CN103417518B - Oxidized resveratrol is as the application of preparation tumor - Google Patents
Oxidized resveratrol is as the application of preparation tumor Download PDFInfo
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Abstract
The invention discloses the application of oxidized resveratrol in the medicine of preparation treatment tumor.In vitro cell experiment shows that oxidized resveratrol all has good growth inhibited effect to this 7 strain of Leukemia K562 cell, Proliferation of Human Ovarian Cell SK-OV-3, human breast cancer cell line Bcap-37, human liver cancer cells Hep G2, human lung cancer cell A549, KB cell HNE-1 and human colon cancer cell SW480 humanized tumor cell line, and the IC50 value of 48 hours is between 0.047 ~ 0.277mM; Especially obvious to the growth inhibited effect of human liver cancer cells Hep G2, Proliferation of Human Ovarian Cell SK-OV-3 and human breast cancer cell line Bcap-37 cell.Zoopery shows that the growth of oxidized resveratrol to mice transplantability tumor S180 sarcoma and mice transplantability tumor H22 hepatocarcinoma has obvious inhibitory action, and toxic and side effects is low.
Description
Technical field
The present invention relates to the purposes of oxidized resveratrol, the purposes particularly in pharmacy.
Background technology
Tumor is a kind of disease of serious harm human health, is the clinical tangible tumor block detected by means such as X-ray, CT scan, B ultrasonic or palpation, point optimum, pernicious two classes.Malignant tumor block is also called cancer usually, and wherein clinical common malignant solid tumor comprises pulmonary carcinoma, neuroma, colorectal cancer, sarcoma etc.
At present, China has every year and dies from cancer more than 1,500,000 people, on average often in dead 5 people, just has 1 people to die from cancer.Over nearly 30 years, world's cancer morbidity with average annual 3% ~ 5% increased number, in China, over nearly 20 years, the overall incidence of cancer and mortality rate are all in obvious rising.
The treatment means of current cancer is still with excision, the general treatment measures of chemoradiation therapy treatment is main, but postoperative recurrence, primary tumor transfer and chemicotherapy bring huge toxic and side effects, make treatment of cancer poor effect, the curative effect of traditional chemicotherapy means in cancer patient allows all the more people be unsatisfied with.
Existing pharmacological research and clinical trial show, natural origin compound demonstrates great advantage in the clinical treatment of tumor, and compared with the chemotherapeutics of routine, its toxic and side effects is less.Oxidized resveratrol (Oxyresveratrol) is a kind of natural active matter, be present in various plants, can from Moraceae (Moraceae), Myrtaceae (Myrtaceae), Liliaceae (Liliaceae), extracts in the plants such as Gnetaceae (Gnetaceae).Bibliographical information oxidized resveratrol has anthelmintic, whitening, antiviral, antioxidation and herpes effect.Experiment in vitro confirms that oxidized resveratrol is tyrosinase inhibitor, basophil histamine can be suppressed to discharge (KimDH, KimJH, BaekSH.Enhancementoftyrosinaseinhibitionoftheextractofve ratrumpatulumusingcellulase.BiotechnoologyandBioengineer ing, 2004, 87 (7): 849-854), contraction for smooth muscle also has inhibitory action (Kan Qiming, healthy and free from worry, Tian Haitao, Deng. the antitussive and antiasthmatic effect of mulberry skin glucoside. Shenyang Pharmaceutical University's journal, 2006, 23 (6): 388-391), but the report that there is not yet about oxidized resveratrol anti-tumor activity.
Summary of the invention
The object of the invention is to prove that oxidized resveratrol has anti-tumor activity, can apply in the medicine of preparation treatment tumor, the treatment for tumor provides a kind of novel drugs.
The present invention is proved by In vitro cell experiment: the humanized tumor cell line of oxidized resveratrol to this 7 strain separate sources of Leukemia K562 cell, Proliferation of Human Ovarian Cell SK-OV-3, human breast cancer cell line Bcap-37, human liver cancer cells Hep G2, human lung cancer cell A549, KB cell HNE-1 and human colon cancer cell SW480 all has good growth inhibited effect, and the IC50 value of 48 hours is between 0.047 ~ 0.277mM; Especially obvious to the growth inhibited effect of human liver cancer cells Hep G2, Proliferation of Human Ovarian Cell SK-OV-3 and human breast cancer cell line Bcap-37 cell, the IC50 value of 48 hours is respectively 0.058,0.047 and 0.097mM.Illustrate that the anti-tumor activity of oxidized resveratrol is good, antitumor spectra wide, therefore oxidized resveratrol can be applied in the medicine of preparation treatment tumor.
