CN101485791B - Application of Ampelopsis grossedentata total flavone in preparing medicament for preventing and treating adverse reaction of tumor chemoradiotherapy - Google Patents

Abstract

The invention discloses application of total flavone of ampolopsis grossedentata in preparing medicines for preventing and treating untoward reaction in tumor radiation treatment and chemo-treatment, and in particular relates to application of the total flavone of ampolopsis grossedentata in preparing medicines for preventing and treating the untoward reaction in tumor radiation treatment and chemo-treatment, preventing and treating arrest of bone marrows and alopecia, having anti-mutagenic function, preventing and treating generation of secondary tumor and tumor metastasis, and preventing and treating breast carcinoma, cervical carcinoma, intestinal carcinoma and the like. The invention creatively finds out that the total flavone of ampolopsis grossedentata eliminates free radicals, inhibits free radical reaction chains, uses THT for anti-mutation, inhibits tumor gene expression and initiates tumor cell apoptosis so as to prevent and treat tumor generation, secondary tumor and tumor metastasis, uses the THT to inhibit virus transfection so as to prevent and treat infection, and realizes prevention, control or elimination of hazards or toxic hazards of chemical medicines and radiation on histiocyte and organs.

Description

Ampelopsis grossedentata total flavone is in the application of the medicine of preparation control adverse reaction of tumor chemoradiotherapy

Technical field

The present invention relates to the Ampelopsis grossedentata total flavone new medical use; Particularly relating to the application of Ampelopsis grossedentata total flavone at the medicine of preparation control adverse reaction of tumor chemoradiotherapy, specifically is as the untoward reaction of control tumor chemoradiotherapy and toxic and side effects, prophylaxis of tumours generation and neoplasm metastasis, medicine or health food such as prevent and treat that concurrency in the oncotherapy catches.

Background technology

Ampelopsis grossedentata (A.grossedentata) is the Vitaceae ampelopsis, and stem and leaf that its children is tender and bud are Ramulus et Folium Mussaendae Pubescentis through processing and dry, are also referred to as Ampelopsis grossedentata, field mother-in-law's tea etc.The geographic Ramulus et Folium Mussaendae Pubescentis in Guangdong and Guangxi Provinces removes Ampelopsis grossedentata, also comprises ampelopsis cantoniensis (Acantoniesis).

Ampelopsis grossedentata is distributed widely in provinces and regions such as the Guangdong, Guangxi, Yunnan, Hunan, Hubei, Jiangxi of China, and content reaches more than 20% in its young young stem and leaf THT dry weight, is one of plant that the physiologically active ingredient content of monomer is the highest in the natural plants.According to investigations, Ampelopsis grossedentata is " Ampelopsis grossedentata ", " Ramulus et Folium Mussaendae Pubescentis ", is the special product of Qingyuan City, Guangdong Province, edible more than 1000 year history that records among the people in Guangdong and Guangxi Provinces.Ramulus et Folium Mussaendae Pubescentis has effects such as heat-clearing and toxic substances removing, expelling wind and removing dampness, the broken knot of dissipating blood stasis.

Total flavones in the Ampelopsis grossedentata (Ramulus et Folium Mussaendae Pubescentis) has multiple pharmacological effect effect, and application number 01109641.1 the invention discloses a kind of treatment hepatitis that has, hepatoprotective, the flavonid composition of anti-inflammation and the effect of enhance immunity systemic immunity power; Application number 200810071687.2 provides the total flavone valid target that is rich in ampelopsin to prevent and treat the application in the carcinoma of prostate medicine in preparation; Application number 01106978.3 open Ampelopsis grossedentata total flavone is treated medicine of oral ulcer and preparation method thereof as smelting; Application number 200410048628.5 discloses ampelopsis and the application of extract in preparation control sleep disorder medicine and health product thereof.But above-mentioned patent application is not illustrated Ampelopsis grossedentata total flavone and is being suppressed and removing the chemical damage of free radical and the chemistry of preventing and treating body is poisoned, prevention sudden change and secondary tumors take place and neoplasm metastasis, does not set forth medicine and the application in the health food of the diseases such as untoward reaction, the infection of prophylaxis of tumours treatment concurrency of THT control tumor chemoradiotherapy treatment yet.

