CN104706649A - Application of oroxyloside to preparation of anti-tumor drugs - Google Patents

Abstract

The invention belongs to the field of anti-tumor drugs and discloses an application of oroxyloside to the preparation of the anti-tumor drugs. According to the application of oroxyloside to the preparation of the anti-tumor drugs, oroxyloside can be prepared into various medical forms of tumor-treating drugs, so that better choices are provided for tumor patients.

Description

Oroxylin glycosides is preparing the application in antineoplastic agent

Technical field

The invention belongs to antineoplastic agent field, relate to oroxylin glycosides and preparing the application in antineoplastic agent.

Background technology

Chemotherapeutics, by different mechanisms play antineoplastic actions, is divided into alkylating agent, antimetabolite, antitumor antibiotic, plant amedica, hormone and miscellany by traditional classification if any document by antitumor drug; Have and be categorized as the direct DNA of destruction medicine by mechanism of action, insert DNA profiling medicine, interfere RNA synthetic drug, suppress microtubule to assemble medicine, affect protein synthesis medicine, other mechanism of action class medicines etc.The mechanism of action of understanding antineoplastic agent is more conducive to the feature selecting properly relative medicine when clinical treatment tumour according to tumor and medicine.

Oroxylin is one of active component of Radix Scutellariae, has obvious antitumor action, is a natural antitumor medicine with broad prospect of application.But oroxylin pharmacological action is extensive, and the mechanism of its antitumor action is illustrated at present not yet completely.Oroxylin A-7-glucuronide (oroxylin glycosides, Oroxyloside, OAG) be also one of active component of Radix Scutellariae, be not same substance with oroxylin, less to the research of oroxylin glycosides at present, there is not been reported in the research of relevant pharmacological action.

Summary of the invention

The object of this invention is to provide oroxylin glycosides and prepare the application in antineoplastic agent.

The object of the invention is to be realized by following technical measures:

Oroxylin glycosides is preparing the application in antineoplastic agent, and especially for the antineoplastic agent of hepatocarcinoma, its mechanism playing antitumor action is that oroxylin glycosides has growth inhibited and lethal effect for tumor cell.Its preparation can be any one dosage form pharmaceutically allowed, and includes but not limited to tablet, granule, pill, oral liquid, injection, membrane, capsule, liposome etc.The consumption of oroxylin glycosides can according to changes such as route of administration, patient age, body weight, body surface area, the disease type for the treatment of and the order of severity and the arrangements course for the treatment of, and its daily dose can be 200-8000mg, can use by one or many.

Beneficial effect of the present invention:

Oroxylin glycosides passes through tumor cell generation growth inhibited and lethal effect thus plays antitumor action, has good effect, can be prepared into the anti-tumor drug of various dosage form, for tumour patient provides better selection.

Accompanying drawing explanation

Fig. 1. oroxylin glycosides is to the apoptosis-induced effect of HepG2.

Fig. 2. oroxylin glycosides is to the quantification of the apoptosis-induced effect of HepG2.

Fig. 3 .OAG is to the growth inhibited effect of human liver cancer cell HepG2 transplanted tumor in nude mice.

The impact that Fig. 4 .OAG is heavy on human liver cancer cell HepG2 transplanted tumor in nude mice tumor.

Fig. 5 .OAG is on the impact of human liver cancer cell HepG2 nude mouse xenograft tumor laboratory animal body weight.

Detailed description of the invention

The invention will be further elaborated by the following examples.

Test examples 1:OAG is to the apoptosis-induced effect of human liver cancer cell HepG2 cell

1, experiment material

(1) medicine

Oroxylin A-7-glucuronide (oroxylin glycosides, Oroxyloside, OAG) is buied from market, buff powder.Content: 90%, is made into mother solution with DMSO, is placed in-20 DEG C of preservations.Desired concn is made into before use with RPMI-1640 culture fluid.

(2) cell strain

Human liver cancer cell HepG2, from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences's biochemistry and Institute of Cell Biology cell bank, cultivates with the RPMI-1640 containing 10% calf serum.

(3) reagent

1) culture fluid: RPMI-1640 culture medium, U.S. GIBCO Products.Get RPMI-1640 powder 10.4g to be dissolved in 1000mL sterilizing tri-distilled water, use NaHCO ₃adjust pH to 7.3 ~ 7.4, cylindric style filter filtration sterilization, subpackage, 4 DEG C of Refrigerator stores.10% hyclone, 100U/mL penicillin and 100mg/L streptomycin is added before using.

