CN102000117B - Use of nutgall extract in preparing medicine for inhibiting cell proliferation of colon cancer - Google Patents
Use of nutgall extract in preparing medicine for inhibiting cell proliferation of colon cancer Download PDFInfo
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- CN102000117B CN102000117B CN2010105680272A CN201010568027A CN102000117B CN 102000117 B CN102000117 B CN 102000117B CN 2010105680272 A CN2010105680272 A CN 2010105680272A CN 201010568027 A CN201010568027 A CN 201010568027A CN 102000117 B CN102000117 B CN 102000117B
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Abstract
The invention relates to use of a nutgall extract in preparing a medicine for inhibiting cell proliferation of colon cancer. In the invention, by adopting experimental methods such as methyl thiazolyl tetrazolium (MTT), flow cytometry and the like, a human colon cancer HT-29 cell is taken as an experimental carrier, and functional characteristics of the nutgall extract on the human colon cancer HT-29 cell are described in the aspects of proliferation and apoptosis of cancer cells; and by means of cell proliferation inhibition and cell apoptosis induction, the growth of the colon cancer cells is inhibited, which has a concentration-effect dependence relation. In the use of the invention, an anti-colon cancer action site (that is, a new target point) of the nutgall extract in a molecular biology level is disclosed and a novel natural medicine for prevention and treatment of colon cancer is provided.
Description
Technical field
The present invention relates to the purposes of a kind of Galla halepensis extract in the medicine of preparation inhibition Colon Cancer Cells.
Background technology
Uighur medicine is that uighur ' s culture is in long-term life, produce and with the experience and the crystallization of wisdom that accumulate in the disease struggle, it is an important component part of motherland's medicine, no matter aspect rationale, or all taking the course of its own aspect therapy and the medication rule, and formed more complete, unique theoretical system.Galla Turcica (Galla Helepensis) is regarded as treating the good medicine of various Acute and chronic inflammation diseases since ancient times in Uygur's medical science, have the effect of antiinflammatory, hemostasis, astringent or styptic treatment for spontaneous sweating, convergence, is used for the treatment of that periodontitis, dysentery are more than, habitual enteritis etc.The main component of Galla Turcica (Galla Helepensis) is tannin, tannin claims again tannin (tannin), be the polyhydric phenols that is present in a natural class formation more complicated, have the effects such as convergence, antiinflammatory, antiallergic, antibacterial, antiviral, antitumor, hemostasis, analgesia, blood fat reducing, blood sugar lowering, enhancing renal function.Because tannin has more ortho position phenolic hydroxyl group, easily oxidized, therefore, be difficult to obtain monomer, also to the research of tannin chemical constituent seldom both at home and abroad.
Ulcerative colitis is the not bright inflammatory bowel relevant with autoimmune of a kind of reason, take diarrhoea, mucosa pyemia, stomachache and tenesmus as cardinal symptom, digestive tract commonly encountered diseases and difficult disease take mucous membrane of colon chronic inflammatory disease and ulcer as pathological characteristic.Research is found, has contact between ulcerative colitis and colon cancer occur, and thinking has part ulcerative colitis case finally can develop into colon cancer.The canceration of colitis depends mainly on occurrence degree, happening part and the course of disease length of colitis.Although clinical employing nonsteroidal anti-inflammatory drug treatment can reduce the canceration rate of colitis, but long-term taking, side effects of pharmaceutical drugs make patient be difficult to tolerance, and nonsteroidal anti-inflammatory drug has increased digestive tract ulcer and hemorrhage probability simultaneously, in the patient that intestinal Repeated Hemorrhage and distal ileitis are shown effect repeatedly, the use of such medicine is relatively taboo.Therefore, seek new medicine, effectively control the development of colitis, and stop the canceration of colitis, be the focus that researcheres are paid close attention to always.
Research finds that it is tumorigenic main mechanism that the abnormality proliferation of cell and differentiation are suppressed.Apoptotic research is found the relatively stable dynamic equilibrium that is actually between cell proliferation, differentiation and the apoptosis of normal structure.The propagation of tumor cell, apoptosis not only are subject to the impact of the many factors such as medicine, lonizing radiation and approach, and some oncogene or tumor suppressor gene also play important regulating and controlling effect to apoptosis.
