter, merge 3 filtrates, lyophilization,
Every gram of dry powder 3.98g Han crude drug；Measure through HPLC method, containing berberine 39.85% in Zuojin prescription lyophilized powder, bar horse
Spit of fland 9.19%, jateorhizine 4.86%, rutaecarpin 0.17%, rutaecarpine 0.19%.
It is prepared as the method for the Zuojin prescription of administration concentration in test: be dissolved in deionized water by Zuojin prescription lyophilized powder,
It is configured to the mother solution of 1mg/mL, during experiment, is diluted to desired concn with cell culture fluid.
5-FU: purchased from Tianjin KingYork Amino Acid Co., Ltd., specification: 10ml:0.25g, lot number: 1412011.
It is prepared as the method for the 5-FU of administration concentration in test: take appropriate 5-fluorouracil injection stock solution and add cell
Culture fluid is configured to 1mg/ml mother solution, is diluted to desired concn with cell culture fluid during experiment.
Two, detection method and data statistical approach
1, CCK-8 detects cytoactive
CCK-8 detection kit is used to observe Zuojin prescription or 5-FU or Zuojin prescription associating at the appointed time
The impact of the 5-FU survival rate on cultivating FHC cell, wherein by the survival rate of undressed cellular control unit
It is set as 100%.
2, flow cytomery apoptosis rate
Adjusting HT-29 cell density is 2 × 105/ mL, enters 6 orifice plates with 1.5mL/well kind.The often multiple hole of group 6.
After medicine effect 24h, cytology brush scraping cells, 800r min-1 is centrifuged 5min, removes supernatant, adds PBS weight
Outstanding, adjusting cell concentration is 1 × 106/mL.Take appropriate in streaming pipe, add working solution 500uL, add
Fluorescent dye, 350 mesh nylon wire membrane filtrations remove cell mass, after room temperature lucifuge dyeing 15min, up flow type
(excitation wavelength 488nm, with the passband filter inspection that a wavelength is 515nm for cell instrument detection apoptosis rate
Survey FITC fluorescence, another wavelength filter detection PI more than 560nm).Determination data is joined by flow cytometer
The software put carries out data process, the middle and late stage apoptosis rate that the early apoptosis rate represented with bottom right represents plus upper right
Summation represent natural death of cerebral cells rate.
3, morphological observation
By HT-29 cell (2 × 105/ mL) it is inoculated on the coverslip in 6 orifice plates, each experimental group is given on request
After giving different process factors, add freshly prepared 4% paraformaldehyde (pH is 7.4) fixing thin in 37 DEG C
Born of the same parents 30min, PBS liquid is washed twice, adds 5mg/L Hochest 33258 dyeing liquor dyeing 30min, PBS liquid
After washing, with mounting liquid (20mmol/L citric acid, 50mmol/L disodium hydrogen phosphate, 50% glycerol, pH 5.5)
Fluorescence microscope after mounting, take the photograph sheet.
4, statistical procedures
All of the above data all use SPSS10.0 statistical software to carry out statistical analysis, data mean ± standard deviationRepresent, compare between group with one factor analysis of variance (One-way ANOVA) inspection.
[embodiment 1] Zuojin prescription or the 5-FU independent medication impact on FHC cell survival rate
The impact on FHC cell survival rate of 1.1 Zuojin prescriptions
Take people's Normal Colon mucous membrane of rectum epithelial cell FHC exponential phase cell, by DMEM culture medium system
Become cell suspension (5 × 104/mL) to add 96 orifice plates, 100 μ L/ holes, be administered after cultivating 12h, experimental group
Being separately added into variable concentrations for reagent thing, cultivate 24 respectively, 48h, the CCK-8 adding 10 μ L to every hole is molten
Liquid, hatches culture plate in incubator 4 hours, measures the absorbance (OD) at 450nm by microplate reader.
Cell inhibitory rate=(1-experimental group mean OD value/matched group mean OD value) × 100%.
