CN101485655A - Application of dihydromyricetin in preparing medicament for preventing and treating adverse reaction of tumor chemoradiotherapy - Google Patents
Application of dihydromyricetin in preparing medicament for preventing and treating adverse reaction of tumor chemoradiotherapy Download PDFInfo
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Abstract
The invention discloses application of dihydromyricetin in preparing a medicine for preventing and treating untoward reactions in radiotherapy and chemotherapy of tumor. In particular, the application is to prepare medicines for preventing and treating untoward reactions in radiotherapy and chemotherapy of tumor, preventing and treating arrest of bone marrow and baldness, resisting mutation, preventing and treating the generation of secondary tumors, preventing and treating tumor transfer, and preventing and treating breast cancer, cervical carcinoma, intestinal cancer and the like. The invention creatively finds that the dihydromyricetin has the effects of removing free radicals, inhibiting reaction chains of the free radicals, resisting the mutation, inhibiting the expression of the tumor gene, causing the death of tumor cells, preventing and treating the tumor generation, preventing and treating the secondary tumors and the transfer of the secondary tumors and preventing and treating infection. Therefore, as the medicine for preventing and treating untoward reactions in radiotherapy and chemotherapy of tumor, the dihydromyricetin can inhibit and alleviate chemical damage, prevent and treat damage of a mutagen and further prevent gene mutation or the secondary tumors; and by combination with the radiotherapy and chemotherapy, the effects of preventing untoward reactions in radiotherapy and chemotherapy of tumor and preventing generation of tumors are achieved.
Description
Technical field
The present invention relates to the dihydromyricetin new medical use, particularly relates to as the control untoward reaction of tumor chemoradiotherapy and toxic and side effects, prophylaxis of tumours generation and neoplasm metastasis, medicine such as prevent and treat that concurrency in the oncotherapy catches.
Background technology
Dihydromyricetin (dihydromyricetin) claims ampelopsin again, and formal name used at school is 3,5,7,3`, 4`, 5`-quercetagetin.Dihydromyricetin is a kind of compound known, document Walter Karrarr, and Birkhauser Verlag undStuttgart (1958), P.652, NO:1640 has announced that its structural formula is:
Application number 03123879 discloses the purposes of compositions in preparation broad-spectrum antiviral medicament and preparation raising immunity medicine that contains dihydromyricetin and ampelopsin; Application number 200410052153 discloses the application of ampelopsin in the medicine of preparation angiogenesis inhibitor; Application number 200410062289 discloses the purposes of ampelopsin in the medicine of preparation and aldose reductase relevant disease; Application number 03114133 discloses ampelopsin and has prevented and treated application in the AIDS-treating medicine in preparation; The application of application number 00117225 ampelopsin in preparation leukemia and anti-medicine for nasopharyngeal.But the unexposed dihydromyricetin of above-mentioned patent application is suppressing and is removing the chemical damage of free radical and the chemistry of preventing and treating body is poisoned, the medicine of diseases such as the untoward reaction of also unexposed dihydromyricetin control tumor chemoradiotherapy treatment, the infection of prophylaxis of tumours treatment concurrency and the application in the health food take place and neoplasm metastasis for prevention sudden change and secondary tumors.
Both at home and abroad in the antineoplastic clinical medicine, mainly comprise cytotoxic drug, hormone medicine and biological targeting curative, cell toxicant based chemotherapy medicine wherein comprises that the medicine of effect and DNA chemical constitution is (as mitomycin, cyclophosphamide, cisplatin etc.), influence the synthetic medicine of nucleic acid (as cytosine arabinoside, fluorouracil, mercaptopurine etc.), the medicine that acts on nucleic acid delivery is (as actinomycin D, Bleomycin A5 etc.), the topoisomerase I depressant is (as hydroxycamptothecin, topotecan etc.), act on the medicine (vincristine of tubulin, paclitaxel etc.) etc., these chemotherapeutics often produce many serious adverse reactions to tumor patient, comprise bone marrow depression, leucocytes reduction, myocardial toxicity, liver dysfunction, hypoimmunity, resistance reduces and easily causes antibacterial and viral infection, cell mutation and secondary tumors, neoplasm metastasis etc.In the radiotherapy in the treatment of tumor,, also caused many-sided untoward reaction of tumor patient because radiation produces significantly injury to body.Chemotherapeutics and radiotherapy can antitumor, but itself all more or less produces some untoward reaction, also are difficult to control and eliminate untoward reaction, especially body sudden change and secondary tumors etc.In the chemicotherapy treatment, chemotherapeutics causes free radical in vivo, or itself can be converted into the chemical constitution of radical form, attack cells membrane lipid, protein and nucleic acid molecules, destroy these macromolecular conformation and structural stabilities, cause macromolecular function to be compromised or inactivation gradually; Because the functionally inactive of high molecular weight protein, the fracture of the linear structure of nucleic acid (DNA and RNA) cause gene mutation to take place, the cytothesis function seriously descends, and the accumulation of gene mutation will cause new secondary tumors; Organ emergency reaction function and cell adverse circumstance repair ability reduce, the accumulation of chemical toxic and side effects will cause tissue and organ injury as bone marrow depression and low leukocyte counts cause immunity degradation and the concurrency infection is difficult to control, hair follicle and skin corium and damages and cause alopecia or erythra, digestive tract and digestive organs often are more vulnerable to the toxic action of cell toxicant based chemotherapy medicine, cause anorexia, vomiting, nauseating, the serious degradation down of muscle power.
The general hypoimmunity of tumor patient, ability to the reparation of the emergency reaction of exogenous chemical substances and toxic action is also very low, therefore press in the clinical therapy of tumor and can tumour progression and neoplasm metastasis take place, control prophylaxis of tumours, can control, alleviate or prevent and treat the untoward reaction of tumor pharmacother again, itself but seldom produce the novel control tumour medicine of adverse effect or toxic and side effects.And the dihydromyricetin that provides among the present invention is as the medicine of control adverse reaction of tumor chemoradiotherapy, again can antitumor, prevention cell mutation and secondary tumors and neoplasm metastasis, infection or bone marrow depression.
Summary of the invention
The purpose of this invention is to provide the application of dihydromyricetin at the medicine of preparation control adverse reaction of tumor chemoradiotherapy.Its chemotherapy is preferably the cytotoxic drug chemotherapy.Described cell toxicant based chemotherapy medicine is preferably the medicine that acts on the DNA chemical constitution, the medicine that acts on nucleic acid delivery, topoisomerase I depressant or acts on the medicine of tubulin.
The described synthetic medicine of nucleic acid that acts on is a fluorouracil.The described medicine that acts on tubulin is vincristine or paclitaxel.
The medicine of the described DNA of acting on chemical constitution is cyclophosphamide, mitomycin or cisplatin.
Described control adverse reaction of tumor chemoradiotherapy is bone marrow depression, hemocyte reduction, cell mutation, alopecia or secondary tumors.
Described adverse reaction of tumor chemoradiotherapy is breast carcinoma, cervical cancer or the untoward reaction of intestinal cancer chemicotherapy, and dihydromyricetin and chemotherapy drugs in combination medication improve the oncotherapy curative effect.
Described dihydromyricetin and chemotherapy drugs in combination medication are meant patch, ointment, gel, soft capsule or the suppository with the acceptable accessories preparation.
Described medicine is oral solid formulation, oral liquid, injection, lyophilized injectable powder or the infusion solutions dosage form with the acceptable accessories preparation.