The present invention is proved by zoopery: when oxidized resveratrol dosage is 40mg/kg, to the growth of mice transplantability tumor S180 sarcoma and mice transplantability tumor H22 hepatocarcinoma, there is obvious inhibitory action, and the body weight of mice is had no significant effect, to the immune organ of mice without overt toxicity, illustrate that the toxicity of the more conventional anti-tumor medicine of oxidized resveratrol is low.Therefore can by the application of oxidized resveratrol in the medicine of preparation treatment tumor.
The application of above-mentioned oxidized resveratrol in the medicine of preparation treatment tumor, described tumor is leukemia, colon cancer, ovarian cancer, breast carcinoma, pulmonary carcinoma, hepatocarcinoma, nasopharyngeal carcinoma or sarcoma.
The application of above-mentioned oxidized resveratrol in the medicine of preparation treatment tumor, is prepared into the solution use that concentration is 1.56mg/mL ~ 100mg/mL by oxidized resveratrol.
The present invention has following beneficial effect:
1, the invention provides a kind of new medical application of known compound oxidized resveratrol, open up the application of oxidized resveratrol.
2, the present invention is that the treatment of tumor provides a kind of new medicine, and its anti-tumor activity is good and toxic and side effects is low, has obvious social benefit.
Accompanying drawing explanation
Fig. 1 is that various dose oxidized resveratrol described in embodiment 1 is to Leukemia K562 cell and Proliferation of Human Ovarian Cell SK-OV-348h growth inhibited effect curves;
Fig. 2 is that various dose oxidized resveratrol described in embodiment 1 is to human breast cancer cell line Bcap-37 and human lung cancer cell A549 48h growth inhibited effect curves;
Fig. 3 is that various dose oxidized resveratrol described in embodiment 1 is to human liver cancer cells Hep G2, human colon cancer cell SW-480 and KB cell HNE-148h growth inhibited effect curves;
Fig. 4 is the tumor photo of each group S180 tumor-bearing mice after oxidized resveratrol medication in first time test described in embodiment 2;
Fig. 5 is the tumor photo of each group S180 tumor-bearing mice after oxidized resveratrol medication in second time test described in embodiment 2;
Fig. 6 is the tumor photo of each group S180 tumor-bearing mice after oxidized resveratrol medication in third time test described in embodiment 2;
Fig. 7 respectively organizes S180 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in first time test described in embodiment 2;
Fig. 8 respectively organizes S180 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in second time test described in embodiment 2;
Fig. 9 respectively organizes S180 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in third time test described in embodiment 2;
Figure 10 respectively organizes S180 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in first time test described in embodiment 2;
Figure 11 respectively organizes S180 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in second time test described in embodiment 2;
Figure 12 respectively organizes S180 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in third time test described in embodiment 2;
Figure 13 is the tumor photo of each group H22 tumor-bearing mice after oxidized resveratrol medication in first time test described in embodiment 3;
Figure 14 is the tumor photo of each group H22 tumor-bearing mice after oxidized resveratrol medication in second time test described in embodiment 3;
Figure 15 is the tumor photo of each group H22 tumor-bearing mice after oxidized resveratrol medication in third time test described in embodiment 3;
Figure 16 respectively organizes H22 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in first time test described in embodiment 3;
Figure 17 respectively organizes H22 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in second time test described in embodiment 3;
Figure 18 respectively organizes H22 tumor-bearing mice gross tumor volume with the change curve observing natural law after oxidized resveratrol medication in third time test described in embodiment 3;
Figure 19 respectively organizes H22 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in first time test described in embodiment 3;
Figure 20 respectively organizes H22 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in second time test described in embodiment 3;
Figure 21 respectively organizes H22 tumor-bearing mice body weight with the change curve observing natural law after oxidized resveratrol medication in third time test described in embodiment 3.