CCK-8 experimental result is as shown in table 1.Table 1 lists the Zuojin prescription of variable concentrations, and to act on FHC thin
The survival rate (n=6) of FHC cell after born of the same parents 24h and 48h.
The impact (n=6) on FHC cell survival rate of the Zuojin prescription effect different time of table 1 variable concentrations
*: p < 0.05 compared with matched group；*: p < 0.01 compared with matched group；
Fig. 1 is the curve chart of table 1, can find out Zuojin prescription and FHC cell survival rate more intuitively from Fig. 1
Relation.
CCK-8 experimental result shows: 1) Zuojin prescription is the least on the impact of FHC cell viability, i.e. works as administration concentration
After increasing to a certain degree, along with the increase of administration concentration, the prolongation of action time, cell viability slightly weakens；2)
Low dose of Zuojin prescription can promote propagation and the division of people's Normal Colon mucous membrane of rectum epithelial cell.
1.2 5-FU impact on FHC cell survival rate
Take people's Normal Colon rectal mucosa epithelial cell FHC exponential phase cell, by DMEM culture medium system
Become cell suspension (5 × 104/mL) to add 96 orifice plates, 100 μ L/ holes, be administered after cultivating 12h, experimental group
Being separately added into variable concentrations for reagent thing, cultivate 24 respectively, 48h, the CCK-8 adding 10 μ L to every hole is molten
Liquid, hatches culture plate in incubator 4 hours, measures the absorbance (OD) at 450nm by microplate reader.
Wherein the survival rate of undressed cellular control unit is set as 100%.Cell inhibitory rate=(1-experimental group is put down
All OD value/matched group mean OD value) × 100%.
CCK-8 experimental result is as shown in table 2.Table 2 lists the 5-FU of variable concentrations and acts on FHC cell
The survival rate (n=6) of FHC cell after 24h and 48h.
The impact (n=6) on FHC cell survival rate of the table 2 variable concentrations 5-FU effect different time.
*: p < 0.05 compared with matched group；*: p < 0.01 compared with matched group
Fig. 2 is the curve chart of table 2, can find out 5-FU Yu FHC cell survival rate more intuitively from Fig. 2
Relation (n=6).
Knowable to result of the test: 1) impact of the vigor of FHC cell presents " Concentration-time dependency " by 5-FU
Relation, i.e. along with the increase of administration concentration, the prolongation of action time, cell viability gradually weakens；2) with left
Gold side is different, and low dose of 5-FU can cause cytotoxicity, and along with Drug level increases, its toxicity also strengthens.
[embodiment 2] Zuojin prescription or the 5-FU independent medication impact on HT-29 apoptosis rate
The impact on HT-29 apoptosis rate of 2.1 Zuojin prescriptions
Taking HT-29 exponential phase cell, adjusting cell density by DMEM culture medium is 2 × 105/ mL, with
1.5mL/well kind enters 6 orifice plates.Often the multiple hole of group 6, cultivates 12h, is subsequently adding corresponding medicine effect 24h,
Cytology brush scraping cells, 800r min-1 is centrifuged 5min, removes supernatant, and PBS is resuspended in addition, adjusts cell concentration
It is 1 × 106/mL.Take appropriate in streaming pipe, add working solution 500uL, add fluorescent dye, 350 mesh Buddhist nuns
Dragon net membrane filtration removes cell mass, after room temperature lucifuge dyeing 15min, and flow cytometer detection apoptosis
Rate.The software that determination data is configured by flow cytometer carries out data process, the early apoptosis represented with bottom right
Rate represents natural death of cerebral cells rate plus the summation of the middle and late stage apoptosis rate that upper right represents.
Result of the test is as shown in table 3.Table 3 lists the Zuojin prescription of variable concentrations and acts on HT-29 cell 24h
After, the apoptosis rate (n=6) of HT-29 cell.
