CN110433150A - Acetylshikonin prevents and treats the application in colon cancer drug in preparation - Google Patents

Abstract

The invention discloses Acetylshikonins to prevent and treat the application in colon cancer drug in preparation.The present invention demonstrates Acetylshikonin and fastens in source of people colon cancer cell through targeting and TOPK, and then plays remarkable effect in preventing and treating colon cancer.Inventor is by handling the source of people colon cancer PDX mouse model that colon carcinoma cell line and TOPK are overexpressed for Acetylshikonin, when terminating experiment, it was found that Acetylshikonin is able to suppress the growth of colon cancer cell, the gross tumor volume of Acetylshikonin drug-treated group mouse has significant statistical difference compared with the control group, to show that Acetylshikonin has significant effect on preventing and treating colon cancer by TOPK.By giving patient's Acetylshikonin drug, it can clinically prevent and treat the highly expressed colon cancer of TOPK.Acetylshikonin of the present invention can targeting in kinases TOPK, be applicable not only to colon cancer and apply also for other cancers.

Description

Acetylshikonin prevents and treats the application in colon cancer drug in preparation

Technical field

The invention belongs to field of medicaments, and in particular to a kind of Acetylshikonin prevents and treats in colon cancer drug in preparation Application.

Background technique

Cancer has become the Chinese the first cause of death, is serious public health problem.According to statistics the results show that Chinese knot Intestinal cancer is the most common cancer types, and most common cancer mortality reason.Cancer is by environment, diet and life style With inherent cause synergistic effect as a result, by carcinogenic substance effect combination cell inherent cause lead to cytogenetics gene mutation and by Gradually development is cancer.The clinical symptoms of cancer early stage are unobvious, when clinical symptoms are more prominent, during the state of an illness of patient has been in Phase or advanced stage, so selection safely and effectively prevents and treats prognosis and quality of life of the method for cancer for improvement patient All it is of great significance.

Acetylshikonin is one of lithospermum euchromum Royle important component.Asian puccoon belongs to Boraginaceae herbaceos perennial, tool There is the effects of antibacterial, anti-inflammatory, antiviral and antitumor.Main component of the Acetylshikonin as Asian puccoon is by lithospermum euchromum Royle cell The naphthoquinones class pigment compound of large-scale culture technology production, has anticancer activity.Some researches show that Acetylshikonins to pass through The phosphorylation of the JNK and p38 of inducing cell induce cell apoptosis, and then inhibit the occurrence and development of Stomatocyte carcinoma squamosum. The researchs such as ChoSeok-Cheol find that Acetylshikonin can mainly be led to by the growth of inhibition human pancreatic cancer cell PANC-1 Cross the activation plays effect for inhibiting NF-1B.And some researches show that Acetylshikonins can inhibit Human gastric cancer SGC-7901 cells Hyperplasia.

TOPK (T-LAK cell-originated protein kinase) is one of serine threonine kinases, Belong to mapk kinase family.TOPK plays many important functions in the cell, may participate in the occurrence and development of cancer, and cell increases It grows, apoptosis.Data show that expression quantity of the TOPK in colon cancer, lung cancer and breast cancer is all significantly higher than normal index.Research Show that the occurrence and development of lung cancer can be effectively suppressed in TOPK inhibitor.

Summary of the invention

The purpose of the present invention is to provide a kind of Acetylshikonins to prevent and treat the application in colon cancer drug in preparation.

Based on above-mentioned purpose, the present invention is adopted the following technical scheme that:

Acetylshikonin (Acetylshikonin, molecular formula: C₁₈H₁₈O₆, molecular weight: 330.3) in preparation prevent and treat Application in colon cancer drug has the function of described in (1) or (2): (1) being used as kinases TOPK inhibitor, (2) inhibit cancer cell Proliferation.

Further, kinases TOPK mass is 2 μ g, and Acetylshikonin dose concentration is 0.6~1.25 μM.

Further, colon cancer cell quantity is 1000, and Acetylshikonin inhibits the dosage of growth to colon cancer cell Range is 1.25~10 μM, and in non-anchor experiment, tumour cell quantity is 8000, every hole, and Acetylshikonin measures range is 0.6~1.25 μM.