Detailed description of the invention
Embodiment 1: the extracorporeal anti-tumor function of oxidized resveratrol
1. experiment material
1.1 cell strains: Leukemia K562 cell, Proliferation of Human Ovarian Cell SK-OV-3, human breast cancer cell line Bcap-37, human liver cancer cells Hep G2, human lung cancer cell A549, KB cell HNE-1, human colon cancer cell SW480, all purchased from Shanghai cell bank, and by Sichuan University's preclinical medicine and pharmacology teaching and research room of legal medical expert institute frozen.
1.2 trial drugs and positive control drug
1.2.1 trial drug: oxidized resveratrol, purity >=98%, wishes love purchased from ladder and changes into industrial (Shanghai) Co., Ltd.;
1.2.2 positive control drug: hydrochloride for injection doxorubicin (amycin), Haizheng Medicine Stock Co., Ltd., Zhejiang Prov, lot number 090204A.
1.3 reagent and preparation
RPMI-1640 culture medium, Chengdu Harry biotech firm subpackage; Calf serum, Chengdu Harry biotech firm, lot number 090625; Penicillin and streptomycin liquid, Chengdu Harry biotech firm subpackage; Tetramethyl azo azoles salt (MTT), Sigma company; Dimethyl sulfoxide (DMSO), the special Chemical Company in Rui Jin, Tianjin; Trypan blue, Sigma company; Trypsin, Chengdu Harry biotech firm subpackage.
Phosphate buffer (PBS solution): PBS powder (potassium dihydrogen phosphate: 0.27g, sodium hydrogen phosphate: 1.42g, sodium chloride: 8g, potassium chloride 0.2g) is dissolved in distilled water, and adjust ph is 7.4, after be settled to 1L, autoclave sterilization is for subsequent use;
MTT solution: take 250mgMTT, adding 50mL concentration is 0.1mol/LPBS solution, is mixed with the MTT stock solution of 5g/L, and 0.22 μm of filtering with microporous membrane is degerming, after subpackage, 4 DEG C of preservations, in two weeks effectively.
1.4 instrument and equipment
CO
2incubator, Japanese SANYO company; Superclean bench, SuZhou Antai Air Tech Co., Ltd.; Full-automatic microplate reader, Beijing Pu Lang Technew SA, model DNM-9602G;-20 DEG C of refrigerators; Liquid nitrogen container; Vertical pressure steam sterilizer; Electric-heated thermostatic water bath; Heat-insulating type electro-heating standing-temperature cultivator; Tissue Culture Flask; 96 hole flat undersides.
2. experimental technique
2.1 cellar culture cells
2.1.1 cell recovery
In this step, respectively to Leukemia K562 cell, Proliferation of Human Ovarian Cell SK-OV-3, human breast cancer cell line Bcap-37, human liver cancer cells Hep G2, human lung cancer cell A549, KB cell HNE-1, human colon cancer cell SW480 carries out cell recovery operation, and concrete operations are as follows:
Before experiment, by the table top of superclean bench ultraviolet radiation 30min, water-bath is added water and is preheated to 37 DEG C, then by complete medium (containing 10%(v/v) calf serum and 1%(v/v) the RPMI-1640 culture medium of penicillin and streptomycin) be placed in water-bath preheating.Take out frozen cell strain, rapidly cryopreservation tube is put in the water-bath of preheating and thaw rapidly, and constantly shake, liquid in pipe is melted rapidly, after about 1 ~ 2min, frozen liquid in pipe dissolves completely, take out, with containing 75%(percentage by volume) outer wall of the cotton balls wiping cryopreservation tube of ethanol.Draw cryopreservation tube inner cell, be transferred in 15mL centrifuge tube, add 5mL preheating complete medium simultaneously, the centrifugal 3 ~ 5min of 500g, inhale and abandon supernatant, add 10mL complete medium in centrifuge tube, cell suspension is made in soft piping and druming.After carrying out vitality test, cell suspension is added in 10cm culture dish, in 5%CO by Trypan Blue cell count
2, saturated humidity, the CO of 37 DEG C
2overnight incubation in incubator.