The table 3 variable concentrations Zuojin prescription effect 24h impact (n=6) on HT-29 apoptosis rate
*: p < 0.05 compared with matched group；*: p < 0.01 compared with matched group
Fig. 3 is the curve chart of table 3, can find out 5-FU Yu HT-29 apoptosis rate more intuitively from Fig. 3
Relation (n=6).
Knowable to result of the test: 1) impact of HT-29 apoptosis rate presents " Concentration-time dependency " by Zuojin prescription
Relation, i.e. along with the increase of administration concentration, the prolongation of action time, apoptosis rate is gradually increased；2) little
The Zuojin prescription of dosage just can promote the apoptosis of HT-29 cell.
2.2 5-FU impact on HT-29 apoptosis rate
Taking the logarithm trophophase cell, adjusting cell density by DMEM culture medium is 2 × 105/ mL, with 1.5mL/well
Plant 6 orifice plates.Often the multiple hole of group 6, cultivates 12h, is subsequently adding corresponding medicine effect 24h, cell brushing
Taking cell, 800r min-1 is centrifuged 5min, removes supernatant, and PBS is resuspended in addition, and adjusting cell concentration is 1 × 106/mL。
Take appropriate in streaming pipe, add working solution 500uL, add fluorescent dye, 350 mesh nylon wire membrane filtrations
Remove cell mass, after room temperature lucifuge dyeing 15min, flow cytometer detection apoptosis rate.Determination data
The software configured by flow cytometer carries out data process, adds upper right generation with the early apoptosis rate that bottom right represents
The summation of the middle and late stage apoptosis rate of table represents natural death of cerebral cells rate.
Result of the test is as shown in table 4.Table 4 lists after the 5-FU of variable concentrations acts on HT-29 cell 24h
The apoptosis rate (n=6) of HT-29 cell.
The 5-FU effect 24h of the table 4 variable concentrations impact (n=6) on HT-29 apoptosis rate
*: p < 0.05 compared with matched group；*: p < 0.01 compared with matched group
Fig. 4 is the curve chart of table 4, can find out 5-FU Yu HT-29 apoptosis rate more intuitively from Fig. 4
Relation.
Knowable to result of the test: 1) impact of HT-29 apoptosis rate presents " Concentration-time dependency " by 5-FU
Relation, i.e. along with the increase of administration concentration, the prolongation of action time, apoptosis rate is gradually increased；2)
50.00ug·mL-1With 100.00ug mL-1Apoptosis rate after 5-FU effect HT-29 cell 24h is respectively
(50.40 ± 1.45) % and (65.30 ± 1.27) %.
[embodiment 3] Zuojin prescription and 5-FU drug combination
Selecting of 3.1 drug combination concentration
When Zuojin prescription concentration is 1.00ug mL-1, acting on FHC cell 24h, cell survival rate is
(106.68 ± 1.65) %, 48h is (100.16 ± 1.23) %, acts on HT-29 cell 24h, apoptosis rate
For (37.90 ± 1.06) %, selected 1.00ug mL-1Concentration for Zuojin prescription drug combination.
Selecting of 3.2 5-FU drug combination concentration
When 5-FU concentration is 50.00ug mL-1, acting on FHC cell 24h, cell survival rate is (47.76 ± 1.48)
%, 48h are (37.69 ± 1.62) %, act on HT-29 cell 24h, and apoptosis rate is (50.40 ± 1.45)
%.
When 5-FU concentration is 100.00ug mL-1, acting on FHC cell 24h, cell survival rate is
(42.42 ± 1.74) %, 48h is (32.79 ± 1.95) %, acts on HT-29 cell 24h, and apoptosis rate is
(65.30 ± 1.27) %, selected 50.00,100.00ug mL-1 are the concentration of 5-FU drug combination.