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 comprise effect with the medicine (like mitomycin, cyclophosphamide, cisplatin etc.) of DNA chemical constitution, influence the synthetic medicine of nucleic acid (like cytosine arabinoside, fluorouracil, mercaptopurine etc.), act on nucleic acid delivery medicine (like actinomycin D, Bleomycin A5 etc.), topoisomerase I depressant (like hydroxycamptothecin, TPT etc.), act on the medicine (vincristine, paclitaxel etc.) of tubulin etc.; These chemotherapeutics often produce many serious adverse reactions to tumor patient, comprise that bone marrow depression, leucocytes reduction, myocardial toxicity, liver dysfunction, hypoimmunity, resistance reduce easy antibacterial and viral infection, cell mutation and secondary tumors, the neoplasm metastasis etc. of causing.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 Ampelopsis grossedentata total flavone 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 Ampelopsis grossedentata total flavone (being called for short THT) at the medicine of preparation control adverse reaction of tumor chemoradiotherapy.Its chemotherapy is preferably the cytotoxic drug chemotherapy, and said 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.Saidly be preferably that to act on the synthetic medicine of nucleic acid be fluorouracil.The said medicine that acts on tubulin is preferably vincristine or paclitaxel.The medicine of the said DNA of acting on chemical constitution is preferably cyclophosphamide, mitomycin or cisplatin.

Said control adverse reaction of tumor chemoradiotherapy is bone marrow depression, hemocyte reduction, cell mutation, alopecia or secondary tumors.

Said adverse reaction of tumor chemoradiotherapy is breast carcinoma, cervical cancer or the untoward reaction of intestinal cancer chemicotherapy, improves the oncotherapy curative effect with the chemotherapy drugs in combination medication.

Said medicine is oral solid formulation (comprising tablet, capsule, pill and granule), oral liquid, injection, lyophilized injectable powder or infusion solutions dosage form with the acceptable accessories preparation.

Said medicine is with patch, ointment, gel, liniment, spray, soft capsule or the suppository of acceptable accessories preparation.

The method for preparing of THT and the method for assay of also providing of the present invention.

The invention property discovery Ampelopsis grossedentata total flavone 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; Find that also anti-curing oncoma generation, control secondary tumors and transfer find that also THT suppresses virus transfection and prevents and treats infection to the THT mutation with suppressing oncogene expression and triggering tumor cell apoptosis.Therefore; THT 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 the generation of adverse reaction of tumor chemoradiotherapy and prophylaxis of tumours with the chemicotherapy drug combination.The dead immediate cause of most tumor patients 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, through 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 THT integrates multiple pharmacology and drug action are that single medicine is not available in the present clinical therapy of tumor, will make it in oncotherapy, be used widely.

Description of drawings

Fig. 1 (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. 1 (b) adds the O that THT 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, specify the present invention through following embodiment, but should be appreciated that these embodiment just explain the present invention, rather than manner in office limits scope of the present invention.

The extraction of embodiment 1:THT with separate preparation

Process for extracting: with the young tender leaf of exsiccant porcelain ampelopsis and the mixture of tender shoots; Weight ratio by 1: 8～10 (raw material: water) add purified water; Be heated to 70 ℃ and soak after 0.5-1 hour, be heated to boiling, insulation was extracted 30～60 minutes; With ceramic membrane (30KD holds back) cross-flow ultrafiltration, collect extracting solution while hot; Press 1: 5～1: 8 weight ratio (dried raw material: water) add purified water, be heated to boiling, be incubated and extracted 20～40 minutes,, merge the collection extracting solution once more while hot with ceramic membrane (30KD holds back) cross-flow ultrafiltration; Extracting solution is evaporated to volume when being 20%～40% (or relative density is 1.06-1.10) of original volume, and adding edible ethanol to ethanol final concentration is 50%～75%, is heated to boiling reflux insulation 20～60 minutes; Filter pressing or the centrifugal deposition of going; After reclaiming ethanol, the cooling of will filtrating was left standstill 1～2 day, collected deposit and (supernatant was poured out; Intermediary turbid solution filter pressing or centrifugal collecting precipitation), this deposit is the THT CE; In the total flavones CE, add an amount of purified water, stirring and evenly mixing left standstill 1 day, collected deposit, (decompression) below 85 ℃ drying, and the dry thing of acquisition is THT.It is light grey to the yellow-white powder pulverizing the back.

The colorimetric method for determining of the flavones content of embodiment 2:THT

1. principle: the phenolic hydroxyl group in the chromocor compound can and Al ³⁺Ion forms yellow complex, and characteristic absorption wavelength is 314nm.Colour developing concentration and flavones content are linear within the specific limits.

2. instrument and reagent

Analytical balance, sensitivity 0.0001g; Ultraviolet-visible spectrophotometer;

Absolute methanol (AR); 5%AlCl3 solution; Dihydromyricetin (reference standards).

3. detection method

(1) preparation of standard solution: the dihydromyricetin standard substance are put in the tared dish, be dried to constant weight in 105 ℃, take out and put natural cooling in the exsiccator, precision takes by weighing 0.0504g immediately, and dissolve with methanol is settled to 50ml.