The invention discovery dihydromyricetin is removed free radical, is suppressed the radical reaction chain, thereby prevention, control or elimination chemicals and radiation are to the injury or the murder by poisoning of histiocyte and organ, also find dihydromyricetin mutation and the expression of inhibition oncogene and triggering tumor cell apoptosis and prevent curing oncoma generation, control secondary tumors and transfer, also find dihydromyricetin inhibition virus transfection and prevent and treat infection.Therefore, dihydromyricetin is as the medicine of control adverse reaction of tumor chemoradiotherapy, suppress and alleviate the infringement of chemistry injury, control mutagenic agent and prevent gene mutation or secondary tumors, will realize preventing and treating adverse reaction of tumor chemoradiotherapy and prophylaxis of tumours with the chemicotherapy drug combination and take place.The immediate cause of most tumor patient death is not a tumor itself, but the untoward reaction of tumor chemoradiotherapy and concurrency infection etc.Therefore, the target of clinical therapy of tumor is in conjunction with resection operation, by Drug therapy, effectively to control and eliminate tumor, the untoward reaction in the treatment of reduction chemicotherapy, the complication in the elimination oncotherapy, raising patient's life quality and late result.The multi-functional characteristics that dihydromyricetin integrates multiple pharmacology and drug action are that single medicine is not available in the present clinical therapy of tumor, will make it be used widely in oncotherapy.
Description of drawings
Fig. 1 is that dihydromyricetin is to the protection of Hb A hemoglobin adult and the inhibitory action figure that the metahemoglobin that TBHP causes is generated.
Fig. 2 a is the ESR wave spectrum with the hydroxyl radical free radical of the Fenton reaction system generation of DMPO (0.1mol/L) capture.
Fig. 2 b adds the ESR wave spectrum of dihydromyricetin to the hydroxyl radical free radical of this system generation in the system.
Fig. 3 a is the O that the system of the illumination riboflavin that captures with DMPO/EDTA) produces
-2The ESR characteristic wave spectrum of free radical.
Fig. 3 b adds the O that dihydromyricetin produces this system in the system
-2The ESR characteristic wave spectrum of free radical.
The specific embodiment
In order to understand essence of the present invention better, describe the present invention in detail by the following examples, but should be appreciated that these embodiment just illustrate the present invention, rather than in office where face limits the scope of the invention.
Embodiment 1: extraction and purification prepares dihydromyricetin from porcelain ampelopsis
With 1 kilogram in the young tender leaf of porcelain ampelopsis or tender shoots or young tender leaf and tender shoots mixture, by the weight ratio of 1:8～1:12 (raw material: water) add entry, soak 30 minutes-4 hours after, be heated to boiling, insulation was extracted 30～60 minutes, and extracting solution is collected in filtered while hot or centrifugalize; (raw material: water) add entry, be heated to boiling, insulation was extracted 20～40 minutes, and filtered while hot or centrifugalize merge and collect extracting solution by the weight ratio of 1:5～1:8 once more; Concentrated extracting solution is to 20%～50% of original volume, adding edible ethanol to concentration of alcohol is 40%～75%, be heated to boiling back insulation 10～60 minutes, a little after the cooling, filter or the centrifugal precipitation of removing, reclaim ethanol or directly collecting precipitation (dry sediment promptly gets the porcelain ampelopsis extractive of general flavone, or the crude extract of dihydromyricetin) was left standstill in the filtrate cooling 1～2 day.With the weight ratio water heating for dissolving of precipitation with 1:5～10, the crystal that obtained dihydromyricetin in 1～2 day, separation and purification dihydromyricetin are left standstill in cooling; Again crystal is added the suitable quantity of water thermosol, the recrystallization thing that obtained dihydromyricetin in 1～2 day is left standstill in cooling; Can prepare content above the dihydromyricetin more than 99% by purification through 2～4 recrystallization.
Embodiment 2: the structural identification of dihydromyricetin
The dihydromyricetin of embodiment 1 preparation.
(1) nuclear magnetic resonance spectroscopy
The magnetic resonance detection instrument is an INOVA500 superconduction pulse fourier transform nmr spectrometer.
Compose check and analysis through proton nmr spectra, carbon spectrum, DEPT spectrum, gCOSY spectrum, gHMQC spectrum, gHMBC, determine itself and 3,5,7,3,, 4,, 5, the structure of-hexahydroxy 2,3 flavanonols conforms to.
1. hydrogen spectrum, gCOSY compose
Spectrum width: 6711.4Hz (～13ppm)
GCOSY spectrum: spectrum width 4598.2 * 4598.2Hz; Data point 2048 * 256.
Table 1,1H nuclear magnetic resoance spectrum data and analysis
Chemical shift COSY is relevant
Spectrum peak sequence number is split branch peak number coupling constant proton number remarks
δ (ppm) honeybee
4.90，
10.5,613 CH of a 4.40 dd
5.69
10.5 1 4.40 2 CH of b 4.90 d
61 4.40 3 CH of c 5.69 d
21 5.90 8 CH of d 5.85 d
E 5.90 d 21 5.85 6` position CH
F 6.40 S 2 2`/6
CH
G 8.11 S 1 4` position OH
H 8.81 S 2 3`/5` position
OH
17 OH of i 10.74 br s
15 OH of j 11.86 S
2. carbon spectrum, DEPT spectrum, gHMQC spectrum, gHMBC compose
Spectrum width: 30911.9Hz (～240ppm)
GCOSY spectrum: spectrum width 4598.2 * 4598.2Hz; Data point 2048 * 256
Table 2,13C nuclear magnetic resoance spectrum data and analysis
The chemical shift multiplicity
Spectrum peak sequence number carbon number remarks
δ(ppm) (DEPT)
13 CH of A 71.55 d
12 CH of B 83.15 d
18 CH of C 94.85 d
16 CH of D 95.84 d
E 100.38 s 1 10` position C
F 106.85 d, 2 2` (6`) position CH
G 127.02 s 1 1` position C
H 133.36 s 1 4` position C
I 145.60 s, 2 3` (5`) position
C
19 C of J 162.40 s
15 C of K 163.23 s
17 C of L 166.69 s
M 197.40 s 1 4` position C=0
(2) mass spectral analysis of dihydromyricetin
Detecting instrument is a VG ZAB-HS chromatograph mass spectrometer.
The determining molecular weight of dihydromyricetin is 321M/Z, and is consistent with theoretical molecular and related fragment peak.
(3) ultraviolet spectral analysis
The ultraviolet absorption peak of dihydromyricetin in methanol is λ 1=292nm, λ 2=208nm.
Embodiment 3: the inhibitory action that the metahemoglobin that dihydromyricetin causes the special butane of peroxidating (TBHP) generates
1, experimental technique
Packed red cells is added distilled water, make 1% erythrocytic complete hemolysis liquid, add 37 ℃ of temperature of each sample component respectively and incubate 30min, add TBHP and make its final concentration reach 250uM, continue temperature and incubate 30min, measure OD
630Detect the relative variation of ferrihemoglobin content.High ferritin generates the calculating of suppression ratio:
Generate suppression ratio=(oxidative damage group OD
630-sample sets OD
630)/(oxidative damage group OD
630-matched group OD
630).