3.3 Zuojin prescriptions and the impact on FHC cell survival rate of the 5-FU drug combination
Take people's Normal Colon rectal mucosa epithelial cell FHC exponential phase cell, by DMEM culture medium system
Become cell suspension (5 × 104/mL) to add 96 orifice plates, 100 μ L/ holes, be administered after cultivating 12h, experimental group
Being separately added into variable concentrations for reagent thing, cultivate 24 respectively, 48h, the CCK-8 adding 10 μ L to every hole is molten
Liquid, hatches culture plate in incubator 4 hours, measures the absorbance (OD) at 450nm by microplate reader.
Cell inhibitory rate=(1-experimental group mean OD value/matched group mean OD value) × 100%.
Result of the test is as shown in table 5, table 6.Table 5, table 6 list matched group, 1.00ug mL-1Zuojin prescription,
50.00ug·mL-1After 5-FU and the two drug combination are respectively acting on FHC cell 24h and 48h, FHC is thin
The survival rate (n=6) of born of the same parents.
Table 5 Zuojin prescription and the impact (n=6) on FHC cell survival rate of the 5-FU drug combination effect different time
*: drug combination group and Zuojin prescription 1.00ug mL-1Group compares p < 0.05；#: drug combination group and 5-FU 50.00ug mL-1Group phase
Than p < 0.05
Fig. 5 is the curve chart of table 5, can find out Zuojin prescription and 50.00ug mL more intuitively from Fig. 5-15-FU
Drug combination 24h and the relation of FHC cell survival rate.
Table 6 lists matched group, 1.00ug mL-1 Zuojin prescription, 100.00ug mL-15-FU and the two associating
Medication is respectively acting on the survival rate (n=6) of FHC cell after FHC cell 24h and 48h.
Table 6 Zuojin prescription and the impact (n=6) on FHC cell survival rate of the 5-FU drug combination effect different time
*: drug combination group and Zuojin prescription 1.00ug mL-1Group compares p < 0.05；#: drug combination group and 5-FU 100.00ug mL-1Group phase
Than p < 0.05
Fig. 6 is the curve chart of table 6, can find out Zuojin prescription and 100.00ug mL more intuitively from Fig. 6-15-FU
Drug combination 24h and the relation of FHC cell survival rate.
Knowable to result of the test: Zuojin prescription and 5-FU drug combination can be significantly increased people's Normal Colon rectum and stick
The cell survival rate of film epithelial cell FHC.Compared with the medication independent with Zuojin prescription and 5-FU of drug combination group
P < 0.05, has significant significant difference, the i.e. cytoprotection of drug combination group to be better than each independent medication group.
3.4 Zuojin prescriptions and the impact on HT-29 apoptosis rate of the 5-FU drug combination
Taking the logarithm trophophase cell, adjusting cell density by DMEM culture medium is 2 × 105/ mL, with 1.5mL/well
Plant 6 orifice plates.Often the multiple hole of group 6, cultivates 12h, is subsequently adding corresponding medicine effect 24h, cell brushing
Taking cell, 800r min-1 is centrifuged 5min, removes supernatant, and PBS is resuspended in addition, and adjusting cell concentration is 1 × 106/mL。
Take appropriate in streaming pipe, add working solution 500uL, add fluorescent dye, 350 mesh nylon wire membrane filtrations
Remove cell mass, after room temperature lucifuge dyeing 15min, flow cytometer detection apoptosis rate.Determination data
The software configured by flow cytometer carries out data process, adds upper right generation with the early apoptosis rate that bottom right represents
The summation of the middle and late stage apoptosis rate of table represents natural death of cerebral cells rate.
Result of the test is as shown in table 7, table 8.Table 7, table 8 list matched group, the Zuojin prescription of selected concentration,
The 5-FU of selected concentration, and the Zuojin prescription of selected concentration combines the 5-FU of selected concentration, and to act on HT-29 thin
After born of the same parents 24h, the apoptosis rate (n=6) of HT-29 cell.