2.1.2 cell culture with go down to posterity
Complete medium is adopted to be cultivated by 2.1.1 gained cell.Original fluid in the culture dish growing to 80% ~ 90% degree of converging cell is inhaled and abandons, add 2 ~ 3mLPBS buffer, softly rock culture dish, then inhale and abandon, add 0.25%(w/v) Trypsin-EDTA solution (addition: 10cm culture dish is 1mL), culture dish floor cells is all immersed in Trypsin-EDTA solution, after 30s, puts observation of cell under inverted microscope.Along with passage of time, former adherent cell is tending towards circular gradually, is discarded by Trypsin-EDTA solution, add 10mL complete medium and stop digestion when also non-levitating.Adherent cell is blown and beaten into suspension, divides dish by 1:2, in 5%CO
2, saturated humidity, the CO of 37 DEG C
2overnight incubation in incubator.
2.1.3 cell cryopreservation
Prepare fresh cell cryopreservation culture fluid, the composition (v/v) of described cell cryopreservation culture fluid is: 60%RPMI-1640 culture medium, 30% hyclone (FBS), 10%DMSO.To take the logarithm trophophase cell, with 0.05%(w/v) trypsinization, attached cell piping and druming is moved in 15mL centrifuge tube after suspending.Inhale after the centrifugal 5min of 1000g and abandon supernatant, add appropriate cell cryopreservation culture fluid, blow and beat gently make cell evenly and count with suction pipe, the final densities regulating cell in cell cryopreservation culture fluid is 1 × 10
6individual/mL.Cell is distributed in cryopreservation tube, often pipe 1mL.Preserve under moving to-80 DEG C of conditions after program temperature reduction box freeze-stored cell.
In the present embodiment, each IC50 pH-value determination pH experiment all uses the cell of same generation.
2.2 draft and configure drug dose
According to preliminary result, draft trial drug (oxidized resveratrol) as shown in table 1 and the Dosages of positive control drug (amycin).
Table 1 trial drug and positive control drug are to the Dosages of various cell
2.3 experiment groupings
2.3.1 blank group: not celliferous complete medium;
2.3.2 negative control group: without the normal cultured cell of drug treating;
2.3.3 positive controls: the cultured cell adding amycin process by the dose gradient in table 1;
2.3.4 oxidized resveratrol experimental group: the cultured cell adding oxidized resveratrol process by the dose gradient in table 1.
In above-mentioned oxidized resveratrol experimental group and positive controls, each dosage establishes 3 multiple holes.
2.4MTT method test cell vigor
The step of mtt assay test cell vigor is as follows:
(1) collect exponential phase cell, every hole adds 100 μ L cell suspension, and every porocyte number is for optimizing rear quantity (about 5 × 10
3individual/hole), the aseptic PBS solution in bore edges hole is filled;
(2) by the cell in above-mentioned steps (1) at 5%CO
2, saturated humidity, the CO of 37 DEG C
2hatch in incubator and be paved with at the bottom of 96 flat-floored holes, hole to cell monolayer, add the RPMI-1640 culture medium of drug containing after 24h by the dose gradient in table 1 respectively to positive controls, oxidized resveratrol experimental group, every hole adds 200 μ L;
(3) step (2) is added the cell after medicine at 5%CO
2, saturated humidity, the CO of 37 DEG C
2hatching in incubator, observing with inverted microscope when hatching 24h and 48h respectively;
(4) after adding the full 48h of drug incubation, inhale the RPMI-1640 culture medium of abandoning drug containing, every hole adds the MTT solution that 100 μ L are 5mg/mL without phenol red RPMI-1640 culture medium, 20 μ L concentration, at 5%CO
2, saturated humidity, the CO of 37 DEG C
2continue in incubator to cultivate 4h;
(5) after the incubation time of step (4) expires, stop cultivating, careful suction abandons culture fluid and MTT solution in hole;
(6) every hole adds 100 μ L dimethyl sulfoxide, puts low-speed oscillation 30min on shaking table, crystal is fully dissolved;
(7) in full-automatic microplate reader, the light absorption value at OD570nm place is measured, simultaneously with isopyknic blank DMSO setting zeroing hole.
2.5 data analysis
Inhibitory rate of cell growth=(1-OD sample/OD is negative) × 100%, wherein OD sample is the OD value of medicine feeding hole or Positive control wells, and OD feminine gender is the OD value of not medicine feeding hole, adopts SPSS17.0 data processing software process data.