(2) normal concentration gradient appearance liquid preparation: draw above-mentioned standard solution 0 (as blank), 2.0,4.0,6.0,8.0,10.0ml respectively, put in the 10ml volumetric flask, be settled to 10ml, shake up the back room temperature and place for use with absolute methanol.

(3) colorimetric operation: with the normal concentration gradient appearance liquid 0.3ml that all accurately takes a sample, put in the 10ml volumetric flask, pipette 3.0ml5%AlCl ₃Solution mixes, and adds methanol constant volume to scale, changes 10ml tool plug after shaking up in vitro, and room temperature is placed.Behind the 40min, be reference with the blank, measure each sample light absorption value, formulate standard curve at wavelength 314nm.

(4) sample determination: precision takes by weighing the about 0.0406g of THT, dissolve with methanol standardize solution 50ml.Measure according to standard substance colorimetric process.

4. result: the Concentraton gradient of standard reference material dihydromyricetin (mg/ml) has good linear relationship with light absorption value (OD314nm), and its relational expression is Y=2.0574x-0.0012 [R ²=0.9999, wherein x is concentration (mg/ml), and y is light absorption value (OD314nm)].The OD314 of the THT sample of measuring is 1.598, and through calculating, the concentration of its total flavones is 0.7761mg/ml, and content of total flavone is 95.58%.

The inhibitory action that the metahemoglobin that embodiment 3:THT causes the special butane of peroxidating (TBHP) generates

1, experimental technique

Packed red cells is added distilled water, process 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 through 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 receives TBHP was attacked, the part HbO2 Oxyhemoglobin converted metahemoglobin rapidly to, and the highest absworption peak is arranged near 630nm.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).As shown in table 1, the inhibitory action that the addition of THT and its generate metahemoglobin be proportionate and dose-effect relationship obvious.After explaining that THT joins in the erythrocytic complete hemolysis liquid, TBHP has received inhibitory action to the destruction of HbO2 Oxyhemoglobin and the promotion generation of metahemoglobin, and the absworption peak of HbO2 Oxyhemoglobin raises, and the absworption peak of metahemoglobin reduces.

The influence that the metahemoglobin that table 1, THT cause TBHP generates

THT (μ g/ml) metahemoglobin generates suppression ratio (%)

10 9.3

20 17.6

40 31.5

80 37.2

THT is to measured by esr technique for embodiment 4:ESR technology for detection

The ESR technology is the 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, thus can obtain can with the ESR spectrometer at normal temperatures the detected life-span than the free radical of growing.

1. material and method

Material: DMPO (5,5-imethyl-1-pyrroline-1-oxide) purify back free from admixture ESR signal before use with active carbon.Acetone, ethyl acetate, riboflavin, diphenyl picryl phenylhydrazine reagent such as (DPPH) are analytical pure.THT 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 PBS 5 μ L, and mixing sucks in the quartz capillary rapidly, and illumination was tested after 90 seconds.

2. result

It is 1: 2: 2 that the ESR wave spectrum of the hydroxyl radical free radical that is produced by Fenton reaction is formed aspect ratio by 4 spectral lines: and 1 typical collection of illustrative plates (g=2.0045, aN=aH=1.49mT).Add hyperfine splitting constant and the g value not influence of THT to spectroscopic signal, along with the increase of THT concentration, its ESR signal intensity successively decreases.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 THT that adds variable concentrations removes the hydroxyl radical free radical that this system produces, and the ESR wave spectrum after the removing is as shown in Figure 1.After adding the THT of variable concentrations, the ESR signal intensity obviously reduces, and has tangible dose-dependent effect.

Shown in Fig. 1 a, figure 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 the hyperfine splitting constant not influence of THT to spectroscopic signal, only signal intensity is different.As shown in table 2, add the THT of variable concentrations after, 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 100 μ g/ml, with the O that produces ^-2Free radical 100% is removed.

Table 2 ESR method detects the removing ability of THT to free radical

Add THT concentration (μ g/ml) 20 40 100 200 contrasts and be PBS

(percent % 27.4 50.3 65.5 100 0 to remove the OH free radical

Add THT concentration (μ g/ml) 3 15 30 100 contrasts and be PBS

Remove O ²Free radical (%) 27.2 60.9 79.4 100 0

Add THT concentration (μ g/ml) contrast and be anhydrous second

2 5 10 20

Alcohol

Remove DPPH free radical (%) 40.5 50.7 85.5 100 0

Annotate: clearance rate can be used the peak height calculating of the peak height of certain sample concentration divided by contrast.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.