2, experimental result
TBHP discharges H gradually at aqueous phase
2O
2, H
2O
2By bonded ferrum catalysis in free or the haemachrome, produce superoxide anion and hydroxyl radical free radical by the Fenton reaction.The maximum light absorption value of the α of normocyte hemoglobin, β peptide chain respectively 540,577nm, after the oxidation that erythrocyte is subjected to TBHP was attacked, the part HbO2 Oxyhemoglobin converted metahemoglobin rapidly to, and the highest absworption peak is arranged near 630nm.As shown in Figure 1, under the effect of TBHP, HbO2 Oxyhemoglobin (its maximum absorption band is at 540nm and 575nm place) is destroyed, and part is converted into metahemoglobin (its maximum absorption band is at the 630nm place).And after dihydromyricetin joined in the erythrocytic complete hemolysis liquid, TBHP generated the promotion of the destruction of HbO2 Oxyhemoglobin and metahemoglobin and has been subjected to inhibitory action, and the absworption peak of HbO2 Oxyhemoglobin raises, and the absworption peak of metahemoglobin reduces.
As shown in table 3, the inhibitory action that the addition of dihydromyricetin and its generate metahemoglobin be proportionate and dose-effect relationship obvious.
The influence that the metahemoglobin that table 3, dihydromyricetin cause TBHP generates
Embodiment 4, ESR technology for detection dihydromyricetin are to measured by esr technique
The ESR technology is the most direct effective method of research freedom base and antioxidant.In order to detect and recognize the short life free radical, need a undersaturated diamagnetic substance-scavenger of free radicals is added reaction system, catch instantaneous free radical, thereby can obtain using ESR spectrometer long free radical of detected life-span at normal temperatures.
1. material and method
Material: DMPO (5,5-imethyl-1-pyrroline-1-oxide) purify the back free from admixture ESR signal of purifying before use with active carbon.Acetone, ethyl acetate, riboflavin, diphenyl picryl phenylhydrazine reagent such as (DPPH) are analytical pure.Dihydromyricetin is the separation and purification preparation.
Brucker 200 type ESR spectrometers are used in the ESR test, and test condition is: X-band, and microwave power 20mW, modulation 100kHz, amplitude modulation 0.1mT sweeps wide 20mT, and central magnetic field 324.5mT detects under the room temperature.
Utilize the Fenton reaction to produce the OH free radical, capture the OH free radical with DMPO: reaction system is totally 50 μ L, and pH=7.4 wherein contains 0.04M DMPO, 0.01%H
2O
2, 0.025mM FeSO
4, sample solution or PBS (phosphate buffer pH=7.4) 5 μ L, mixing sucks in the quartz capillary ESR wave spectrum when writing down a minute rapidly.Illumination riboflavin produces O
2 -Free radical, DMPO captures O
2 -Free radical.Reaction system is totally 30 μ L, and pH=7.4 wherein contains 0.08M DMPO, 0.3M riboflavin, 5mM EDTA sodium salt, 2mM DETAPAC, sample or PBS5 μ L, and mixing sucks in the quartz capillary rapidly, and illumination was tested after 90 seconds.
2. result
The ESR wave spectrum of the hydroxyl radical free radical that produces by Fenton reaction (Fig. 2 a) by 4 spectral lines form the typical collection of illustrative plates that aspect ratio is 1:2:2:1 (g=2.0045, aN=aH=1.49mT).Adding dihydromyricetin does not influence the hyperfine splitting constant and the g value of spectroscopic signal, along with the increase of dihydromyricetin concentration, and its ESR signal intensity successively decrease (Fig. 2 b).So peak heights h (mm) is represented the relative intensity of ESR signal with the peak at spectroscopic signal second peak.In the Fenton reaction system, the dihydromyricetin that adds variable concentrations is removed the hydroxyl radical free radical that this system produces, and the ESR wave spectrum after the removing as shown in the figure.After adding the dihydromyricetin of variable concentrations, the ESR signal intensity obviously reduces, and has tangible dose-dependent effect (table 4).
Shown in Fig. 3 a, Fig. 3 b, with the O of DMPO trapped light according to riboflavin/EDTA system generation
-2The typical collection of illustrative plates that the ESR wave spectrum of free radical is made up of 12 spectral lines (aN=1.42mT, aBH=1.12mT, aCH=0.13mT).Add or do not add dihydromyricetin to the hyperfine splitting constant of spectroscopic signal not influence, only signal intensity difference.After adding the dihydromyricetin of variable concentrations, wherein (b) (c) the wave spectrum peak height of (d) significantly reduce, along with the increase of sample concentration, remove O
-2The effect of free radical is also more and more obvious.When sample concentration is 100ug/mL, with the O that produces
-2Free radical 100% is removed.
Table 4, ESR method detect the removing ability of dihydromyricetin to free radical
Annotate: clearance rate can be calculated divided by the peak height that contrasts with the peak height of certain sample concentration.The computational methods of peak height: hydroxyl radical free radical is got the height at second peak, and superoxide radical is got first peak, and DPPH gets the 3rd peak.Diphenyl picryl phenylhydrazine (DPPH) free radical be a kind of stable be the free radical at center with nitrogen.
Dihydromyricetin can be removed superoxide radical, hydroxyl radical free radical and contain nitrogen free radical, be owing to his molecular structure own, not only but chelating is eliminated the initiator of metal ion as radical chain reaction, stop and suppress the generation of free radical, and can remove, capture free radical its reaction chain is interrupted, bring into play antioxidation in cell in body and the biochemical metabolism reaction thereof, participate in, mediate and regulate body widely, bring into play its pharmacology and drug action with this.Free radical is the main cause that many chemical substances and radiation produce injury or poison body cell.The radical pair cell membrane produces damage, the molecular conformation of lipid, protein and nucleic acid is implemented non-specific hydrogen extracting and chemical addition and formed high molecular free radical, these high molecular free radicals will form high molecular polymer or cause the fracture of nucleic acid linear molecule with other protein high molecular, thereby will destroy high molecular structure and function.
Embodiment 5, dihydromyricetin are to H
2O
2The inhibitory action of inductive cellular oxidation damage
The Hela cell is with new-born calf serum, 100U/ml penicillin, 100mg/ml streptomycin that contains 10% deactivation and the DMEM culture medium that contains the 2mmol/ml glutamate, Glu, at 5%CO
2, cultivate under 37 ℃, relative humidity 95% condition.The cultured cell monolayer growth, when the coverage of bottle floor cells reaches 90%, with 0.25% trypsinization, the cultivation of going down to posterity.Changed liquid once in per two days, only the cell of exponential phase of growth is used for experimentation.
The cell of exponential phase of growth is made single cell suspension with 0.25% trypsinization, with the DMEM culture medium dilution that contains 10% new-born calf serum, adjusts cell density to 2 * 10
4/ ml is inoculated in 96 well culture plates with every hole 100ul, 37 ℃, 5%CO
2Condition was cultivated 24 hours.Adding dihydromyricetin makes its final concentration be respectively 5ug/ml, 10ug/ml and 15ug/ml.Incubation adds the H of various dose in the Hela cell after 30 minutes
2O
2As the oxidative damage agent;
At each different H
2O
2Under the concentration, H
2O
2Can both significantly reduce the vigor of Hela cell.Work as H
2O
2Concentration when being equal to or less than 1.2mM, the dihydromyricetin processed group is with respect to adding the dihydromyricetin group, the final concentration that the Hela cell viability is significantly increased and increase degree and dihydromyricetin mostly be proportionate (table 5).
Table 5, dihydromyricetin are to H
2O
2The influence of inductive Hela cellular oxidation damage
Embodiment 6, dihydromyricetin are to the test of pesticide effectiveness of bone marrow depression mouse model:
1, material and method
Cyclophosphamide; Dihydromyricetin.Kunming mouse, 18～22g, 30,8～12 ages in week.Male and female are regardless of, and are provided by Zhongshan University's Experimental Animal Center.