3. experimental result
3.1 oxidized resveratrols are to the growth inhibited effect of each strain tumor cell
Various dose oxidized resveratrol to the growth of human tumor cells suppression ratio of 7 strain separate sources and IC50 value (48h) as shown in table 2; Various dose oxidized resveratrol to the human tumor cells 48h growth inhibited effect curves of 7 strain separate sources as shown in Figures 1 to 3.
Table 2 oxidized resveratrol is external to growth of tumour cell inhibitory action
As can be seen from Table 2, the IC50 value of oxidized resveratrol to the human tumor cells of 7 strain separate sources is all less than 0.3mM, and represent that its anti-tumor activity is good, antitumor spectra is wide.
Various dose oxidized resveratrol to the detailed growth inhibited effect of the human tumor cell line of 7 strain separate sources, in table 3 ~ 9.
Table 3 oxidized resveratrol is to the effect of K562 Leukaemia growth inhibited and IC50 value
Table 4 oxidized resveratrol is to ovarian cancer cell SK-OV-3 growth inhibited effect and IC50 value
Table 5 oxidized resveratrol is to breast cancer cell MCF-7 growth inhibited effect and IC50 value
Table 6 oxidized resveratrol is to the effect of Hepatocellular carcinoma cell line growth inhibited and IC50 value
Table 7 oxidized resveratrol is to the effect of lung cell A549 growth inhibited and IC50 value
Table 8 oxidized resveratrol is to nasopharyngeal carcinoma cell HNE-1 growth inhibited effect and IC50 value
Table 9 oxidized resveratrol is to colon cancer cell SW-480 growth inhibited effect and IC50 value
Embodiment 2: oxidized resveratrol is to the growth inhibited effect of mice transplantability tumor S180 sarcoma
1. experiment material
1.1 laboratory animals: Kunming mouse, male, body weight 18 ~ 22g, totally 180, animal quality certification SCXK(river) 2008-24, purchased from Da Shuo bio tech ltd, Chengdu.
1.2 mouse S180 sarcoma cell: purchased from Shanghai cell bank, by Sichuan University's preclinical medicine and pharmacology teaching and research room of legal medical expert institute frozen.
1.3 Experimental agents and reagent
1.3.1 trial drug: oxidized resveratrol, purity >=98%, wishes love purchased from ladder and changes into industrial (Shanghai) Co., Ltd.;
1.3.2 positive control drug: cyclophosphamide injectable powder, purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov., lot number 11030621;
1.3.3 reagent
0.9% sodium chloride injection (normal saline): purchased from ChengDu QingShan LiKang Pharmacy Co., Ltd, lot number A1201082.
1.4 Experimental agents preparations
Cyclophosphamide: be that the cyclophosphamide injectable powder of 0.2g/ bottle is dissolved in 10mL normal saline by specification, be mixed with the solution that concentration of cyclophosphamide is 20mg/mL, extraction this solution of 1.5mL and 3.5mL normal saline mix before use, obtain the solution that concentration of cyclophosphamide is 6mg/mL;
Oxidized resveratrol: be dissolved in DMSO by 4mg/10 μ L before using, after dissolving completely, slowly adds in 1mL normal saline, oxidized resveratrol final concentration 4mg/mL.
2. experimental technique
The preparation of 2.1 lotus tumor models
The mice being vaccinated with transplantability S180 sarcoma cell is fed 7 ~ 10d under the condition of water and feedstuff abundance, after ascites grows, aseptically extract tumor liquid, be also fully mixed to get cell suspension by tumor liquid and the dilution proportion tumor liquid of the volume ratio=1:2 of normal saline.After expecting blue dyeing with platform, hematimeter counts, and wherein viable count is greater than 95%, is 1 × 10 by the concentration of normal saline adjustment cell suspension
7individual oncocyte/mL.Then the cell suspension adjusting concentration is only inoculated in tested animal oxter by 0.2mL/, builds solid tumor animal model, obtain S180 tumor-bearing mice.