Table 7 Zuojin prescription and the 5-FU drug combination effect impact on HT-29 apoptosis rate in 24 hours (n=6)
*: drug combination group and Zuojin prescription 1.00ug mL-1Group compares p < 0.05；#: drug combination group and 5-FU 50.00ug mL-1Group phase
Than p < 0.05
Fig. 7 is the curve chart of table 7, from Fig. 7 can find out more intuitively Zuojin prescription and 5-FU drug combination with
The relation of HT-29 apoptosis rate.
Table 8 Zuojin prescription and the 5-FU drug combination effect impact on HT-29 apoptosis rate in 24 hours (n=6)
*: drug combination group and Zuojin prescription 1.00ug mL-1Group compares p < 0.05；#: drug combination group and 5-FU 100.00ug mL-1Group phase
Than p < 0.05
Fig. 8 is the curve chart of table 8, from Fig. 8 can find out more intuitively Zuojin prescription and 5-FU drug combination with
The relation of HT-29 apoptosis rate.
Can be seen that from result of the test, 1.00ug mL-1Zuojin prescription respectively with 50.00ug mL-1、100.00ug·mL-1
After 5-FU drug combination acts on HT-29 cell 24h, apoptosis rate increases substantially, drug combination group with
P < 0.05 is compared in Zuojin prescription medication independent with 5-FU, has significant significant difference, i.e. drug combination group induction to wither
The effect died is better than each independent medication group.
3.5 Zuojin prescriptions and the impact on Bcl-2, Bax and Survivin albumen of the 5-FU drug combination
Extract albumen: PMSF 10 μ L is joined 1mL cell pyrolysis liquid RIPA, thin after medicine effect of learning from else's experience
Born of the same parents about 2.4 × 106This RIPA of individual addition, after cracking 30min on ice, 10 000r min-1Centrifugal 10min, takes
Clearly.
BCA measures protein concentration, and it is A562 that microplate reader measures wavelength, calculates protein concentration according to standard curve.
Recording 12% separation gel, 4% concentrates the PAGE gel of glue.Draw 80 μ g total proteins, constant voltage 100V electrophoresis
Until bromophenol blue arrives bottom separation gel.Electricity of wet process turns constant voltage 100V, and 120min, by albumen from SDS-PAGE
Gel goes to pvdf membrane.The defatted milk powder of 3%/PBS-T closes, and dilutes Rabbit by 1:500
Anti-Bcl-2/-Bax/-Survivin, 4 DEG C of overnight incubation.1:3000 dilutes two anti-Goat anti-Rabbit IgG, hatches
1h, enhanced chemiluminescence (enhanced chemiluminescence, ECL) develops.UVI gel imaging system
Shooting.
Fig. 9 lists blank, 1.00 μ g/mL Zuojin prescriptions, 100.0 μ g/mL 5-FU and 1.00 μ g/mL
Zuojin prescription is combined after 100.0 μ g/mL 5-FU act on HT-29 24h respectively, in regulation cell BAX, Bcl-2 and
The gel electrophoresis figure (n=6) of the Western Blotting of Survivin protein expression.
1.00ug mL is can be seen that from result of the test-1Zuojin prescription, 100.00ug mL-15-FU acts on HT-29 respectively
After cell 24h, the expression of Bax increases, and the expression of Bcl-2 and Survivin albumen reduces；Zuojin prescription is combined
5-FU (1.00+100.00) drug combination effect 24h, the expression of Bax increases further, and Bcl-2 and Survivin
The expression of albumen reduces further, compares P < 0.05, significant difference with each independent medication group.
3.6 Zuojin prescriptions and the impact on HT-29 morphocytology of the 5-FU drug combination
By HT-29 cell (2 × 105·mL-1) be inoculated on the coverslip in 6 orifice plates, each experimental group gives on request
Corresponding medicine, adds freshly prepared 4% paraformaldehyde (pH is 7.4), in 37 DEG C of fixing cell 30min,
PBS liquid is washed 2 times, after adding the dyeing of 5mg/L Hochest 33258 dyeing liquor 30min, PBS liquid washing, uses mounting liquid
(20mmol·L-1Citric acid, 50mmol L-1Disodium hydrogen phosphate, 50% glycerol, pH 5.5) fluorescence shows after mounting
Micro mirror is observed, is taken the photograph sheet.