Modeling method: cyclophosphamide 175mg/kg intraperitoneal injection of mice, 0.2ml/ time, once a day, continuous three times.
Animal grouping and processing: 10/group,
1. modeling group: cyclophosphamide 175mg/kg intraperitoneal injection of mice, once a day, continuous three times.
2. normal control group: injecting normal saline is 0.2ml/ time when other group injection cyclophosphamide.
3. dihydromyricetin group: cyclophosphamide 175mg/kg intraperitoneal injection of mice, once a day, continuous three times.Irritate stomach once every day according to dihydromyricetin 120mg/kg, continuous 10 days.The observation sign changes.
Handling animal the next day behind the dihydromyricetin the last time observes.Adopt tail vein blood, carry out cytometry.After mice is put to death, take out femur, reject muscle and connective tissue, cut off femur two, go out medullary cell with RPMI 1640 liquid, detect the bone marrow nucleated cell number with No. 6 syringe needles.
2, experimental result
Dihydromyricetin has significant protective effect (table 6) to the bone marrow depression of cyclophosphamide, and the bone marrow nucleated cell number of cyclophosphamide group is compared obvious minimizing with matched group, and the recovery of dihydromyricetin acceleration loss injury of the bone marrow hemopoietic function.
Table 6, respectively organize peripheral hemogram and sign and change
The antimutagenic effect of embodiment 7, dihydromyricetin
1, experimental technique:
Ring phosphorus phthalein amine (CyCloPhosphamide is called for short CP), ametycin (Mitomyein is called for short MMC), N-methyl-N '-nitro-N-nitrosoguanidine (N-methyl-N '-nltro-N-nitrosogua-nidine be called for short MNNG), colchicine.
(1) white mice bone marrow PCE micronucleus test: the NIH white mice is divided into 5 groups at random, i.e. negative control group, 3 dihydromyricetin test group and 1 positive controls.Negative control group mice every ig0.3ml normal saline every day, 3 dihydromyricetin administration groups are respectively with 20,40 and the dihydromyricetin dosage ig mice administration of 60mg/kg, the CP of lumbar injection 100mg/kg dosage when being administered to the 7th day, again with the CP intraperitoneal injection of mice of same dose, put to death mice behind the 6h behind the 24h.The positive controls mice is the ig normal saline only, and the time of lumbar injection CP, dosage had both been handled consistent with the administration group.Preparation mouse Bone marrow cells micronucleus slide sample, under optical microscope, count polychromatic erythrocyte (PCE) sum and the PCE number that contains micronucleus, 2 above micronucleus in 1 PCE, occur all by 1 cytometer, and calculate the micronucleus cell rate (MNCF) of respectively organizing by following formula:
MNCF=(the PCE number/observed PCE sum that contains micronucleus) * 1000%.
(2) the rugged change of mouse marrow cell chromosome (CA) test: animal grouping and dihydromyricetin dosage are all identical with " bone marrow cells in mice PCE micronucleus test ", different is at the MMC of administration administration group and 1 2mg/kg dosage of the equal lumbar injection of positive controls mice in the 7th day, before putting to death mice 3h again the colchicine solution of lumbar injection 4mg/kg dosage carry out pretreatment, legal system is equipped with the bone marrow cell chromosome slide sample routinely.Each experimental group microscopy is observed 500-1000 metaphase phase, record chromosomal aberration cell number and distortion type, the main chromatid break of observing, akinetic chromosome part and ring chromosome etc. calculate distortion cell rate of each group=contain cell number metaphase of cell division of cell number/observation of CA then.
2, experimental result
(1) dihydromyricetin is to being brought out the inhibitory action of mouse Bone marrow cells micronucleus by CP
Dihydromyricetin shows the depression effect experiment of the mouse bone marrow cells PCE micronucleus that brought out by CP, bone marrow cells in mice MNCF high, that the dihydromyricetin mutation test group CP of low 3 dosage of neutralization brings out significantly is lower than positive controls, the dihydromyricetin test group is respectively 68.5% to the suppression ratio of the MNCF that brought out by CP, 61.0% and 42.6% (suppression ratio=[MNCF of (MNCF of MNCT one test group of positive controls)/positive controls] * 100%) shows certain dose-effect relationship in the dosage range of being surveyed.
(2) dihydromyricetin is to the inhibitory action of the bone marrow cells in mice cA that brought out by MMc
The CA cell rate (CAF) of dihydromyricetin test group compares with the CAF of MMC positive controls, all reach significant difference (P＜0.05), the dihydromyricetin mutation test group of low, high 3 dosage of neutralization is respectively 65.1% to the suppression ratio of the CAF that brought out by MMC, 56.8% and 49.1%, a little more than high dose group, dosage and effect relation are negative correlation to low dosage dihydromyricetin test group to the depression effect of CA.
The antimutagenic effect of dihydromyricetin shows that it has the carcinogenic effect of inhibition, promptly has carcinogenesis and anti-mutation effect, thereby the prevention body cell is undergone mutation and canceration.The generation of tumor at present mostly is the mutagenesis chemical substance greatly and causes body injury or inducing cell sudden change and canceration, therefore, dihydromyricetin has the drug actions such as canceration formation under prophylaxis of tumours generation, the high carcinogenic condition of secondary tumors, control and the minimizing of prophylaxis of cancer patient in long-term chemotherapy.
Embodiment 8, dihydromyricetin are to the protective effect of tumor-bearing mice radiation damage
1, experimental technique
The NIH white mice; Dihydromyricetin.
(1) dihydromyricetin is to the influence of NIH white mice irradiation
Age in NIH white mice 6-7 week, 10 every group,, the male and female dual-purpose, grouping comprises:
1. normal control group: drink-service normal saline;
2. irradiation group: 60Co radiation gamma;
3. according to preceding drink-service 0.1% dihydromyricetin normal saline solution, refuse to obey dihydromyricetin according to the back;
4. irritate stomach (ig) 100mg/kg according to the back;
5. according to back ig200mg/ (bwd) hemopoietic treasured.The equal free drink-service of pre-irradiation 7d.
The equal free drink-service of pre-irradiation 7d, irradiation back 7d ig.100mg/kg every day, femoral artery was got blood in the 10th day, and counting WBC and RBC put to death back counting thymus and index and spleen index with each group Mus again.Irradiation: the 60Co source is provided by south China agricultural university irradiation center, the disposable irradiation of gamma-rays, and total absorbed dose is 4.5Gy, absorbed dose rate is 0.97Gy/min.
(2) dihydromyricetin is to the influence of tumor-bearing mice irradiation
The mice in 18 ages in week, body weight 22～24g is divided into 6 groups at random, and 10 every group, A group: CK; B group: lotus tumor; C group: lotus tumor+dihydromyricetin+irradiation; D group: lotus tumor+irradiation+dihydromyricetin; E group: lotus tumor+irradiation; F group: lotus tumor+dihydromyricetin.After raising 7d, all the other press the inoculation of transplanted tumor organon except that the A group.The 5ml disposable syringe extracts S180 ehrlich ascites carcinoma mouse ascites, after normal saline dilutes 5 times, and every injected in mice 0.3ml (except the A group).Beginning in the 2nd day, C group and F group mice ig. every day dihydromyricetin, dosage is 100/kg.Behind the 15d, C, D and E group are carried out the 60Co radiation gamma, and absorbed dose are 100cGy.The irradiation back is to D group mice ig dihydromyricetin (dosage is the same), and the C group then stops the ig. dihydromyricetin.Behind 15d, take off neck and put to death again, dissect, press preceding method and detect erythrocyte, leukocyte, immune organ index.