Further, the Acetylshikonin is obtained by following methods:

(1) extracting solution for being obtained alkanet to alkanet raw material refluxing extraction using ethyl alcohol is carried out gained extracting solution dense Contracting obtains crude extract；

(2) crude extract obtained by step (1) is diluted with water, is successively extracted with petroleum ether, ethyl acetate, n-butanol It takes, obtains effective component；

It (3) is that 200-300nm large pore resin absorption column separates by aperture by step (2) resulting effective component, Eluent is positive hexane-chloroform-methanol three's mixed solution, and n-hexane, chloroform and methanol volume ratio are 25:6:1, and utilization is thin Layer chromatography identified, is merged and is collected the eluent of same ingredient and be simultaneously concentrated, and obtains 7 kinds of extract component, ingredient 1,2, 3,4 and 5 be the crude product of effective component, is the plant residue in alkanet in ingredient 6 and 7.

Further, the detailed process of the step (1) are as follows: 90% or more of 60L is added into 10kg alkanet raw material Ethanol solution, extract 2~4 times, every time flow back 1.5~3h, filtering, collect liquid, be evaporated off solvent to get；The step (2) Detailed process are as follows: by crude extract obtained by step (1) after the dilution of 2L distilled water, first use petroleum ether extraction 2~4 times, then use Ethyl acetate extracts 2~4 times, finally carries out extraction 2~4 times through n-butanol again, the petroleum ether, ethyl acetate and n-butanol Dosage be 3L, merge petroleum ether, ethyl acetate and n-butanol layer, be evaporated off solvent to get.

Further, extract component 3 is separated through sephadex-LH20 Sephadex-LH20 column, Separation column diameter is 5cm, is highly 1.20 meters, and eluent is the petroleum ether and ethyl acetate mixtures of volume ratio 20:1, is obtained Two kinds of compound beta, beta-dimethyl acry-lalkannins and Acetylshikonin.

Further, by extract component 4 flow through aperture be 200-300nm, diameter 5cm, highly be 20cm macropore inhale Attached resin column, flow velocity are 1 second/drop, and eluent is that volume ratio is 25:6:1 n-hexane-chloroform-methanol mixed solution, obtains acetyl Alkannin.

A kind of side using PDX mouse model detection Acetylshikonin effective dose in preparation treatment colon cancer drug Method, by the colon cancer sample taken plantation in will be obtained in immunodeficient mouse body, after 2-5 week be immunized mouse Colon tumor, It takes resulting colon cancer tumours and passes in immunodeficient mouse, until obtaining colon cancer PDX mouse model, and benefit after 3-4 generation PDX model mice Acetylshikonin is fed with the method for stomach-filling, detects the growing state of colon cancer mouse interior tumor.

Further, Acetylshikonin is dissolved in the Acetylshikonin solution that 40~60mg/kg is made in PEG400 and water, Daily with 10 μ L/g mouse weight stomach-filling Acetylshikonin solution.

The present invention relates to Acetylshikonins can inhibit cell by targeting and TOPK in colon carcinoma cell line Growth, and be inoculated on the colon cancer PDX model that immunodeficient SCID mice is subcutaneously established and have using people's fresh tumor tissue Significant neoplasm growth effect, can play the effect of colon cancer recurrence prevention and treatment.

The Acetylshikonin voluntarily extracts preparation by Sino-U.S. (Henan) He Meier research institute, and purity is up to 97%.This medicine The dosage range that object uses in vitro experiment is 0.6125~2.5 μM, is 60 in the in-vivo tumour treatment concentration of SCID mice ~120mg/kg weight.Oral solution, beverage can be made with the dosage of 5.4~10.8mg/kg in the drug, drink for patient. Present invention utilizes synergistic effect of the inhibitor Acetylshikonin of TOPK in the prevention and treatment of colon cancer, significantly improve knot The prevention and therapeutic effect of intestinal cancer.The compound has clinical prevention and treats the potential using value of colon cancer.By giving patient Acetylshikonin can clinically prevent and treat colon cancer.

Acetylshikonin of the invention and suitable pharmaceutically acceptable carrier can be combined.This kind of pharmaceutical composition contains There are the compound and pharmaceutically acceptable carrier or excipient of therapeutically effective amount.This kind of carrier includes (but being not limited to): salt Water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Pharmaceutical preparation should match with administration mode.Second of the invention Acyl alkannin can be made into injection form, such as the aqueous solution with physiological saline or containing glucose and other adjuvants passes through often It is prepared by rule method.The pharmaceutical composition of such as tablet and capsule etc can be prepared by conventional method.Active constituent Dosage be therapeutically effective amount, such as about 5.4~10.8mg/kg weight daily.Certainly, specific dosage is also contemplated that administration way The factors such as diameter, patient health situation, within the scope of these are all skilled practitioners technical ability.In addition, Acetylshikonin of the invention It can also be used together with other therapeutic agents.