2.2 animal grouping and medications
Mouse inoculation S180 sarcoma cell, after 1 day, represents by random digit and mice is divided into negative control group, positive controls, oxidized resveratrol group, in first time test, often organizes 11 mices, and second time is tested and in third time test, often organized 10 mices.Each group according to following medication administration:
Negative control group: lumbar injection gives equal-volume normal saline, the volume giving normal saline is identical with the volume of oxidized resveratrol solution, injects 1 every day, injects 6 times weekly, amounts to injection 12 times, raises 14 days continuously;
Positive controls: lumbar injection gives cyclophosphamide solution (concentration 6mg/mL), the dosage of cyclophosphamide is 60mg/kg Mouse Weight, Per-Hop behavior 1 time, amounts to administration 2 times, raises 14 days continuously;
Oxidized resveratrol group: lumbar injection gives oxidized resveratrol solution (concentration 4mg/mL), and oxidized resveratrol dosage is 40mg/kg Mouse Weight, injects 1 every day, injects 6 times weekly, amounts to injection 12 times, raises 14 days continuously.
2.3 Testing index and method
2.3.1 ordinary circumstance
The reaction observed the autonomic activities of mice, the mental status, hair, breathing, diet, feces character every day and stimulate to external world.
2.3.2 tumor volume
Use the major diameter a of vernier caliper measurement tumor block, minor axis b every other day, by V=1/2 × a × b
2calculate tumor volume, draw mouse tumor block growth curve.
2.3.2 body weight
Claim Mouse Weight every other day, draw Mouse Weight with the change curve observing natural law.
2.3.3 the mensuration of tumor control rate and immune organ coefficient
In the 15th day, de-cervical vertebra put to death mice, took the body weight of each mice.Mice right axil tumor location skin is lived with tweezers tweezer, skin cut off by operation scissors, expose tumor, weigh by operating scissors blunt separation tumor, then abdominal cavity is cut off with operation, be separated spleen and the thymus of each group of mice successively, take the weight of spleen and thymus respectively, by following various calculating tumor control rate, Spleen coefficient and thymus coefficient.
Tumor control rate (%)=[1-(average experiment group tumor weight/mean negative control group tumor weight)] × 100%
Spleen coefficient=[spleen weight (mg)/body weight (g)] × 100%
Thymus coefficient=[thymic weight (mg)/body weight (g)] × 100%
2.4 statistical method
Altogether in triplicate, result represents with mean+SD in the zoopery of the present embodiment, and sided t inspection checking each group of difference, P≤0.05 thinks to have statistical significance.
3. experimental result
The tumor control rate of 3.1 oxidized resveratrol medications on mouse S 180 sarcoma and the impact of mortality rate
In three tests, the tumor of each group is heavy, tumor control rate data are as shown in table 10 ~ 12, the tumor photo of each group of mice as shown in figures 4-6, as can be seen from table 10 ~ 12 and Fig. 4 ~ 6: the growth of oxidized resveratrol to mice transplanted tumor S180 sarcoma has obvious inhibitory action.
The tumor that table 10 first time tests each group of mice weighs and tumor control rate
The tumor that table 11 second time tests each group of mice weighs and tumor control rate
The tumor that table 12 third time tests each group of mice weighs and tumor control rate
3.2 oxidized resveratrol medications are to the growth inhibited effect of mouse S 180 sarcoma
In three tests, each group of mice to give during medicine gross tumor volume as shown in table 13 ~ 15, Fig. 7 ~ 9 are that the tumor volume of each group of mice is with the change curve observing natural law, it can thus be appreciated that, oxidized resveratrol can slow down the speed of growth of mice transplanted tumor S180 sarcoma, has inhibition to tumor growth curve.
Gross tumor volume during table 13 tests each group of mice administration for the first time
Gross tumor volume during table 14 second time tests each group of mice administration
Gross tumor volume during table 15 tests each group of mice administration for the third time
3.3 oxidized resveratrol medications are on the impact of S180 tumor-bearing mice body weight
In three tests, average weight during each group of mice administration is as shown in table 16 ~ 18, Figure 10 ~ 12 are each group of Mouse Weight change curves with observation natural law, and shown in table 16 ~ 18, Figure 10 ~ 12 are known: the body weight of oxidized resveratrol to mice has no significant effect.