THT can remove superoxide radical, hydroxyl radical free radical and contain nitrogen free radical; Be owing to his molecular structure own, but not only 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:THT is to H ₂O ₂Cause the effect that erythrocyte hemolysis and MDA generate

1. method

Get fresh anticoagulant people venous blood, the centrifugal 10min of 3000r/min abandons supernatant, and (the centrifugal 10min of 3000r/min repeats 2 times, obtains packed red cells for PBS, pH=7.4) washing with PBS; With pH7.4PBS packed red cells is made into 1%.Respectively get 1ml 1% red cell suspension and add THT to 10,20,40,80ug/ml respectively, 37 ℃ of incubation 30min, adding final concentration then respectively is 300mmol/L H ₂O ₂, 37 ℃ of temperature were incubated 3 hours, and the centrifugal 10min of 3000r/min gets supernatant and measures OD ₅₄₀(A); With packed red cells, become 1% erythrocyte with distilled water diluting, as the contrast of complete hemolysis, the same processing measured OD ₅₄₀(B).By formula (A/B) * 100% calculates hemolytic variation.Get the content (pressing the assay method of MDA test kit) that above-mentioned reaction mixture detects malonaldehyde (MDA) again.

2. experimental result

THT is to H ₂O ₂The generation of inductive erythrocyte hemolysis and MDA all has significant inhibitory effect.In the oxidative damage group, the erythrocyte hemolysis rate is up to 65.12 ± 3.13%, and the MDA growing amount is up to 9.35 ± 0.20nmol/ml; When THT concentration was 10ug/ml, the erythrocyte hemolysis rate dropped to 52.60 ± 2.91%, and the MDA growing amount drops to 7.98 ± 0.19nmol/ml; The erythrocyte hemolysis rate dropped to 36.75 ± 1.43% when THT concentration was elevated to 20ug/ml; The MDA growing amount drops to 3.94 ± 0.14nmol/ml; Between adjacent THT concentration; Erythrocyte hemolysis rate and MDA growing amount all have utmost point significant difference (P＜0.01), and the THT group all has utmost point significant difference (P＜0.01) with the oxidative damage group.

Table 3 THT is to H ₂O ₂The influence that erythrocyte hemolysis that causes and MDA generate

The actual content of MDA has reflected the concentration of MDA, the major reason of old degenerative disease development.

The growing amount of erythrocytic hemolysis rate and MDA has close getting in touch, and in the process that THT concentration raises, their range of decrease is consistent.Erythrocyte membrane causes lipid peroxidation under the attack of free radical, MDA is the intermediate product of lipid peroxidation process, and its growing amount is the important indicator of level of lipid peroxidation; And along with the aggravation of membrane lipid peroxide injury, the integrity of erythrocyte membrane is destroyed, and has finally caused haemolysis.Because erythrocyte hemolysis rate and MDA growing amount all are the reflection of lipid peroxidation injury on different levels, so have also shown suitable concordance between them.

Embodiment 6:THT is to H ₂O ₂The inhibitory action of inductive cellular oxidation damage

The Hela cell is with NBCS, 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 processed single cell suspension with 0.25% trypsinization, with the DMEM culture medium dilution that contains 10% NBCS, adjustment 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 THT 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 THT processed group is with respect to adding the THT group, the final concentration that the Hela cell viability is significantly increased and increase degree and THT mostly be proportionate (table 4).

Table 4 THT is to H ₂O ₂The influence of inductive Hela cellular oxidation damage

Embodiment 7:THT is to the test of pesticide effectiveness of bone marrow depression mouse model:

1, material and method

Cyclophosphamide; THT.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 divides into groups and handles: 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. THT organizes: cyclophosphamide 175mg/kg intraperitoneal injection of mice, once a day, continuous three times.Irritate stomach once every day according to THT125mg/kg, continuous 10 days.The observation sign changes.

Handling animal the next day behind the THT 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 RPMI1640 liquid, detect the bone marrow nucleated cell number with No. 6 syringe needles.

2, experimental result

THT has significant protective effect (table 5) 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 THT acceleration loss injury of the bone marrow hemopoietic function.

Peripheral hemogram respectively organized by table 5 and sign changes

The body weight of counting WBC RBC PLT nucleated cell presses down fur and takes off

Group

(* 10 ⁹/ L) (* 10 ¹²/ L) (* 10 ¹¹/ L) (* 10 ⁶/ L) system (%) falls

Normal control group 5.4 ± 0.9 4.6 ± 0.7 7.35 ± 0.113 2.08 ± 0.14--few

7.2 many, be prone to

Modeling group 2.7 ± 0.6 3.0 ± 0.6 2.45 ± 0.101 1.2 ± 0.3

Come off

THT group 4.3 ± 0.5 ^*4.0 ± 0.5 ^*4.78 ± 0.118 ^*1.89 ± 0.16 ^*1.5 it is few

The antimutagenic effect of embodiment 8:THT

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 THT test group and 1 positive controls.Negative control group mice every ig 0.3ml every day normal saline; 3 THT administration groups are respectively with 20,40 and the THT 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; Counting polychromatic erythrocyte (PCE) sum and the PCE number that contains micronucleus under optical microscope; 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 THT 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, prepare the bone marrow cell chromosome slide sample by conventional method.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) THT is to being brought out the inhibitory action of mouse Bone marrow cells micronucleus by CP