The present invention is on the basis according to acquired " preventing and treating oral-cavity cleaning liquor and the preparation method of oral pathology " (patent No. is ZL 97116074.0), develop the solid gum liquid of Galla Turcica (Galla Helepensis) (Xi Payi), and be put into for " Drug Standard of Ministry of Public Health of the Peoples Republic of China-Uigurs medicine fascicle " version in 1999 and record kind (numbering: 02CJ-15-02-13).The solid gum liquid of Galla Turcica (Galla Helepensis) (Xi Payi) is increased new indication by changing its route of administration, and the treatment that is used in the ulcer colon cancer in the mode of coloclysis has obtained good clinical efficacy.
Studies show that the main component of the solid gum liquid of medicine Galla Turcica (Galla Helepensis) (Xi Payi) is Galla halepensis extract, and is evident in efficacy to stomachache, diarrhoea and the pyemia etc. of chronic ulcerative colitis; Common pathogen to oral diseases such as staphylococcus aureus, beta hemolytic streptococcus, Diplococcus pneumoniaes has very strong inhibitory action; Obviously suppress carrageenin induced mice pedal swelling and rat abdominal cavity leukocytoplania; Suppressing acetic acid induced mice capillary permeability increases; Suppress granuloma induced by implantation of cotton pellets; Rat temperature raises due to inhibition acetic acid induced mice writhing response and the yeast; Prolong the mice swimming time; Suppress the mice delayed hypersensitivity that DNCB induces; Clotting time of mice is obviously shortened, prove that reaction all has remarkable inhibitory action to Acute and chronic inflammation for it, and the permeability of energy establishment inflammation phase blood capillary, alleviate and ooze out reaction, stronger antipyretic-antalgic and anti-fatigue effect are arranged simultaneously.
The present invention is directed to the solid gum liquid of medicine Galla Turcica (Galla Helepensis) by name in the Clinical efficacy for the treatment of ulcerative colitis and the internal relation between ulcerative colitis and the colon cancer generation, the application that main component Galla halepensis extract in the solid gum liquid of medicine Galla Turcica (Galla Helepensis) suppresses human colon cancer cell HT-29 propagation and induces its accent to die is set forth, increased the clinical scope of application of said preparation, for the control of colon cancer provides new natural drug.Find not yet that at home and abroad the solid gum liquid of Galla Turcica (Galla Helepensis) is applied to human body and suppresses HT-29 propagation and induce the report of its accent in dying.
Summary of the invention
The object of the invention is to, the purposes of a kind of Galla halepensis extract in the medicine of preparation inhibition colon cancer cell HT-29 is provided, the present invention is on the basis of " preventing and treating oral-cavity cleaning liquor and the preparation method of oral pathology " (patent No. is ZL 97116074.0), provides new purposes to the oral-cavity cleaning liquor of preventing and treating oral pathology.The present invention is take human colon carcinoma HT-29 cell as the experiment carrier, adopt the experimental techniques such as MTT, flow cytometry, set forth from cancer cell multiplication and apoptosis angle " prevent and treat oral pathology oral-cavity cleaning liquor " the main component Galla halepensis extract to the effect characteristics of human colon carcinoma HT-29 cell, by suppressing the mode of cell proliferation and cell death inducing, suppress the growth of colon cancer cell, this effect has concentration-effect dependency relationships.The inhibitor against colon carcinoma cells site of action that has disclosed Galla halepensis extract from the molecular biology level is novel targets, for the control of colon cancer provides new natural drug.
A kind of Galla halepensis extract of the present invention suppresses purposes in the medicine of colon cancer cell HT-29 in preparation, is to be 5 * 10 in concentration
4Add Galla halepensis extract in the exponential phase human colon carcinoma HT-29 cell suspension of individual/ml, its ultimate density is reached: 0.01 μ g/ μ l, 0.025 μ g/ μ l, 0.05 μ g/ μ l, 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l, 2.5 μ g/ μ l or 5 μ g/ μ l, at 5%CO
237 ℃ of temperature, common cultivation 3,6,12,24,48,72 hours, adopt MTT colorimetric method for determining Galla halepensis extract to the inhibitory action of human colon carcinoma HT-29 cell proliferation, the result proves that Galla halepensis extract can suppress in the mode of time dependence and concentration dependent human colon carcinoma HT-29 cell proliferation.