2, experimental result
(1) dihydromyricetin influences the immune organ radiation damage
Behind the radiation gamma, mouse thymus and spleen major injury.Mice is being subjected to all to be significantly higher than matched group according to front or rear dihydromyricetin ig thymus index and index and spleen index.
(2) dihydromyricetin is to the influence of mice hemopoietic function
The erythrocyte of ig. dihydromyricetin group mice, leukocyte are all apparently higher than irradiation group (table 7) behind predose ig. dihydromyricetin group and the irradiation.Show that dihydromyricetin has tangible protection effect to the hemogram damage due to the irradiation.
Table 7, dihydromyricetin are to the effect of irradiation mice hemogram
Annotate: compare * p＜0.05, * * p＜0.01 with the irradiation group; Compare #p＜0.01 with matched group
(3) dihydromyricetin is to the influence of tumor-bearing mice irradiation
The experimental result of mice hemogram and weight gain sees Table 8.With A group relatively, 3 kinds of physical signs of tumor-bearing mice all are lower than the normal saline matched group, index of each group of exposure reaches significance level, mouse-borne tumor be described after, body constitution descends, and is subjected to according to back decline more obvious.3 kinds of physical signs of F group all are higher than the B group, illustrate that dihydromyricetin has certain guarantee to the quality of life of tumor-bearing mice.The leucocyte level of C, D, F group all is higher than the E group, and reaches significant level, illustrates that dihydromyricetin has significant protective effect to the irradiation damage of murine interleukin.Dihydromyricetin has also promoted the recovery of the body weight of mice.
Table 8, dihydromyricetin are to the effect of irradiation mice hemogram
Annotate: compare * p＜0.05 with matched group, compare ☆ p＜0.01 with the B group;
Compare #p＜0.01 with the E group; With C group comparison ▲ p＜0.01
Tumor-bearing mice immune organ experimental result (table 9) shows that the cell number of every gram spleen, thymus and index and spleen index, thymus index significantly are lower than A group blank behind the B group mouse-borne tumor.4 kinds of indexs of C, D and F group tumor-bearing mice all are higher than B group tumor-bearing mice, and significant difference.Illustrate that dihydromyricetin can keep the proliferate of the immunocyte of tumor-bearing mice, and near normal level.4 kinds of indexs of C, D all are higher than the E group, and except that every gram thymocyte cell number of D, other all reach significant level, show that dihydromyricetin keeps body's immunity to have remarkable drug action in the radiation therapy of tumor.
Table 9, dihydromyricetin are to the effect of irradiation mice hemogram
Annotate: compare * p＜0.05 with matched group, compare ☆ p＜0.01 with the B group;
Compare #p＜0.01 with the E group; With C group comparison ▲ p＜0.01
This experimental study shows that dihydromyricetin has and suppresses damage and the toxic action of radiation to immunocyte and immune organ, this body protective for the tumor radiotherapy treatment, alleviates and control the drug action of untoward reaction in the chemotherapy and bone marrow depression, human body immunity improving power.
Embodiment 9, dihydromyricetin are to the inhibition and the apoptotic effect of HeLa Cells and transplanted tumor
1, experimental technique
HeLa Cells; The BALB/c-nu nude mice.Dihydromyricetin.
RPMI-1640 culture medium and pancreatin (EDTA) U.S. Gibco company product.Newborn calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd., and tetrazolium bromide (MTT) dye liquor is Beijing Zhong Shan company product.Annexin V-FITC/PI apoptosis detection kit is a Bender Medsystem company product.
(1) dihydromyricetin is to cervical cancer HeLa cell inhibiting and apoptotic effect
Cervical cancer HeLa cell culture places 5%CO2,37 ℃, the constant-temperature enclosed incubator of 95% saturated humidity to carry out routine cultivation in containing 10% newborn calf serum and two anti-RPMI-1640 culture medium, every day observation of cell growing state.3d goes down to posterity 1 time.When 0-80 μ g/ml dihydromyricetin concentration increased progressively, porous plate was cultivated the HeLa cell, 3 multiple holes, and mtt assay detects cell viability, flow cytometer observation of cell apoptotic effect.
(2) dihydromyricetin is to the inhibitory action of cervical cancer HeLa cell transplantation tumor
The BALB/c-nu nude mice, age in 4-6 week, 18-22g, female, the human cervical carcinoma Hela cell (1 * 10 of the trophophase of taking the logarithm
7/ ml), in the place's subcutaneous vaccination of the nearly right hind of nude mice dorsal part, 0.2ml/ is only under aseptic condition.The 14th day visible tumor growth in inoculation back, tumor is cauliflower form, and color is red, and matter is hard, and is movable, and tumor is grown to about 50-80mm
3, but in time, begin the tumor experiment.
Behind the nude mice subcutaneous vaccination tumor 6d, 32 mouse subcutaneous transplanting places all become tumor, and mice is divided into 4 groups at random.Matched group, normal saline 0.5ml/ are only; The dihydromyricetin group, 60mg/kg, 1 time/day, ip0.5ml; The 5-Fu group, 30mg/kg, ip, 1 time/4d, totally 6 times; Dihydromyricetin-5-Fu group, 30mg/kg, 1 time/day, ip0.5ml, 5-Fu, 30mg/kg, ip, 1 time/4d, totally 6 times.Intraperitoneal injection, continuous use 24d, every injected in mice amount of liquid equates, is 0.4mL, puts to death whole mices and carries out the index detection in the 26th day.
Tumour inhibiting rate is measured: adjust administration by changes of body mass; Inoculation back was measured major diameter (a) of 1 mice Subcutaneous tumor and minor axis (b) in per 2 days, calculated gross tumor volume [V=(a * b2)/2], the drafting tumor growth curve.The inoculation back was taken off cervical vertebra on the 26th day and is put to death mice, and the complete tumor of peeling off is weighed, and calculates tumour inhibiting rate, tumour inhibiting rate=(the average tumor quality of the average tumor quality/matched group of 1-intervention group) * 100%.
2, experimental result
(1) dihydromyricetin is to cervical cancer HeLa cell inhibiting and apoptotic effect
Dihydromyricetin has utmost point significant inhibitory effect (table 10) to the propagation of HeLa Cells.
Table 10, dihydromyricetin are to the inhibitory action of cervical cancer HeLa cytoactive
With the two mark dyeing of AnnexinV-PI flow cytometry analysis dihydromyricetin cervical cancer HeLa apoptosis is shown, behind the dihydromyricetin effect cervical cancer HeLa cell 24,48 of variable concentrations, the 72h, the apoptosis incidence rate obviously increases, strengthen and prolongation action time with dihydromyricetin concentration, apoptosis rate obviously increases, and compares with matched group to have the utmost point significance difference opposite sex.Dihydromyricetin induces HeLa Cells to have tangible drug level dependency, and section increases with the increase apoptosis rate of drug level at one time, both be proportionate (P＜0.01).(24,48,72h) apoptosis rate of cell increases same concentration different time sections gradually, and the apoptosis rate behind the 80 μ g/ml concentration dihydromyricetin effect 72h is the highest, surpasses 70%.
(2) dihydromyricetin is to the inhibitory action of cervical cancer HeLa cell transplantation tumor
Behind the inoculation cervical cancer HeLa oncocyte, tumor formation rate is 100%.Each volume of organizing the subcutaneous transplantation tumor increases gradually, and tumor becomes the growth of local nodositas, and postvaccinal the 1st～6 day, growth of xenografted speed was basic identical, but subsequently the matched group growth of xenografted obviously faster than other administration groups.Behind the 26d, dihydromyricetin processed group gross tumor volume and tumor quality all are lower than matched group (P＜0.01), and with treatment matched group there was no significant difference (P〉0.05) relatively, show that dihydromyricetin has remarkable antitumor action (table 11) to cervical cancer.