Average weight during table 16 tests each group of mice administration for the first time
Average weight during table 17 second time tests each group of mice administration
Average weight during table 18 tests each group of mice administration for the third time
3.4 oxidized resveratrol medications affect S180 tumor-bearing mice immune organ
In three tests, the Spleen coefficient of each group mice and thymus coefficient as shown in table 19 ~ 21, from table 19 ~ 21, oxidized resveratrol to the immune organ of mice without overt toxicity.
Table 19 tests Spleen coefficient and the thymus coefficient of each group of mice for the first time
Table 20 second time tests Spleen coefficient and the thymus coefficient of each group of mice
Table 21 tests Spleen coefficient and the thymus coefficient of each group of mice for the third time
Embodiment 3: oxidized resveratrol medication is to the growth inhibited effect of mice transplantability tumor H22 hepatocarcinoma
1. experiment material
1.1 laboratory animals: Kunming mouse, male, body weight 18 ~ 22g, totally 180, animal quality certification SCXK(river) 2008-24, purchased from Da Shuo bio tech ltd, Chengdu.
1.2 mouse H22 hepatoma carcinoma cell: purchased from Shanghai cell bank, by Sichuan University's preclinical medicine and pharmacology teaching and research room of legal medical expert institute frozen.
1.3 Experimental agents and reagent
1.3.1 trial drug: oxidized resveratrol, purity >=98%, wishes love purchased from ladder and changes into industrial (Shanghai) Co., Ltd.;
1.3.2 positive control drug: cyclophosphamide injectable powder, purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov., lot number 11030621;
1.3.3 reagent
0.9% sodium chloride injection (normal saline): purchased from ChengDu QingShan LiKang Pharmacy Co., Ltd, lot number A1201082.
1.4 Experimental agents preparations
Cyclophosphamide: be that the cyclophosphamide injectable powder of 0.2g/ bottle is dissolved in 10mL normal saline by specification, be mixed with the solution that concentration of cyclophosphamide is 20mg/mL, extraction this solution of 1.5mL and 3.5mL normal saline mix before use, obtain the solution that concentration of cyclophosphamide is 6mg/mL;
Oxidized resveratrol: be dissolved in DMSO by 4mg/10 μ L before using, after dissolving completely, slowly adds in 1mL normal saline, oxidized resveratrol final concentration 4mg/mL.
2. experimental technique
The preparation of 2.1 lotus tumor models
The mice being vaccinated with transplanted hepatoma H22 ascites is fed 7 ~ 10d under the condition of water and feedstuff abundance, after ascites grows, aseptically extract tumor liquid, be also fully mixed to get cell suspension by tumor liquid and the dilution proportion tumor liquid of the volume ratio=1:2 of normal saline.After expecting blue dyeing with platform, hematimeter counts, and wherein viable count is greater than 95%, is 1 × 10 by the concentration of normal saline adjustment cell suspension
7individual oncocyte/mL.Then the cell suspension adjusting concentration is only inoculated in tested animal oxter by 0.2mL/, builds solid tumor animal model, obtain H22 tumor-bearing mice.
2.2 animal grouping and medications
Mouse inoculation H22 hepatoma carcinoma cell, after 1 day, represents by random digit and mice is divided into negative control group, positive controls, oxidized resveratrol group, often organizes 10 mices; Each group according to following medication administration:
Negative control group: lumbar injection gives equal-volume normal saline, the volume giving normal saline is identical with the volume of oxidized resveratrol solution, injects 1 every day, injects 6 times weekly, amounts to injection 12 times, raises 14 days continuously;
Positive controls: lumbar injection gives cyclophosphamide solution (concentration 6mg/mL), the dosage of cyclophosphamide is 60mg/kg Mouse Weight, Per-Hop behavior 1 time, amounts to administration 2 times, raises 14 days continuously;
Oxidized resveratrol group: lumbar injection gives oxidized resveratrol solution (concentration 4mg/mL), and oxidized resveratrol dosage is 40mg/kg Mouse Weight, injects 1 every day, injects 6 times weekly, amounts to injection 12 times, raises 14 days continuously.
2.3 Testing index and method
2.3.1 ordinary circumstance
The reaction observed the autonomic activities of mice, the mental status, hair, breathing, diet, feces character every day and stimulate to external world.
2.3.2 tumor volume
Use the major diameter a of vernier caliper measurement tumor block, minor axis b every other day, by V=1/2 × a × b
2calculate tumor volume, draw mouse tumor block growth curve.