Described a kind of Galla halepensis extract suppresses purposes in the medicine of colon cancer cell HT-29 in preparation, is to be 5 * 10 in concentration
4Add Galla halepensis extract in the exponential phase human colon carcinoma HT-29 cell suspension of individual/ml, its ultimate density is reached: 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l or 2.5 μ g/ μ l, at 5%CO
2, 37 ℃ of temperature were cultivated 48,72 hours jointly, adopted the Flow Cytometry Assay Galla halepensis extract to the inducing action of colon cancer cell line HT-29 apoptosis, and the result proves that Galla halepensis extract can be induced human colon carcinoma HT-29 apoptosis in the concentration dependent mode.
Galla halepensis extract of the present invention is with Galla Turcica (Galla Helepensis) medical material 20g, pulverizes, add water 200ml, soak after 1 hour, decocted 0.5 hour, decoct altogether 3 times, collecting decoction adopts the sheet frame method to filter, and adds successively 2g cyclamate, 1ml Mint Essence and 0.5g sodium benzoate in the filtrate, stirring makes dissolving evenly, add water and make medicine liquid volume be adjusted to 1000ml, packing, sterilization get final product.
The specific embodiment
The technical scheme of the purposes of Galla halepensis extract of the present invention in the medicine of preparation inhibition colon cancer cell HT-29 is to realize by following approach:
1. experiment material
1.1 medicine and reagent
1.2 key instrument
2. experimental technique
2.1 cell culture
Human colon cancer cell strain HT-29 grows in the DMEM culture medium that contains 10% hyclone, 37 ℃ of temperature, 5%CO
2Cultivate in the cell culture incubator of saturated humidity, changed culture fluid once in every 2-3 days, the cell of the trophophase of taking the logarithm is used for experiment.
2.2MTT method detects the effect that Galla halepensis extract suppresses human colon carcinoma HT-29 cell proliferation
(1) collect the logarithmic (log) phase cell, adjusting concentration of cell suspension is 5 * 10
4Individual/ml, every hole adds 100 μ l, bed board;
(2) 5%CO
237 ℃ of temperature are hatched, at the bottom of being paved with the hole to cell monolayer (96 hole flat underside), add Galla halepensis extract, the medicine ultimate density reaches: 0.01 μ g/ μ l, 0.025 μ g/ μ l, 0.05 μ g/ μ l, 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l, 2.5 μ g/ μ l or 5 μ g/ μ l, establish 4 multiple holes, the blank group is set simultaneously for every group;
(3) 5%CO
2, 37 ℃ of temperature are hatched, and continue to cultivate 3h, 6h, 12h, 24h, 48h and 72h, and every hole adds 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h again;
(4) stop cultivating, carefully suck culture fluid in the hole;
(5) every hole adds 150 μ l dimethyl sulfoxide, put low-speed oscillation 10min on the shaking table, crystal is fully dissolved, measure the absorbance (A value) in each hole at enzyme-linked immunosorbent assay instrument OD490nm place, zeroing hole (culture medium, MTT, dimethyl sulfoxide) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide);
(6) experiment repeats 3 times;
(7) calculating of suppression ratio: suppression ratio=[(control group A value-blank group A value)-(experimental group A value-blank group A value)]/(control group A value-blank group A value) * 100%.The results are shown in Table 1
Table 1 Galla halepensis extract is to the inhibitory action of human colon carcinoma HT-29 cell proliferation
Annotate: SPSS 13.0 statistical softwares, the repeated measure variance analysis, (different time sections) comparing difference has statistical significance (F=6.12, P＜0.05) in the group, and (negative control group and variable concentrations KJA) comparing difference has statistical significance (F=12.07 between group; P＜0.05).