THT 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 THT mutation test group CP of low 3 dosage of neutralization brings out significantly is lower than positive controls; The THT test group is respectively 69.5% to the suppression ratio of the MNCF that brought out by CP; 60.1% and 41.5% (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) THT is to the inhibitory action of the bone marrow cells in mice cA that brought out by MMc

The CA cell rate (CAF) of THT test group compares with the CAF of MMC positive controls; All reach significant difference (P＜0.05); The THT mutation test group of low, high 3 dosage of neutralization is respectively 64.6% to the suppression ratio of the CAF that brought out by MMC; 57.3% and 48.5%, a little more than high dose group, dosage and effect relation are negative correlation to low dosage THT test group to the depression effect of CA.

The antimutagenic effect of THT 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 causes body injury or inducing cell sudden change and canceration for the mutagenesis chemical substance mostly; Therefore, THT 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 9:THT is to the protective effect of tumor-bearing mice radiation damage

1, experimental technique

The NIH white mice; THT.

(1) THT 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, divide into groups to comprise:

1. normal control group: drink-service normal saline;

2. irradiation group: 60Co radiation gamma;

3. according to preceding drink-service 0.1%THT normal saline solution, refuse to obey THT according to the back;

4. irritate stomach (ig) 100mg/kg according to the back;

5. precious according to back ig 200mg/ (bwd) hemopoietic.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) THT 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+THT+ irradiation; D group: lotus tumor+irradiation+THT; E group: lotus tumor+irradiation; F group: lotus tumor+THT.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.THT every day, dosage is 100/kg.Behind 15 d, 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 THT (dosage is the same), and the C group then stops ig.THT.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) THT influences the immune organ radiation damage

Behind the radiation gamma, mouse thymus and spleen major injury.Mice is receiving all to be significantly higher than matched group according to front or rear THT ig thymus index and index and spleen index.

(2) THT is to the influence of mice hemopoietic function

The erythrocyte of ig.THT group mice, leukocyte are all apparently higher than irradiation group (table 6) behind predose ig.THT group and the irradiation.Show that THT has tangible protection effect to the hemogram damage due to the irradiation.

Table 6 THT is to the effect of irradiation mice hemogram

Processed group erythrocyte (* 10 ¹²/ L) cell (* 10 ⁹/ L)

① 9.53±0.85 ?7.35±0.59

② 4.45±0.75# 1.89±0.53#

③ 5.47±0.46* 4.31±0.42*

④ 7.61?±0.53** 5.25±0.65**

⑤ 8.15±0.53** 6.27±0.53**

Annotate: compare * p＜0.05, * * p＜0.01 with irradiation group; Compare #p＜0.01 with matched group

(3) THT is to the influence of tumor-bearing mice irradiation

The experimental result of mice hemogram and weight gain is seen table 7.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 receives according to back decline more obvious.3 kinds of physical signs of F group all are higher than the B group, explain that THT 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, explains that THT has significant protective effect to the irradiation damage of murine interleukin.

Table 7 THT is to the effect of irradiation mice hemogram

Processed group erythrocyte (* 10 ¹²/ L) leukocyte (* 10 ⁹/ L) weight average weightening finish (g)

A 5.53±0.40☆ 5.45±1.25☆ 5.90±0.82☆#▲

B 2.45±1.25*# 2.89±1.13*# 1.51±0.95☆#▲

C 3.78±1.10☆* 4.55±0.52☆# 3.57±0.85☆#▲

D 3.21±0.65* 4.19±0.75☆# 2.32±0.81*☆

E 3.55±0.55☆ 3.46±0.85#▲ 2.45±1.32*▲

F 4.18±0.50☆ 4.75±0.50☆ 2.85±1.51*☆

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 8) 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.Explain that THT 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 THT keeps body's immunity to have remarkable drug action in the radiation therapy of tumor.

Table 8 THT is 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 THT has and suppresses damage and the toxic action of radiation to immunocyte and immune organ, this body protective of treating for tumor radiotherapy, alleviates and controls untoward reaction and the bone marrow depression in the chemotherapy, the drug action of human body immunity improving power.

Embodiment 10:THT is to the inhibition and the apoptotic effect of HeLa Cells and transplanted tumor

1, experimental technique

HeLa Cells; The BALB/c-nu nude mice.