2) calf serum: Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation product.Through 56 DEG C of water-bath deactivation 30min, subpackage is also stored in-20 DEG C of cryogenic refrigerators.

3) PBS buffer: take NaCl 8.0g, KCl 0.20g, Na ₂hPO4H ₂o 1.56g, KH ₂pO ₄2.0g, is dissolved in 1000mL tri-distilled water, autoclaving, 4 DEG C of Refrigerator stores.

4) 0.02%EDTA solution: take EDTA 20mg, is dissolved in 100mL PBS buffer, stirring at low speed, adjust pH to 7.3 ~ 7.4, cylindric style filter filtration sterilization, subpackage, 4 DEG C of Refrigerator stores.

5) 0.25% pancreatin: take pancreatin 0.25g, is dissolved in 100mL PBS buffer, autoclaving.

6) DMSO solution: U.S.'s Sigma-Aldrich (St.Louis, Mo) Products

7) the two transfection reagent box of Annexin-V/PI: purchased from Kai Ji company.

2, experimental apparatus:

1) YJ-875 type Medical purification workbench: Suzhou Decontamination Equipment Plant produces.

2) 3111 type water-jacket typ CO ₂incubator: U.S. Thermo electron Products.

3) electronic balance: Beijing Sai Duolisi instrument system company limited product.

4) LD4-2 generic centrifuge: Beijing Medical Centrifugal Machine Factory's product.

5) Centrifuge 5810R type centrifuge: German Eppendorf Products.

6) FACSCalibur flow cytometer: U.S. Becton Dickinson (BD) company produces.

3, experimental technique:

(1) flow cytomery apoptosis rate

By HepG2 cell with concentration for 5 × 10 ⁵individual/L is inoculated in 6 well culture plates, is placed in 37 DEG C, 5%CO ₂after cultivating 24h in incubator, add the oroxylin glycosides process cell 48h that final concentration is 25,50 and 100 μm of ol/L.Digestion collecting cell, the centrifugal 5min of 1000r/min, PBS liquid washs 1 time, centrifugally remove PBS, the 500 μ lBinding Buffer adding ice pre-cooling are resuspended, then add 5 μ Annexin V and PI dyestuff respectively, room temperature dyeing 20min, detect through flow cytometer (Becton – Dickinson FACSCalibur) after dyeing, experimental data is by CellQuest software analysis.。

4, experimental result:

Experimental result shows, and as Fig. 1 and Fig. 2, after oroxylin glycosides (25,50,100 μMs) acts on human hepatoma HepG2 cell 24h, can induce it that apoptosis occurs, apoptosis rate is respectively 18.68%, 30.63%, 48.3%, has dose dependent.

Test examples 2:OAG is to the effect of human liver cancer cell HepG2 nude mouse xenograft tumor growth inhibited

1, experiment material

(1) test medicine: oroxylin A-glucuronide is buied from market, purity is more than 99%, makes freeze-dried powder, with injection normal saline, medicine is made into desired concn before using.

(2) control drug: Fluorouracil Injection, purchased from Tianjin gold credit some company of Pharmaceutical, lot number: 140929, specification: 10ml/ props up.Compound method: accurate absorption Fluorouracil Injection 0.12ml before administration, is dissolved in 0.9% sodium chloride injection (manufacturer: Cisen Pharmaceutical Co., Ltd.; Lot number: 1303207021; Specification: 500ml*0.9%) 0.88ml is mixed with the liquid that concentration is 3mg/ml, and administration volume is 0.2ml/20g, and namely dosage is 30mg/kg.

(3) laboratory animal

Source, germline, strain: BALB/c nude mouse, provided (laboratory animal production licence: SCXK (army) 2007-004) by Chinese military medicine academy of science Animal Experimental Study center.Age in days: 35-40 days, body weight: 18-24g, sex: female.