The result: Galla halepensis extract is on the impact of human colon carcinoma HT-29 cell proliferation
This experiment acts on human colon carcinoma HT-29 cell with the variable concentrations Galla halepensis extract, cultivates respectively 3h, 6h, and 12h, 24h, 48h behind the 72h, observes the impact of medicine on cell proliferation.Draw following result (table 1) through MTT colorimetric method for determining and repeated measure variance analysis: prolong in time, the same concentration Galla halepensis extract presents rising trend to the suppression ratio of human colon carcinoma HT-29 cell, meta-effect dependency relationships when having; The same time, with the increase of concentration, the inhibitory action of Galla halepensis extract on cell proliferation strengthens, and has concentration-effect dependency relationships.The two all shows: after Galla halepensis extract was intervened human colon carcinoma HT-29 cell, living cells quantity reduced, and cell proliferation is suppressed.
2.3 flow cytometer detects the effect that Galla halepensis extract is induced Apoptosis of Colon Cancer Cells
(1) will be in the human colon carcinoma HT-29 cell of exponential phase, trypsinization, making cell concentration is 5 * 10
4Individual/the mL single cell suspension;
(2) above-mentioned cell suspension kind is entered 10 culture bottles, 5ml/ bottle, exponential phase to be entered, make drug level reach 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1 μ g/ μ l or 2.5 μ g/ μ l, continue to cultivate 48h, 72h establishes negative control group simultaneously;
(3) the cell culture fluid sucking-off to centrifuge tube, PBS washing attached cell once, with pancreatin cell dissociation buffer peptic cell;
(4) after cell dissociation gets off, add the cell culture fluid of collecting in the step (3), slightly mixing is transferred in the centrifuge tube, 1000rpm, and centrifugal 5 minutes, abandon supernatant, collecting cell with PBS gently re-suspended cell and counting, reaches 1 * 10
6Individual/mL;
(5) 3000rpm, centrifugal 2 minutes, abandon supernatant, collecting cell adds 400 μ LBanding Buffer re-suspended cells, adds 5 μ l Annexin V-FITC, then puts upside down back and forth mixing cell suspension, with it as for 4 ℃, lucifuge 15 minutes;
(6) add 10 μ l propidium iodide dyeing liquors, put upside down back and forth mixing cell suspension, 4 ℃ of lucifuges were placed 5 minutes;
(7) carrying out flow cytometer detects.
The apoptosis rate of table 248 hour Galla halepensis extract effect human colon carcinoma HT-29 cell
Annotate: △ represents to compare with matched group, and group difference has statistical significance (P＜0.05).★ represents that KJA2.5 μ g/ μ l organizes and all the other each groups are compared, and group difference has statistical significance (P＜0.05).
The apoptosis rate of table 3 Galla halepensis extract effect in 72 hours human colon carcinoma HT-29 cell
Annotate: △ represents to compare with matched group, and group difference has statistical significance (P＜0.05).★ represents that KJA2.5 μ g/ μ l organizes and all the other each groups are compared, and group difference has statistical significance (P＜0.05).■ represents that KJA0.1 μ g/ μ l group compares with 1.0 μ g/ μ l group, and difference has statistical significance (P＜0.05).
2.4 statistical procedures
Adopt the SPSS13.0 statistical software, with mean scholar standard deviation
Variance analysis is adopted in expression.P＜0.05 expression difference has statistical significance.
The result: Galla halepensis extract is on the apoptotic impact of human colon carcinoma HT-29
Behind Galla halepensis extract effect human colon carcinoma HT-29 cell 48h and 72h, adopt Annexin V-FITC/PI double labelling, flow cytometry (FCM) is analyzed apoptotic cell, the result shows: Galla halepensis extract effect 48h, behind the 72h, the cell of early apoptosis and late period apoptosis cell all obviously more than negative control group (table 2,3): the same time period, with the increase of concentration, the Galla halepensis extract cell death inducing has concentration-effect dependency relationships; The isoconcentration Galla halepensis extract is at the effect not statistically significant of different time cell death inducing.