RPMI-1640 culture medium and pancreatin (EDTA) U.S. Gibco Company products.Newborn calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd., and tetrazolium bromide (MTT) dye liquor is Beijing Zhong Shan Company products.Annexin V-FITC/PI apoptosis detection kit is a Bender Medsystem Company products.

(1) THT is to cervical cancer HeLa cell inhibiting and apoptotic effect

Cervical cancer HeLa cell culture places 5%CO in containing 10% newborn calf serum and two anti-RPMI-1640 culture medium ₂, carry out routine in 37 ℃, the constant-temperature enclosed incubator of 95% saturated humidity and cultivate, every day observation of cell growing state.3d goes down to posterity 1 time.When 0-80 μ g/mlTHT 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) THT 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 THT group, 60mg/kg, 1 time/day, ip0.5ml; Fluorouracil (5-Fu) group, 30mg/kg, ip, 1 time/4d, totally 6 times; The THT-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: by changes of body mass adjustment administration; 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) THT is to cervical cancer HeLa cell inhibiting and apoptotic effect

THT has utmost point significant inhibitory effect (table 9) to the propagation of HeLa Cells.

Table 9 THT is to the inhibitory action of cervical cancer HeLa cytoactive

The suppression ratio of cell viability

THT(μg/ml)

(％)

0 0

10 9.80

20 15.7

40 40.6

60 79.1

80 89.5

THT shows cervical cancer HeLa apoptosis with the two mark dyeing of AnnexinV-PI flow cytometry analysis; Behind the THT effect cervical cancer HeLa cell 24,48 of variable concentrations, the 72h; The apoptosis incidence rate obviously increases; Strengthen and prolongation action time with THT concentration, apoptosis rate obviously increases, and compares with matched group to have the utmost point significance difference opposite sex.THT 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 THT effect 72h is the highest, surpasses 70%.

(2) THT 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 1-6 days, growth of xenografted speed was basic identical, but subsequently the matched group growth of xenografted obviously faster than other administration groups.Behind the 26d, THT processed group gross tumor volume and tumor quality all are lower than matched group (P＜0.01), and compare there was no significant difference (P＞0.05) with the treatment matched group, show that THT has remarkable antitumor action (table 10) to cervical cancer.

Table 10 THT is to the effect of cervical cancer inhibition of proliferation

Processed group number of animals tumor body weight (g) suppression ratio (%) sign changes

Negative control 8 1.351 ± 0.373/fur comes off few

THT 8 0.406 ± 0.117 ^*69.9 ^*Fur comes off few

5-Fu 8 0.417 ± 0.121 ^*69.1 ^*Fur comes off more

THT-5-Fu organizes 8 0.365 ± 0.101 ^*72.9 ^*Fur comes off few

^*P＜0.01 is with respect to negative control group.

Embodiment 11:THT is 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 age in 4-6 week, heavy 13-20g is 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 In Situ Cell Death Detection (Pod kit) is available from Roche company.Human breast cancer cell strain MDA-MB-231; THT.

The foundation of human breast cancer cell transplanted tumor in nude mice: human breast cancer cell strain MDA-MB-231 cultivates in containing the RPMIl640 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 divides into groups and medication:

1. matched group: 8, propylene glycol 0.1ml/ only, ip, 15d altogether; 0.9%NS, 0.1ml/, ip, 5d altogether.

2. THT organizes: 8, THT60mg/ (kgd) is dissolved in the 0.1ml propylene glycol, ip, 15d altogether; 0.9%NS 0.1ml/, ip, 5d altogether.

3. 5-Fu organizes: 8,5-Fu 30mg/ (kgd) is dissolved in 0.9%NS 0.1ml, ip, 5d altogether; Propylene glycol 0.1ml/, ip, 15d altogether.

4. THT+5-Fu organizes: 8, THT60mg/ (kgd) is dissolved in the 0.1ml propylene glycol, ip, 15d altogether; 5-Fu 30mg/ (kgd) is dissolved among the 0.9%NS 0.1ml, ip, 5d altogether.

Tumour inhibiting rate is observed: every separated 2d calculates gross tumor volume with the maximum major diameter (a) of vernier caliper measurement tumor, transverse diameter (b), 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 by following formula: tumour inhibiting rate=(the average tumor of the average tumor weight/matched group of 1-experimental group is heavy) * 100%.The observation sign changes.

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 (++), and positive rate＞50% is (+++).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.THT group, 5-Fu group, THT+5-Fu group tumour inhibiting rate are respectively 41.5%, 44.6%, 50.3%, receive to suppress the most obviously (table 11) with drug combination group tumor growth.