Do not saw the drug for targeting in the relevant report of TOPK pairs of clinical treatment tumour.Through the invention The provable drug of related experiment can apply to colorectal cancer clinical treatment.

Detailed description of the invention

Fig. 1 is to extract obtained Acetylshikonin¹HNMR figure；

Fig. 2 is to extract obtained Acetylshikonin¹³CNMR figure；

Fig. 3 is activity suppression effect of the Acetylshikonin to kinases TOPK: where A is that Acetylshikonin handles kinases Its activity change trend after TOPK；B is the combination situation of TOPK albumen in Acetylshikonin and colon cancer cell lysate；C is The Acetylshikonin and kinases TOPK binding site of computer simulation illustrate；

Fig. 4 is Acetylshikonin to normal colon cell line and colon carcinoma cell line growth inhibition situation: where A is Acetylshikonin handles normal colon cell to the toxic effect of cell；B is thin after Acetylshikonin processing colon cancer cell Intracellular growth trend chart；After C is non-anchor experiment 3 weeks, the non-rivet growth change trend of colon cancer cell is (with control group phase Than difference is statistically significant, and * indicates that P < 0.05, * * indicate P < 0.01)；

Fig. 5 is tumour growth therapeutic effect of the Acetylshikonin on colon cancer (number: HJG17P4) PDX mouse model: Wherein, mouse weight trend chart during A is administration；Mouse tumor volume change tendency chart during B is administration；C is control The tumour picture of group and administration group (compared with the control group, difference is statistically significant, and * indicates that P < 0.05, * * indicate P < 0.01)；

Fig. 6 be the Ki-67 after Acetylshikonin handles colon cancer (number: HJG17P4) PDX mouse model, in tumour with And expression of the p-TOPK in tumour.

Specific embodiment

Technical solution of the present invention is further discussed in detail with reference to embodiments, but protection scope of the present invention It is not limited thereto.

Acetylshikonin is native compound, there is antitumor action in many tumours, but some researches show that acetyl purples Careless element targeting proteins during treating colon cancer.Main feature of the present invention with using vitro kinase experiment and cell experiment, Humanized's heteroplastic transplantation model In vivo model studies Acetylshikonin targeting in TOPK.In vitro cell experiment can simulate second Acyl alkannin and TOPK binding mechanism, humanized's heteroplastic transplantation model remain intrinsic gene and phenotype, are a kind of closer Human tumor occurs, the model of development process, to individualized treatment significantly model.Present invention demonstrates that Acetylshikonin Tumour on human colon cancer cell and xenograft tumor transplantation model mouse can be treated or prevented in TOPK by targeting Growth.Acetylshikonin of the present invention can targeting in kinases TOPK, be applicable not only to colon cancer and apply also for other cancers.

Embodiment:

Materials and methods

1 material

1.1.1 Acetylshikonin

The Acetylshikonin that the present invention uses is extracted from alkanet by Sino-U.S. (Henan) He Meier tumor research institute, purity It is 97%.The alkanet that the present invention uses is purchased from Zhengzhou City's Chinese herbal medicine market, and alkanet sample is stored in Sino-U.S. (Henan) He Meier Tumor research institute.The plant is identified through pharmaceutical college, Zhengzhou University chief of office associate professor Pan Chengxue.

In alkanet Acetylshikonin isolate and purify and assay experiment: take the alkanet of 10kg in the 95% of 60L Through reflow treatment 2h in alcohol, the step 3 time is repeated, filtering collects the solution of above-mentioned resulting crude extract, ethyl alcohol is evaporated off and obtains Obtain kermesinus crude extract.Resulting crude extract is dissolved in 2L distilled water and first three times, then with ethyl acetate is extracted with petroleum ether extraction It taking three times, is finally extracted three times through n-butanol again, the dosage of the petroleum ether, ethyl acetate and n-butanol is 3L, Merge petroleum ether, ethyl acetate and n-butanol layer, solvent is evaporated off to get the Asian puccoon root extract of 215g, by extract benefit It is mixed with the filter column and n-hexane-chloroform-methanol (n-hexane, chloroform and methanol volume ratio are 25:6:1) in the aperture 200-300 It closes liquid 4L and carries out elution 7 kinds of Asian puccoon root extracts of acquisition, ingredient 6 and 7 is mainly the plant residue compounds content in alkanet It is lower.Wherein the quality of extract 1,2,3,5 and 5 is 2.856g, 12.56g, 42.34g, 18.37g, 19.25g and 4.765g。