2.3.2 body weight
Claim Mouse Weight every other day, draw Mouse Weight with the change curve observing natural law.
2.3.3 the mensuration of tumor control rate and immune organ coefficient
In the 15th day, de-cervical vertebra put to death mice, took the body weight of each mice.Mice right axil tumor location skin is lived with tweezers tweezer, skin cut off by operation scissors, expose tumor, weigh by operating scissors blunt separation tumor, then abdominal cavity is cut off with operation, be separated spleen and the thymus of each group of mice successively, take the weight of spleen and thymus respectively, be calculated as follows tumor control rate, Spleen coefficient and thymus coefficient.
Tumor control rate (%)=[1-(average experiment group tumor weight/mean negative control group tumor weight)] × 100%
Spleen coefficient=[spleen weight (mg)/body weight (g)] × 100%
Thymus coefficient=[thymic weight (mg)/body weight (g)] × 100%
2.4 statistical method
Altogether in triplicate, result represents with mean+SD in the zoopery of the present embodiment, and sided t inspection checking each group of difference, P≤0.05 thinks to have statistical significance.
3. experimental result
The tumor control rate of 3.1 oxidized resveratrol medications on mice H22 hepatocarcinoma and the impact of mortality rate
In three tests, the tumor of each group is heavy, tumor control rate data are as shown in table 22 ~ 24, the tumor photo of each experimental mice is as shown in Figure 13 ~ 15, and from table 22 ~ 24 and Figure 13 ~ 15, oxidized resveratrol has obvious inhibitory action to mice transplanted tumor H22 hepatocarcinoma.
The tumor that table 22 first time tests each group of mice weighs and tumor control rate
The tumor that table 23 second time tests each group of mice weighs and tumor control rate
The tumor that table 24 third time tests each group of mice weighs and tumor control rate
3.2 oxidized resveratrol medications are to the growth inhibited effect of mice H22 hepatocarcinoma
In three tests, during each group of mice administration, gross tumor volume is as shown in table 25 ~ 27, Figure 16 ~ 18 are that the tumor volume of each group of mice is with the change curve observing natural law, from table 25 ~ 27 and Figure 16 ~ 18, oxidized resveratrol medication can slow down the speed of growth of mice transplanted tumor H22 hepatocarcinoma, has inhibition to tumor growth curve.
Gross tumor volume during table 25 tests each group of mice administration for the first time
Gross tumor volume during table 26 second time tests each group of mice administration
Gross tumor volume during table 27 tests each group of mice administration for the third time
3.3 oxidized resveratrol medications are on the impact of H22 tumor-bearing mice body weight
In three tests, during the administration of each group mice, average weight is as shown in table 28 ~ 30, and Figure 19 ~ 21 are each experimental mice body weight change curves with observation natural law, from table 28 ~ 30 and Figure 19 ~ 21: oxidized resveratrol has no significant effect Mouse Weight.
Average weight during table 28 tests each group of mice administration for the first time
Average weight during table 29 second time tests each group of mice administration
Average weight during table 30 tests each group of mice administration for the third time
3.4 oxidized resveratrol medications affect H22 tumor-bearing mice immune organ
In three tests, the Spleen coefficient of each group mice and thymus coefficient as shown in table 31 ~ 33, from table 31 ~ 33, oxidized resveratrol to the immune organ of mice without overt toxicity.
Table 31 tests Spleen coefficient and the thymus coefficient of each group of mice for the first time
Table 32 second time tests Spleen coefficient and the thymus coefficient of each group of mice
Table 33 tests Spleen coefficient and the thymus coefficient of each group of mice for the third time
Claims (2)
1. the application of oxidized resveratrol in the medicine of preparation treatment tumor, described tumor is Leukemia K562 cell, Proliferation of Human Ovarian Cell SK-OV-3, human breast cancer cell line Bcap-37, human liver cancer cells Hep G2, human lung cancer cell A549, KB cell HNE-1, human colon cancer cell SW480, mice transplantability tumor S180 sarcoma or mice transplantability tumor H22 hepatocarcinoma.
2. application according to claim 1, is characterized in that oxidized resveratrol being prepared into the solution use that concentration is 1.56mg/mL ~ 100mg/mL.
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