Conclusion:
The MTT experiment shows that Galla halepensis extract has certain propagation to colon cancer cell line HT-29 to be suppressed and lethal effect, since 0.5 μ g/ μ l colon cancer cell line HT-29 is had obvious inhibited proliferation (P＜0.05).Along with Galla halepensis extract concentration increases, prolong action time gradually, and the survival rate of cell reduces gradually, and the inhibited proliferation that colon cancer cell line HT-29 is subject to obviously strengthens.Galla halepensis extract was processed colon cancer cell line HT-29 after 72 hours, and the proliferation inhibition rate of colon cancer cell line HT-29 has reached 88.35%.The result has shown that Galla halepensis extract has stronger inhibited proliferation to colon cancer cell line HT-29, and has time, dosage effect dependency.Experimental result shows, (5 μ g/ μ l) can significantly suppress the propagation of colon cancer cell line HT-29 when Galla halepensis extract concentration was low, toxicity to normal cell and normal structure reduces greatly, the medicine that is expected to become a kind of safe and effective treatment ulcerative colitis and prevents its canceration.
The flow cytometer experiment shows, colon cancer cell line HT-29 by Galla halepensis extract process 48h, after 72 hours, dye through AnnexinV/PI pair, flow cytometer detection display: compare with negative control group, Galla halepensis extract processed group apoptosis rate obviously rises, have concentration-effect dependency, maximum apoptosis rate is 85.77 ± 7.03%, is about 10 times of negative control group.The result shows that Galla halepensis extract can significantly induce the people to tie the apoptosis of cancerous cell HT-29.
Show through test: Galla halepensis extract reaches the purpose for the treatment of and prevent colon cancer by suppressing human colon carcinoma HT-29 cell proliferation and cell death inducing.
Claims (2)
1. a Galla halepensis extract suppresses purposes in the medicine of colon cancer cell HT-29 in preparation, it is characterized in that: be 5 * 10 in concentration
4Add Galla halepensis extract in the exponential phase human colon carcinoma HT-29 cell suspension of individual/ml, its ultimate density is reached: 0.01 μ g/ μ l, 0.025 μ g/ μ l, 0.05 μ g/ μ l, 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l, 2.5 μ g/ μ l or 5 μ g/ μ l, at 5%CO
2, 37 ℃ of temperature cultivate 3 jointly, 6,12,24,48,72 hours, adopt MTT colorimetric method for determining Galla halepensis extract to the inhibitory action of human colon carcinoma HT-29 cell proliferation, wherein Galla halepensis extract, with Galla Turcica (Galla Helepensis) medical material 20g, pulverize, add water 200ml, soak after 1 hour, decocted 0.5 hour, decoct altogether 3 times, collecting decoction adopts the sheet frame method to filter, add successively 2g cyclamate, 1ml Mint Essence and 0.5g sodium benzoate in the filtrate, stirring makes dissolving evenly, adds water and makes medicine liquid volume be adjusted to 1000ml, and packing, sterilization get final product.
2. a Galla halepensis extract suppresses purposes in the medicine of colon cancer cell HT-29 in preparation, it is characterized in that: be 5 * 10 in concentration
4Add Galla halepensis extract in the exponential phase human colon carcinoma HT-29 cell suspension of individual/ml, its ultimate density is reached: 0.1 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l or 2.5 μ g/ μ l, at 5%CO
237 ℃ of temperature, the common cultivation 48,72 hours adopts the Flow Cytometry Assay Galla halepensis extract to the inducing action of colon cancer cell line HT-29 apoptosis, Galla halepensis extract wherein, be with Galla Turcica (Galla Helepensis) medical material 20g, pulverize, add water 200ml, soak after 1 hour, decocted 0.5 hour, and decocted altogether collecting decoction 3 times, adopt the sheet frame method to filter, add successively 2g cyclamate, 1ml Mint Essence and 0.5g sodium benzoate in the filtrate, stir to make and dissolve evenly, add water and make medicine liquid volume be adjusted to 1000ml, packing, sterilization get final product.
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| CN101862351A (en) * | 2010-06-30 | 2010-10-20 | 斯拉甫·艾白 | Application of active parts of gallnut in preparing anti-ulcerative colitis medicine |
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