Contain compound deoxidation alkannin in extract component 1, passes through sephadex-LH20 Sephadex- LH20 splitter is separated, separation column diameter be 5cm, be highly 1.20 meters, eluent be 1L methanol, flow velocity be 15 seconds/ Drop obtains deoxidation alkannin, quality 365mg；Extract component 2 is dissolved in volume ratio to mix for the petroleum ether and ethyl acetate of 20:1 Liquid is closed, it is highly 20cm that the large pore resin absorption column for being 200-300nm by aperture, large pore resin absorption column diameter, which is 5cm, Flow velocity is 1 second/drop, and eluent is methanol, and methanol usage 1L obtains a kind of single compound β, beta-dimethyl acryloyl Ah Ka Rather, quality 6.75g；Divided in extract component 3 through sephadex-LH20 Sephadex-LH20 splitter From it is highly 1.20 meters that separation column diameter, which is 5cm, and eluent is the petroleum ether and ethyl acetate mixtures of volume ratio 20:1, is obtained Two kinds of compound β, beta-dimethyl-acry-lalkannin and Acetylshikonin, β are obtained, beta-dimethyl-acry-lalkannin quality is The mass ratio of 12.216g, Acetylshikonin 5.765g, β, beta-dimethyl-acry-lalkannin and Acetylshikonin is 60:40； The large pore resin absorption column diameter that extract component 4 is 200-300nm by aperture is 5cm, is highly 20cm, flow velocity be 1 second/ Drop, eluent are positive hexane-chloroform-methanol mixed solution (volume ratio 25:6:1), obtain Acetylshikonin 4.657g, utilize core Magnetic resonance hydrogen spectrometry and carbon spectrometry identify the structure and molecular size range of separated compound.

The structure of Acetylshikonin is as follows:And by hydrogen spectrum and carbon spectrum to its structure into Go characterization, agents useful for same is deuterated chloroform, as depicted in figs. 1 and 2, δ_H(J in Hz), 6.99 (1H brd), 12.58 (1H, S ,-OH), 7.19 (2H, brs), 12.43 (1H, s ,-OH), 6.03 (1H, m), 2.62 (1H, m), 2.51 (1H, m), 5.12 (1H, M), 1.69 (3H, s), 1.58 (3H, s), 2.14 (3H, s).δ_C178.2,176.7,169.8,167.4,166.9,148.2, 136.1,132.9,132.7,131.4,117.6,111.8,111.5,69.5,32.8,25.7,21.0,17.9.

1.1.2 reagent

RPMI-1640, McCoy ' 5A culture medium and FBS are purchased from BI company, and L-15 culture medium is purchased from Jiangsu Kai Ji company, Mycillin is dual anti-and EDTA- pancreatin is purchased from solarbio, and the above reagent is used to the culture of cell, thiazolyl blue (MTT) and Dimethyl sulfoxide (DMSO) is purchased from Sigma company for cell proliferation experiment, kinases TOPK, substrate c-Jun, TOPK kinase reaction Liquid is purchased from signalchem company, is used for TOPK kinase assay, and the ATP of 10mM is purchased from Promega.Antibody used herein Ki-67 is purchased from Thermo, and p-TOPK is purchased from SIGMA, p-ERK1/2, and p-c-Jun is purchased from CST.

1.1.3 colon normal cell and colon carcinoma cell line

People's normal colon epithelial cells CCD 841CoN, human colon cancer cell HCT-15, the HCT-116 that the present invention uses, SW620, SW480, DLD1 and HT29 are both from ATCC.

1.1.4 tumor tissues

Present invention uses human colon cancer sample number be JG17.JG17 derives from Henan Prov. Tumour Hospital, female Property, 63 years old, gland cancer is broken up in (duodenum) ulcer type, with myxoadenocarcinoma.Outer membrane is invaded, does not invade pancreas and choledochus, stomach And duodenum is clean, mucous membrane of gallbladder chronic inflammation.The JG17 model that the present invention uses was the 4th generation.