As shown in Figure 10, Figure 10 is blank to result of the test, 1.00 μ g/mL Zuojin prescriptions, 50.0 μ g/mL 5-FU,
100.00 μ g/mL 5-FU, 1.00 μ g/mL Zuojin prescriptions combine 50.0 μ g/mL 5-FU and 1.00 μ g/mL Zuojin prescriptions connection
Close 100.0 μ g/mL 5-FU and act on morphocytology microscope observation figure (× 200) of HT-2924h.
Can be seen that from result of the test, after Hoehest33258 fluorescence staining, under fluorescence microscope, see blank group
HT-29 cell membrane is all complete, and nuclear morphology is full, and caryoplasm coloring is shallow, and even density is consistent, sees Figure 10 A；1.00
ug·mL-1Zuojin prescription, 50.00ug mL-15-FU、100.00ug·mL-1After 5-FU effect 24h, there is allusion quotation in cell
Type apoptosis morphological characteristic: cell membrane shrinkage, the nucleus fluorescence that relatively diminishes is remarkably reinforced, and becomes highly block, sees
Figure 10 B, 10C, 10D；Zuojin prescription+5-FU (1.00+50.00), Zuojin prescription+5-FU (1.00+100.00) combine use
Medicine effect 24h, cell is in addition to features described above, it is seen that apoptotic body number is significantly more than each independent medication group, sees figure
10E、10F。
To sum up can obtain, Zuojin prescription and 5-FU drug combination are than Zuojin prescription, or the treatment that the independent medication of 5-FU is to intestinal cancer
Effect is more preferable, Zuojin prescription and 5-FU drug combination, significantly improves the thin of people Normal Colon rectal mucosa epithelial cell FHC
Born of the same parents' survival rate, significantly reduces the cytotoxic effect of 5-FU, by promoting Bax in colon-cancer cell HT-29 cell
The high expressed of albumen and the low expression of Bcl-2, Survivin albumen, promote the apoptosis of colon cancer cell.The present invention
Achievement in research to reduce clinical anti-intestinal cancer chemotherapeutics toxic and side effects, improve therapeutic effect, have great
Meaning.
Claims (7)
1. Chinese Drug Rhizomes of Coptis is combined in the medicine of preparation treatment intestinal cancer with 5-fluorouracil with the extract of Fructus Evodiae
Purposes.
2. purposes as claimed in claim 1, wherein said Chinese Drug Rhizomes of Coptis is mass ratio with the extract of Fructus Evodiae
Chinese Drug Rhizomes of Coptis and the extract of Fructus Evodiae for 6:1.
3. purposes as claimed in claim 2, wherein said extract is water extract.
4. purposes as claimed in claim 1, wherein said combine for by 5-fluorouracil Chinese Drug Rhizomes of Coptis and Wu
Before the extract of the Fructus Evodiae, give patient simultaneously or after.
5. purposes as claimed in claim 1, the extract of wherein said Chinese Drug Rhizomes of Coptis and Fructus Evodiae is according to Chinese medicine
Prepared by the method for compound Zuojin prescription.
6. purposes as claimed in claim 5, the preparation method of wherein said Zuojin prescription includes: take 30g Rhizoma Coptidis
With 5g Fructus Evodiae, add 8 times of distilled water immersions 30 minutes, boiling 3 times, each 30 minutes, filter,
Merge 3 filtrates, lyophilization, every gram of dry powder 3.98g Han crude drug；Measure through HPLC method, Zuojin prescription lyophilizing
Containing berberine 39.85% in powder, palmatine 9.19%, jateorhizine 4.86%, rutaecarpin 0.17%, Fructus Evodiae
Alkali 0.19%.
7. purposes as claimed in claim 1, wherein said intestinal cancer is colon cancer.
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