2, experimental technique:

(1) laboratory animal grouping

(2) modeling method

To take the logarithm the human liver cancer cell HepG2 cell strain of trophophase, be aseptically prepared into 2 × 10 afterwards ⁸/ ml cell suspension, is inoculated in axillary fossa on the right side of nude mouse with 0.1ml subcutaneous.Nude Mice vernier caliper measurement transplanted tumor diameter, treats tumor growth to 100 ~ 300mm ³after by animal random packet.Use the method measuring tumor footpath, dynamically observe tested thing antineoplastic effect.The pendulous frequency of diameter of tumor is that every 2-3 days surveys 1 time.Administration volume is 0.2ml/20g.After 19 days, sacrifice, operation strips tumor block and weighs.The computing formula of gross tumor volume (tumor volume, TV) is:

TV＝1/2×a×b ²

Wherein a, b represent length and width respectively.

Result according to measuring calculates relative tumour volume (relative tumor volume, RTV), and computing formula is: RTV=V _t/ V ₀.Wherein V ₀for (d during point cage administration ₀) measure gained gross tumor volume, V _tgross tumor volume during for measuring each time.The evaluation index of anti-tumor activity is Relative tumor rate of increase T/C (%), and computing formula is as follows:

T/C(％)＝T _RTV/C _RTV×100％

T _rTV: treatment group RTV; C _rTV: negative control group RTV.

Tumour inhibiting rate computing formula is as follows:

Tumour inhibiting rate (%)=(1-T _weight/ C _weight) × 100%

T _weight: the average tumor weight for the treatment of group; C _weight: the average tumor weight of negative control group.

3, result:

The experimental treatment of oroxylin glycosides to people hepatocarcinoma HepG2 Nude Mice the results are shown in Table 1, and 2 and Fig. 3,4,5.Experimental result is as follows, and OAG is respectively with the administration of 90mg/kg, 45mg/kg tail vein injection, and 3 days once, administration 21 days, altogether administration 7 times.The T/C of experimental group to human liver cancer cell HepG2 Nude Mice is respectively 56.74%, 71.57%; Tumour inhibiting rate is respectively 40.23%, and 28.45%.5-FU is with the administration of 30mg/kg tail vein injection, and 3 days 1 time, administration 21 days is altogether 30.74% to the T/C of human liver cancer cell HepG2 Nude Mice after administration 7 times, inhibitory rate 70.11%.Wherein 5-FU 30mg/kg dosage group has pole appreciable impact (P<0.01) to experimental mouse body weight.

Conclusion: oroxylin glycosides has obvious growth inhibited and lethal effect to hepatoma Hep G 2 cells.

Table 1:OAG is on the impact (X ± SD, gross tumor volume: unit mm3) of human liver cancer cell HepG2 nude mouse xenograft tumor growth change in volume

Note: blank group compares, * P<0.05, * * P<0.01

The inhibitory action (X ± SD) that table 2:OAG grows human liver cancer cell HepG2 nude mouse xenograft tumor

Compare with blank group, * P<0.05, * * P<0.01

Example of formulations 1

Get oroxylin glycosides 10g, add injection (comprising lyophilized injectable powder and aseptic subpackaged dry powder injection) suitably adjuvant, be prepared into antineoplastic agent injection by injection (comprising lyophilized injectable powder and aseptic subpackaged dry powder injection) technique, often prop up containing oroxylin glycosides 500mg.

Example of formulations 2

Get oroxylin glycosides 10g, add tablet and (comprise slow-release tablet, matrix tablet, coated tablet, dispersible tablet etc.) suitable adjuvant, (comprise slow-release tablet, matrix tablet by tablet, coated tablet, dispersible tablet etc.) technique is prepared into antineoplastic agent tablet, and every sheet is containing oroxylin glycosides 300mg.

Example of formulations 3

Get Chinese oroxylin glycosides 10g, add the suitable adjuvant of capsule, be prepared into antineoplastic agent capsule by capsule technique, every containing oroxylin glycosides 300mg.

Example of formulations 4

Getting oroxylin glycosides 10g, add the suitable adjuvant of oral liquid, be prepared into antineoplastic agent oral liquid by oral liquid technique, often propping up containing containing oroxylin glycosides 300mg.

Claims (3)

1. oroxylin glycosides is preparing the application in antineoplastic agent.

2. application according to claim 1, is characterized in that described tumor is hepatocarcinoma.

3. application according to claim 1, is characterized in that described antitumor refers to have growth inhibited and lethal effect to tumor cell.