Table 11, dihydromyricetin are to the effect of cervical cancer inhibition of proliferation
*P＜0.01 is with respect to negative control group.
Embodiment 10, dihydromyricetin are to human breast carcinoma transplanted tumor in nude mice cell proliferation and apoptotic effect
1, experimental technique
Material: the purebred nude mice BALB/c of Healthy female (nu/nu) in 4～6 ages in week, weighs 13～20g, available from Zhongshan University's Experimental Animal Center.5-fluorouracil (5-Fu); Ki67, Bcl-2 antibody and related kit step neoplasm technology company limited available from Foochow.Original position apoptosis detectable ln Situ Cell Death Detection (Pod kit) is available from Roche company.
Human breast cancer cell strain MDA-MB-231; Dihydromyricetin.
The foundation of human breast cancer cell transplanted tumor in nude mice: human breast cancer cell strain MDA-MB-231 cultivates in containing the RPMI1640 culture fluid of 10% hyclone, and exponential phase cell preparation suspension is collected in the amplification of going down to posterity, and viable cell concentrations is 1 * 10
7/ ml.The operation that experimentizes under the SPF environment, inoculating cell suspension 0.2ml/ only (contains cell number 2 * 10 in the right breast mammary gland of every nude mice fat pad
6Individual), form obvious visible transplanted tumor, the medication of 32 mice with tumor random packet about 4 weeks.
Animal grouping and medication:
1. matched group: 8, propylene glycol 0.1ml/ only, ip, 15d altogether; 0.9%NS, 0.1ml/, ip, 5d altogether.
2. dihydromyricetin group: 8, dihydromyricetin 60mg/ (kgd) is dissolved in the 0.1ml propylene glycol, ip, 15d altogether; 0.9%NS0.1ml/, ip, 5d altogether.
3. 5-Fu organizes: 8,5-Fu30mg/ (kgd) is dissolved in 0.9%NS0.1ml, ip, 5d altogether; Propylene glycol 0.1ml/, ip, 15d altogether.
4. dihydromyricetin+5-Fu organizes: 8, dihydromyricetin 60mg/ (kgd) is dissolved in the 0.1ml propylene glycol, ip, 15d altogether; 5-Fu30mg/ (kgd) is dissolved among the 0.9%NS0.1ml, ip, 5d altogether.
Tumour inhibiting rate is observed: with the maximum major diameter (a) of vernier caliper measurement tumor, transverse diameter (b), calculate gross tumor volume every 2d, and average tumor volume=(a * b2)/2, and draw the growth of xenografted curve; After experiment finishes, the complete tumor that strips, the weighing tumor is heavy.Calculate the tumour inhibiting rate of medicine as follows: tumour inhibiting rate=(the average tumor of the average tumor weight/matched group of 1-experimental group is heavy) * 100%.
The detection that Ki67, Bcl-2 express: Ki67 antigen adopts SP method immunohistochemical staining, and the positive cell karyon is dyed pale brown color.Press following calculating K i67 index (Ki67-LI): (* 100 times) define 5 visuals field of representational Ki67 stained positive under the low power, several 200 tumor cells in (* 400 times) each visual field under the high power, and wherein the shared percentage ratio of positive cell is Ki67-LI.Bcl-2 adopts SP method immunohistochemical staining, and the positive cell endochylema is dyed pale brown color.Judge by the following result that carries out: observe the percentage ratio of positive cell in 100 tumor cells, its positive rate＜10% is (-), and positive rate 10%～25% is (+), and positive rate 25%～50% is (++), positive rate〉50% be (+++).Negative control all replaces first antibody with PBS.
2, experimental result
The MDA-MB-231 breast cancer cell is inoculated in nude mice mammary gland fat pad, forms obviously visible transplanted tumor about 4 weeks, is the growth of lobulated or nodositas.During the medication, nude mice does not have death.Dihydromyricetin group, 5-Fu group, dihydromyricetin+5-Fu group tumour inhibiting rate are respectively 41.5%, 44.6%, 50.3%, are subjected to suppress the most obvious (table 12) with drug combination group tumor growth.
Table 12, dihydromyricetin are to the inhibitory action of people's mastadenoma
Dihydromyricetin is to the influence of transplanted tumor cell proliferation
Observe tumor tissues under light microscopic, matched group transplanted tumor cell still keeps the original atypia of cancerous cell under the cultivation conditions, and out-of-shape is examined big engrain, nuclear atypia, visible unusual karyokinesis phase.Dihydromyricetin medication group tumor cell presents degeneration in various degree, and the part tumor tissues occurs obviously downright bad.The Ki67 SABC shows: the Ki67 antigen presentation is in breast cancer cell nuclear, and positive cell is sepia dyeing.Matched group Ki67-LI is higher than dihydromyricetin group, 5-Fu group, dihydromyricetin+5-Fu group, significant difference (P＜0.01) (table 13) is arranged, show that dihydromyricetin medication group tumor cell proliferation is subjected to obvious inhibition, in conjunction with spectroscopic analysis, dihydromyricetin also has certain directly cytotoxic effect to tumor cell.
Table 13, medicine are to the influence of tumor cell proliferation, apoptosis
*P＜0.01 is with respect to negative control group; △ P＜0.05 is between 5-Fu and dihydromyricetin processed group.
Dihydromyricetin is to the apoptotic influence of transplanted tumor
The Tunel method detects, and sees that under fluorescence microscope apoptotic cell is the yellow-green fluorescence cell, and cell volume dwindles shrinkage, and the treatment group is than matched group apoptotic cell showed increased.See under the light microscopic that apoptotic nucleus dyes pale brown color.The result shows: dihydromyricetin group, 5-Fu group, dihydromyricetin+5-Fu group apoptotic index are apparently higher than matched group (P＜0.01), and dihydromyricetin+5-Fu group is organized (P＜0.05) apparently higher than dihydromyricetin group, 5-Fu.Dihydromyricetin can be induced breast cancer cell generation apoptosis, and drug combination, the inducing apoptosis of tumour cell effect strengthens.
Bcl-2 SABC testing result in the breast cancer tissue
The Bcl-2 protein expression is starched in breast cancer cell, and dihydromyricetin group, 5-Fu group, dihydromyricetin+5-Fu group are starkly lower than matched group (P＜0.05), show that dihydromyricetin can suppress the protein expression (table 13) of bcl-2 gene.
Table 13, respectively organize the Bcl-2 of breast cancer tissue protein expression
The inhibitory action that embodiment 11, dihydromyricetin are expressed close bone metastatic breast cancer cell tumour metastatic gene bone sialoprotein
1, experiment material and method
The MDA-MB-231-BO cell
DMEM culture fluid dry powder, Gibco company, hyclone.
Matched group and dihydromyricetin processed group are set; Matched group is cultivated the MDA-MB-231-BO cell for containing 10% hyclone culture fluid, and the dihydromyricetin processed group is the dihydromyricetin that adds 20mg/L on the basis of matched group.All the other operations are identical.
The SABC method detects the proteic expression of breast cancer tumour cell bone sialoprotein (BSP).