Table 11 THT is to the inhibitory action of people's mastadenoma

Tumor suppression

Processed group number of animals tumor body counterpoise (g)

Rate (%)

Negative control 8 0.5221 ± 0.131

THT 8 0.3105±0.0703* 40.5

5-Fu 8 0.2890±0.1711* 44.6

THT+5-Fu 8 0.2410±0.1003* 53.8

THT is to the influence of transplanted tumor cell proliferation

Under light microscopic, observe tumor tissues, 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.THT 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 THT group, 5-Fu group, THT+5-Fu group; Significant difference (P＜0.01) (table 12) is arranged; Show that THT medication group tumor cell proliferation receives obvious inhibition, in conjunction with spectroscopic analysis, THT also has certain directly cytotoxic effect to tumor cell.

Table 12 medicine is to the influence of tumor cell proliferation, apoptosis

Processed group number of animals Ki67-LI (%) AI (%)

Negative control 8 65.15 ± 3.6 9.5 ± 2.1

THT 8 42.93±3.11* 27.5±2.1*

5-Fu 8 45.07±2.88* 25.4±1.3*

THT+5-Fu 8 40.10±2.95* 37.8±2.6*△

* P＜0.01 is with respect to negative control group; △ P＜0.05 is between 5-Fu and THT processed group.

THT 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: THT group, 5-Fu group, THT+5-Fu group apoptotic index are apparently higher than matched group (P＜0.01), and the THT+5-Fu group is organized (P＜0.05) apparently higher than THT group, 5-Fu.THT can induce 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 THT group, 5-Fu group, THT+5-Fu group are starkly lower than matched group (P＜0.05), show that THT can suppress the protein expression (table 13) of bcl-2 gene.

Table 13 is respectively organized the Bcl-2 of breast cancer tissue protein expression

Bcl-2 expresses negative control THT 5-Fu THT+5-Fu

- 1

+ 3 7 6 7

++ 4 1 2

+++ 1

The inhibitory action that embodiment 12:THT expresses 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 THT processed group are set; Matched group is cultivated the MDA-MB-231-BO cell for containing 10% hyclone culture fluid, and the THT processed group is the THT that on the basis of matched group, adds 20mg/L.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%, with the PBS of 0.01mol/L flushing slide 3 times, 10% formaldehyde fixed 30min.0.01mol/L PBS washing, hatch 10min under 3% the H2O2 methanol solution room temperature, the PBS washing of 0.01mol/L is dripped 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 dilution in 1: 200, and room temperature is placed 60～90min, and matched group only adds antibody diluent; 0.01mol/L PBS washing, drip biotin labeled SA (sheep anti-mouse igg), incubated at room 10min, the PBS washing of 0.01mol/L; Drip 1 reagent D (streptomycete antibiotic-peroxide enzymatic solution), incubated at room 10min, the PBS washing of 0.01mol/L, DAB colour developing; The tap water flushing, haematoxylin redyeing, 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 through 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 THT handles, 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 gets into 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 dissolves bone property bone resorption, promotes the bone of breast cancer cell to shift process.Experimental result prompting THT can suppress the transfer of breast cancer tumour.

Embodiment 13:THT is to people's colon-cancer cell and transplanted tumor in nude mice inhibited proliferation

1, experimental technique

Human colon adenocarcinoma cell line lovo cell; THT.

Cell culture: the lovo cell is with the NBCS 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/mlTHT concentration increased progressively, porous plate was cultivated the lovo cell, 3 multiple holes, and mtt assay detects cell viability, observes THT 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% NBCS, 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 uses piece of tissue sleeve pipe skill of handling needles subcutaneous transplantation to be solid tumor after the one-tenth tumor.The nude mice of human colon carcinoma subcutaneous transplantation is divided into 4 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) the THT group is irritated stomach with 80mg/kg; (3) 5-Fu group (positive control) is with the 5-Fu30mg/kg lumbar injection; (4) THT-5-Fu group is with 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 THT 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) THT is to the inhibition and the apoptotic effect of colon-cancer cell

The lovo cell is 20,40,60 at the THT 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 THT to the apoptotic THT effect lovo cell 72h that influences variable concentrations of lovo after; The apoptosis incidence rate obviously increases; Strengthen and prolongation action time with THT concentration; Apoptosis rate obviously increases, and compares with matched group to have the significance difference opposite sex.

Table 14 THT is to the apoptotic influence of intestinal cancer lovo (x ± s)

THT (μ g/ml) cell inhibitory rate (%) apoptosis rate (%)

10 1.7±0.3 1.0±0.1

20 4.2±0.4 1.9±0.3

40 18.0±1.1 8.1±0.4

80 46.5±0.5 29.9±0.7

120 89.5±1.1 50.3±0.8

THT 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 THT effect 72h has reached 48.7%.