1.2 experimental animal

Cb-17SCID immunodeficient mouse is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd. in the present embodiment. Licensing number are as follows: SCXK (capital) 2012-0001, SPF grade is led, weight 16-18g, female mice for 5-6 weeks.Mouse feed is purchased from Beijing The biotech inc Hua Fu.Credit number are as follows: experimental animal feeding is in Sino-U.S. (Henan) He Meier tumor research institute Animal facility in, constant temperature (25-27 DEG C), constant humidity (45%-50%), fresh air, dust and bacteria removing without special pathogen It is raised under the conditions of (SPF grades) receptacle, animal is first put in organic plastics box (the limited public affairs of Suzhou City Feng Shi experimental animal equipment Department), then it is placed in the raising of IVC system, 3-4 animal is raised in each raising box, the feed through aseptic process is freely taken the photograph for animal Enter, the padding of high-temperature sterilization is replaced once every three days, and disinfection by ultraviolet light is primary every three days for cage tool and drinking-water, drinks sterile distillation Water.Sterile principle operation is followed strictly when replacement raising articles.Experimental animal was raised under illumination/dark cycle at 12:12 hours It supports, can be freely close to food and water, room temperature is controlled at 21 DEG C.

1.3 preparation of reagents

1.3.1 100mM Acetylshikonin: the DMSO that the Acetylshikonin powder that 20mg is extracted is dissolved in 605 μ L is weighed In.

1.3.2 it the Acetylshikonin of 2.5mM, 5mM, 10mM: extracts the Acetylshikonin that 10 μ L concentration are 100mM and dissolves The Acetylshikonin of 10mM is obtained in the DMSO of 90 μ L；The Acetylshikonin for extracting 20 μ L10mM is dissolved in the DMSO of 20 μ L Obtain the Acetylshikonin of 5mM；The Acetylshikonin for extracting 20 μ L5mM is dissolved in the acetyl that 2.5mM is obtained in the DMSO of 20 μ L Alkannin.

1.3.3 it is molten the Acetylshikonin of 0.6mM, 1.25mM, 2.5mM: to extract the Acetylshikonin that 10 μ L concentration are 10mM Solution obtains the Acetylshikonin of 2.5mM in the DMSO of 30 μ L；The Acetylshikonin for extracting 20 μ L10mM is dissolved in 20 μ L's The Acetylshikonin of 1.25mM is obtained in DMSO；The Acetylshikonin for extracting 20 μ L1.25mM, which is dissolved in the DMSO of 20 μ L, to be obtained The Acetylshikonin of 0.6mM.

1.3.4 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 10 μ L10mM 20 μM of Acetylshikonin: are extracted Acetylshikonin be dissolved in the DMSO of 5mL and obtain 20 μM of Acetylshikonin, the Acetylshikonin for extracting 10 μ L10mM is molten Solution obtains 10 μM of Acetylshikonin in the DMSO of 10mL, extracts the Acetylshikonin that 10 μ L concentration are 5mM and is dissolved in 10mL DMSO in obtain 5 μM of Acetylshikonin, extract the Acetylshikonin that 10 μ L concentration are 2.5mM and be dissolved in the DMSO of 10mL Obtain 2.5 μM of Acetylshikonin；The Acetylshikonin for extracting 10 μ L1.25mM, which is dissolved in the DMSO of 10mL, obtains 1.25 μM Acetylshikonin；The Acetylshikonin for extracting 10 μ L0.6mM is dissolved in the acetyl Asian puccoon of 0.6 μM of acquisition in the DMSO of 10mL The Acetylshikonin of 10 μ L0.6 μM is dissolved in the Acetylshikonin of 0.3 μM of acquisition in the DMSO of 10 μ L by element.

1.3.5 120mg/ml Acetylshikonin: the Acetylshikonin powder for weighing 480g extraction is dissolved in 4ml DMSO.

1.3.6 it is molten that 0.2g yellow Jackets 0.4% yellow Jackets (Sinopharm Chemical Reagent Co., Ltd.): are weighed Solution filters in 50mL sterile distilled water.

1.3.7 mycillin is dual anti-: penicillin and streptomysin (Huabei Pharmaceutic Co., Ltd), the two concentration are 500U/mL

1.3.8 mono- bottle of physiological saline 500mL (Cisen Pharmaceutical Co., Ltd.)

1.3.9 Acetylshikonin is dissolved in PEG400 and H₂In O, make final concentration of 60mg/kg, with the dosage of 10mL/kg To intragastric administration on mice.