Preparation MDA-MB-231-BO cell climbing sheet adopts routine immunization group SP method to detect the expression of BSP.Treat that the degrees of fusion of cell on microscope slide reaches 75～90%, use the PBS of 0.01mol/L to wash slide 3 times, 10% formaldehyde fixed 30min.0.01mol/L PBS washing, hatch 10min under 3% the H2O2 methanol solution room temperature, 0.01mol/L PBS washing, drip reagent B (normal non-immune serum) in the drawn scope of wax crayon, incubated at room 10min adds 50 μ l one anti-(mouse-anti hBSP) through the 1:200 dilution, room temperature is placed 60～90min, matched group only adds antibody diluent, and the PBS washing of 0.01mol/L drips biotin labeled second antibody (sheep anti-mouse igg), incubated at room 10min, 0.01mol/L PBS washing, drip 1 reagent D (streptomycete antibiotic-peroxide enzymatic solution), incubated at room 10min, 0.01mol/L PBS washing, the DAB colour developing, tap water flushing, haematoxylin redyeing, the tap water flushing, the dehydration of ethanol gradient, clarifier is handled 2 * 10min, gummy mounting.Microscopically is observed, and occurring brown yellow granule in the cell cytoplasm is that BSP expresses the positive.
2, experimental result and discussion
Detect the proteic expression of BSP by the SABC method, in the close bone metastatic breast cancer cell MDA-MB-231-BO kytoplasm brown particle is arranged, show MDA-MB-231-BO cellular expression BSP.And all not having brown particle in the cell cytoplasm that dihydromyricetin is handled, prompting cell BSP expresses and is negative.
(bone sialoprotein BSP) is a kind of acidoglycoprotein in the extracellular matrix to the bone sialoprotein, mainly is distributed in the tissue of mineral nitrogenization, by osteoblast, osteoclast and chondrocytes expressed and secretion.The expression that increases BSP in the existing research report breast cancer cell promotes the generation that the breast cancer cell bone shifts.The BSP positive cancer cell breaks away from blood circulation and enters medullary cavity, stick the surface that is positioned bone mineral nitrogen substrate, the RGD sequence of BSP combines with α V β 3, causes breast cancer cell and bone trabecular sticking, the activation osteoclast produces molten bone bone resorption, promotes the bone of breast cancer cell to shift process.Experimental result prompting dihydromyricetin can suppress the transfer of breast cancer tumour.
Embodiment 12, dihydromyricetin are to people's colon-cancer cell and transplanted tumor in nude mice inhibited proliferation
1, experimental technique
Human colon adenocarcinoma cell line lovo cell; Dihydromyricetin.
Cell culture: the lovo cell is with the new-born calf serum that contains 10% deactivation, two 1640 anti-culture medium, at 5%CO
2, cultivate under 37 ℃, relative humidity 95% condition.The cultured cell monolayer growth, when the coverage of bottle floor cells reaches 90%, with 0.25% trypsinization, the cultivation of going down to posterity.Cell with exponential phase of growth is used for experimentation.
When 0-120 μ g/ml dihydromyricetin concentration increased progressively, porous plate was cultivated the lovo cell, 3 multiple holes, and mtt assay detects cell viability, observes dihydromyricetin to the tumor cell proliferation inhibitory action.
The foundation of human colon adenocarcinoma cell's transplanted tumor in nude mice: human colon adenocarcinoma cell's strain lovo cultivates in containing the RPMI1640 culture fluid of 10% new-born calf serum, and exponential phase cell preparation suspension is collected in the amplification of going down to posterity.The operation that experimentizes under the SPF environment, abdomen drosal part subcutaneous injection contains 5 * 10
6The suspension 0.5ml of cancerous cell, becoming after the tumor with piece of tissue sleeve pipe skill of handling needles subcutaneous transplantation is solid tumor.The nude mice of human colon carcinoma subcutaneous transplantation is divided into 3 groups at random, 8 every group, male and female half and half.(1) model group (negative control), normal saline 0.5ml/ only irritates stomach; (2) dihydromyricetin group is irritated stomach with 80mg/kg; (3) 5-Fu group (positive control) is with the 5-Fu30mg/kg lumbar injection; (4) dihydromyricetin-5-Fu group is with dihydromyricetin 40mg/kg filling stomach and with the 5-Fu30mg/kg lumbar injection.Beginning administration in the 6th day after transplanting, 1 time/d of 5-Fu group administration, drug withdrawal after shared 6 days, 1 time/d of dihydromyricetin group administration, in totally 6 weeks, matched group gives normal saline.24h puts to death after the drug withdrawal, peels off tumor mass and weighs, and observes antitumor action.
2, experimental result (1) dihydromyricetin is to the inhibition and the apoptotic effect of colon-cancer cell
The lovo cell is 20,40,60 at the dihydromyricetin final concentration, during 80ug/ml, cell viability obviously descend and each concentration under cell viability and the poor heteropole of negative control group significantly (p＜0.01) (table 14).
The two mark dyeing of AnnexinV-PI flow cytometry analysis dihydromyricetin to the apoptotic dihydromyricetin effect lovo cell 72h that influences variable concentrations of lovo after, the apoptosis incidence rate obviously increases, strengthen and prolongation action time with dihydromyricetin concentration, apoptosis rate obviously increases, and compares with matched group to have the significance difference opposite sex.
Table 14, dihydromyricetin are to the apoptotic influence of intestinal cancer lovo (x ± s)
Dihydromyricetin induces people's intestinal cancer lovo cell to have tangible drug level dependency, and section increases with the increase apoptosis rate of drug level at one time, both be proportionate (P＜0.01).The apoptosis rate of same concentration different time sections cell increases gradually.Apoptosis rate behind the 120 μ g/m concentration dihydromyricetin effect 72h has reached 48.7%.
(2) dihydromyricetin is to colon-cancer cell transplanted tumor in nude mice inhibited proliferation
Observe tumor tissues under light microscopic, matched group transplanted tumor cell still keeps the original atypia of cancerous cell under the cultivation conditions, out-of-shape, visible unusual karyokinesis phase.Dihydromyricetin administration group tumor cell presents degeneration in various degree, and the part tumor tissues occurs obviously downright bad, shows that dihydromyricetin is to intestinal cancer tumor cell proliferation significant inhibitory effect (table 15).
Table 15, dihydromyricetin are to the inhibitory action of intestinal cancer tumor proliferation
*P＜0.01 is with respect to negative control group.
The bacteriostasis of embodiment 13, dihydromyricetin is observed
1, material
For the examination antibacterial
Bacillus subtilis (Bacillus subtilis)
Staphylococcus aureus (Staphylococcus aureus)
Salmonella (Salmonella typhi)
Diplococcus pneumoniae (Pneumococcus)
Escherichia coli (Escherichia coli)
Bacillus pyocyaneus (Pseudomonas aeruginosa)
Ordinary culture medium: Carnis Bovis seu Bubali cream 3g, peptone 10g, sodium chloride 5g, tap water (or distilled water) 1000ml transfers Ph7.2-7.4 (solid medium adds 2% agar in addition).Be used for the cultivation of staphylococcus aureus, escherichia coli, bacillus pyocyaneus, Salmonella.
Enriched medium: in the aseptic liquid nutrient medium of ordinary culture medium, add 5% aseptic calf serum.Be used to cultivate Diplococcus pneumoniae.