(2) THT is to colon-cancer cell transplanted tumor in nude mice inhibited proliferation

Under light microscopic, observe tumor tissues, matched group transplanted tumor cell still keeps the original atypia of cancerous cell under the cultivation conditions, out-of-shape, visible unusual karyokinesis phase.THT administration group tumor cell presents degeneration in various degree, and the part tumor tissues occurs obviously downright bad, shows THT to intestinal cancer tumor cell proliferation significant inhibitory effect (table 15), and drug combination improves curative effect.

Table 15 THT is to the inhibitory action of intestinal cancer tumor proliferation

Processed group number of animals tumor body weight (g) suppression ratio (%)

Negative control 8 0.951 ± 0.473/

THT 8 0.293±0.151* 69.1*

5-Fu 8 0.277±0.142* 70.8*

THT-5-Fu organizes 8 0.210 ± 0.122* 77.8

* P＜0.01 is with respect to negative control group.

The bacteriostasis of embodiment 14:THT is observed

1, material

Supply 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 kinds of 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 THT 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 culture bacteria liquid coating Agar Plating, observation has or not growth to judge after 37 ℃ of cultivations.With the high dilution of THT that can bacteria growing inhibiting is minimum inhibitory concentration (MIC), is minimum bactericidal concentration (MBC) with the THT greatest dilution that does not still have bacteria growing

3, measure the result

THT all has antibacterial action (table 16) to several kinds of antibacterials, and the mensuration result of minimum inhibitory concentration and minimum bactericidal concentration shows that THT all has sterilizing ability to these antibacterials.

Table 16 THT all has antibacterial action to several kinds of antibacterials

Antibacterial MIC (mg/ml) MBC (mg/ml)

Bacillus subtilis 1.0 1.0

Staphylococcus aureus 0.50 0.50

Salmonella 1.0 2.0

Escherichia coli 0.50 1.0

Bacillus pyocyaneus 0.50 0.50

Diplococcus pneumoniae 0.50 1.0

Embodiment 15:THT is to the therapeutical effect of mice CCl4 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% liquid paraffin solution), secondary weekly, totally eight times.

Experiment is divided into three groups, and the THT 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

CCl4 contamination group; Inflammatory cell infiltration is obvious around the lobules of liver, visible proliferation of fibrous tissue, and big the hepatic necrosis in lobule center is obvious; Part of hepatocytes fat becomes; The apparition of cavity appearance: the liver histological change of THT treatment group and CCl4 contamination group 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 THT is to CCl ₄The chronic injury mice has significant protective effect.

Table 17 THT is to CCl ₄Chronic injury mice serum transaminase and sero-abluminous influence (x ± s, n=10)

Handle ALT (U/100ml) serum albumin (g/L)

Normal control group 29.7 ± 2.03 48.53 ± 6.33

CCl ₄Group 105.95 ± 8.36 33.22 ± 4.31

CCl ₄+THT 37.54±5.16** 42.85±5.78**

* P＜0.01 and CCl ₄Group relatively.

CCl ₄After getting in the body, activate, claim trichloromethyl free radical (CCl through liver cytochrome P 450 ₃), attack the phospholipid molecule on the endoplasmic reticulum through 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 that cytotoxic is dead.THT can improve the degeneration of chronic hepatic injury hepatic tissue cavity appearance, 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 16:THT tablet

Tablet formulation: THT30-80%, medical starch 2-10%, lactose 10%-60%, aspartame 0.2-1%, magnesium stearate 0.5-1%

With THT1000g 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 the aspartame 0.5% and 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.

The capsular preparation of embodiment 17:THT

Capsule prescription: THT30-80%, medical starch 5-30%, lactose 0%-60%, magnesium stearate 0.5-1%

With the dry powder blend of THT1000g, 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 18:THT patch

100g is cut into strip with raw rubber, is pressed into reticular film with glue pressing machine, removes static, puts coldly, immerses in the gasoline about 24 hours, makes its abundant swelling; Moved into the interior stir about of ingredients pot again 3 hours, and added Colophonium 80g, zinc oxide 80g, lanoline 15g, vaseline 15g, liquid paraffin 10g successively, mixing adds THT100g; Stir, filter, be coated with cream, cutting through 80 order copper wire screen clothes; The lid lining, stripping and slicing is processed 1000 and is pasted, and promptly gets the THT patch.

Claims (1)

1. Ampelopsis grossedentata total flavone is characterized in that in the application of preparation control radiotherapy and chemotherapy medicine untoward reaction medicine said control adverse reaction of tumor chemoradiotherapy is bone marrow depression, hemocyte reduction, cell mutation or alopecia.

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