2, to the mensuration of several antibacterial minimum inhibitory concentrations and minimum bactericidal concentration
Get the 20ml triangular flask and organize into groups numbering respectively, except that the 1st bottle, each bottle adds the 3ml culture fluid; Add in the 1st bottle and contain dihydromyricetin culture fluid 6ml, draw 3ml and be added in the 2nd bottle, fully behind the mixing, sucking-off 3ml to the 3 manages, and increases progressively successively to be diluted to the 6th bottle, and sucking-off 3ml mixed liquor discards behind the abundant mixing of the 6th pipe.(bacterial concentration is 10 to add bacteria suspension 50 μ l respectively in each bottle
6-10
7Individual/as ml), to shake up rearmounted 37 ℃ of shaking tables and cultivate 24h, take out observed result.Positive control and negative control are provided with a flask culture.From each bottle of bacterial growth few (cultivating not muddy), continue to cultivate after one day, get and cultivate bacterium liquid coating Agar Plating, observation has or not growth to be judged after 37 ℃ of cultivations.With the high dilution of dihydromyricetin that can bacteria growing inhibiting is minimum inhibitory concentration (MIC), is minimum bactericidal concentration (MBC) with the dihydromyricetin greatest dilution that does not still have bacteria growing
3, measurement result
Dihydromyricetin all has antibacterial action (table 16) to several antibacterials, and the measurement result of minimum inhibitory concentration and minimum bactericidal concentration shows that dihydromyricetin all has sterilizing ability to these antibacterials.
Table 16, dihydromyricetin all have antibacterial action to several antibacterials
Embodiment 14, dihydromyricetin are to the therapeutical effect of mice CC14 chronic injury hepatitis
1, material and method
The female BALB/C mice of animal: 18-22g, random packet.After the fasting 6 hours, press 0.5ml/kg body weight lumbar injection CCl
4(10% paraffin oil solution), secondary weekly, totally eight times.
Experiment is divided into three groups, and the dihydromyricetin group is irritated stomach by the 120mg/kg body weight every day, and normal control group and damage matched group are given and corresponding normal saline.Last administration was killed Mus on the 3rd day, got hematometry alanine aminotransferase (ALT) and serum albumin (g/L).Get a fritter hepatic tissue of hepatomegaly leaf same area simultaneously, after 10% formalin fixed, do check pathological section.
2, experimental result
CC14 contamination group, inflammatory cell infiltration is obvious around the lobules of liver, as seen proliferation of fibrous tissue, big the hepatic necrosis in lobule center is obvious, part of hepatocytes fat becomes, the apparition of cavity sample: the liver histological change of dihydromyricetin extract for treating group and CC1 4 contamination groups have obviously different, and liver proliferation of fibrous tissue and obvious hepatic necrosis are not seen in most visuals field, and inflammatory cell infiltration and fat-like degeneration are lighter.
Serum zymetology and serum albumin check result (table 17) show that dihydromyricetin is to CC1
4The chronic injury mice has significant protective effect.
Table 17, dihydromyricetin are to CCl
4Chronic injury mice serum transaminase and sero-abluminous influence (x ± s, n=10)
* P＜0.01 and CCl
4Group relatively.
CCl
4After entering in the body, activate, claim trichloromethyl free radical (CCl through liver cytochrome P 450
3), attack phospholipid molecule on the endoplasmic reticulum by the absorption of hydrogen, cause the lipid peroxidation of film, CCl
3Then carry out covalent bond with membrane lipid and protein macromolecule, cause the destruction of membrane structure and functional completeness, CCl
3The activity of calcium ion pump on film also capable of inhibiting cell and the microsomal membrane increases flow of calcium ions, thereby causes cytotoxic death.Dihydromyricetin can improve the degeneration of chronic hepatic injury hepatic tissue cavity sample, liver fatization and Fibrotic morphological change, obviously reduces in the serum liver transaminase and promotes albuminous content in the serum, liver function protecting.
The preparation of embodiment 15, dihydromyricetin tablet
Tablet formulation: dihydromyricetin 30-80%, medical starch 2-10%, lactose 10%-60%, aspartame 0.2-1%, magnesium stearate 0.5-1%
With dihydromyricetin 1000g and the lactose 1000g dry powder blend that 100 orders sieve, cross 80 mesh sieves 2-3 time; Add an amount of distilled water, with after the medical starch 60g heating gelatinizing, be sprayed in the mixed powder, and constantly stir, granulate, dry after crossing 16 mesh sieves; Add aspartame 0.5% and the magnesium stearate 0.5% that 100 orders sieve, mixing, tabletting according to dry granular weight.Sheet hardness is 0.9-1.2kg, and sheet heavily is the 0.5-1.0g/ sheet.
Embodiment 16, the capsular preparation of dihydromyricetin
Capsule prescription: dihydromyricetin 30-80%, medical starch 5-30%, lactose 0%-60%, magnesium stearate 0.5-1%.With the dry powder blend of dihydromyricetin 1000g, medical starch 100g, lactose 200g, cross 80 mesh sieves 2-3 time; Add an amount of distilled water, with after an amount of medical starch heating gelatinizing, be sprayed in the mixed powder, stir, granulate, dry after crossing the 16-24 mesh sieve; Add magnesium stearate 0.5%, mixing according to dry granular weight.Filled capsules, the heavy 0.25-0.75g/ grain of grain.
The preparation of embodiment 17, dihydromyricetin patch
100g is cut into strip with raw rubber, is pressed into reticular film with glue pressing machine, remove static, put cold, immersed in the gasoline about 24 hours, and made its abundant swelling, moved in the ingredients pot stir about again 3 hours, add Colophonium 80g, zinc oxide 80g, lanoline 15g, vaseline 15g, liquid paraffin 10g successively, mixing adds dihydromyricetin 100g, stir evenly, filter, be coated with cream through 80 order copper wire screen clothes, cutting, lid lining, stripping and slicing, make 1000 and paste, promptly get the dihydromyricetin patch.
Claims (10)
1, dihydromyricetin is in the application of the medicine of preparation control adverse reaction of tumor chemoradiotherapy.
2, dihydromyricetin according to claim 1 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine described chemotherapy is the cytotoxic drug chemotherapy.
3, dihydromyricetin according to claim 2 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine described cell toxicant based chemotherapy medicine is the medicine that acts on the DNA chemical constitution, the medicine that acts on nucleic acid delivery, topoisomerase I depressant or the medicine that acts on tubulin.
4, dihydromyricetin according to claim 2 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine the described synthetic medicine of nucleic acid that acts on is a fluorouracil.
5, dihydromyricetin according to claim 2 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine the described medicine that acts on tubulin is vincristine or paclitaxel.
6, dihydromyricetin according to claim 2 is in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine, and the medicine that it is characterized in that the described DNA of acting on chemical constitution is cyclophosphamide, mitomycin or cisplatin.
7, dihydromyricetin according to claim 1 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine described control adverse reaction of tumor chemoradiotherapy is bone marrow depression, hemocyte reduction, cell mutation, alopecia or secondary tumors.
8, dihydromyricetin according to claim 1 is in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine, it is characterized in that described adverse reaction of tumor chemoradiotherapy is breast carcinoma, cervical cancer or the untoward reaction of intestinal cancer chemicotherapy, dihydromyricetin and chemotherapy drugs in combination medication improve the oncotherapy curative effect.
9, dihydromyricetin according to claim 8 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine described dihydromyricetin and chemotherapy drugs in combination medication are meant patch, ointment, gel, soft capsule or the suppository with the acceptable accessories preparation.
10, dihydromyricetin according to claim 1 is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine described medicine is oral solid formulation, oral liquid, injection, lyophilized injectable powder or infusion solutions dosage form with the acceptable accessories preparation.
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| CN1293825C (en) * | 2002-08-30 | 2007-01-10 | 广州拜迪生物医药有限公司 | Application of dihydromyricetin in preparation of food, cosmetics or medicine |
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