1.3.10 antigen retrieval buffers: weighing 2.94g trisodium citrate and 2.1g citric acid be dissolved in 1L water, adjusts pH and is 6.0。

1.4 instrument and equipment:

1.4.1 microplate reader (BD company)

1.4.2 inverted microscope (OLYPUMS)

1.4.3 Haier's Medical low-temperature storage box (Qingdao Haire Special Electrical Appliances Co., Ltd)

1.4.4 assay balance (Mei Tele-support benefit instrument Shanghai Co., Ltd)

1.4.5 Thermo superclean bench

1.4.6 IVC system (Suzhou City Feng Laboratory Animal Equipment Co., Ltd)

1.4.7 liquid-transfering gun, pipette (specification is respectively 5ml, 10ml, 25ml), (specification is respectively 15ml/ to centrifuge tube 50ml), 6 orifice plates, 96 orifice plates, 10cm Tissue Culture Dish, eye scissors, surgical forceps, sterile petri dish, scalpel, molten medicine needle, 1mL Syringe

2.1.1 the combination of TOPK kinase assay Acetylshikonin and TOPK kinases and Inhibition test: the TOPK of 2 μ g is taken to swash Centrifuge tube is placed on ice by the TOPK kinase reaction liquid of enzyme and 17 μ L in 1.5mL centrifuge tube, then with 1 μ L various concentration Acetylshikonin mixes in centrifuge tube, makes its final concentration of 0,0.6,1.25 and 2.5 μM, is incubated at room temperature 10 minutes, is being centrifuged The ATP and substrate c-Jun of the 10mM of 5 μ L are added in pipe, 40 DEG C are incubated for 30 minutes, pass through using immunoblot assay to silk- The contents level of threonine phosphorylation antibody test substrate phosphorylation horizontal detection substrate albumen and kinases in turn；It collects and cracks Colorectal cancer sensitive cells obtains cell protein, detects colorectal cancer cell concentration using BCA kit, prepares final concentration of 10 The albumen sample of μ g.Using sepharose 4B Binding experiment, detection compound Acetylshikonin and TOPK swash under Western blotting technique The combination effect of enzyme, is incubated for corresponding antibody test；Meanwhile copolymer, 4 DEG C of mistakes are coupled using compound and 4B agarose microbeads After night, using protein immunoblot technology detection compound and ATP competitive binding effect, concrete outcome is detailed in Fig. 3.

2.1.2 it cell culture and cell proliferation experiment research: Colon and rectum normal cell CCD 841 CoN and has screened It is capable of the culture of colorectal cancer cell HCT-15, HCT-116, SW620 and the DLD1 cell of great expression TOPK, wherein CCD + 10% volume FBS+1% volume mycillin of RPMI-1640 culture medium is dual anti-in the culture medium of 841 CoN, HCT-15 and DLD1 Culture medium used in cell is that RPMI-1640+10% volume FBS+1% volume mycillin is dual anti-, and SW620 cell uses Culture medium is that L15+10% volume FBS+1% volume mycillin is dual anti-, and culture medium used in HCT-116 cell is McCoy ' 5A+10% volume FBS+1% volume mycillin is dual anti-, the colorectal cancer cell kind for capableing of great expression TOPK that will have been screened It plants in 96 orifice plates, every hole cell number is 1000, after 48 hours adherent, gives the acetyl Asian puccoon of every 100 μ L of hole cell Element is handled, wherein the concentration for giving the Acetylshikonin of 841 CoN of colon normal cell CCD processing is respectively 0,2.5, 5,10,20 μM, colon cancer cell HCT-15, the concentration difference for the Acetylshikonin that HCT-116, SW620 and DLD1 are handled are given Be 0,2.5,5,10 μM, then respectively at 0h, for 24 hours, 20 μ l thiazolyl blues (MTT) be added after 48h and 72h be put into 37 DEG C of trainings immediately It supports and is incubated for 2 hours in case, discard the liquid in 96 orifice plates after taking-up, 100 μ l dimethyl sulfoxides (DMSO) are added, utilize enzyme Mark instrument detects cell proliferative conditions in the case where wavelength is 570nm and 620nm, and concrete outcome is detailed in Fig. 4 B, wherein with normal human colonic 841 CoN of epithelial cell CCD is detailed in Fig. 4 A as control.

2.1.3 non-anchor qualitative experimental study: non-anchor growth is one of key property of growth of tumour cell；Inventor sees The inhibiting effect that Acetylshikonin (0,0.3,0.6,1.2 μM) grows every 8000 colorectal cancer cells in hole is examined, lower layer's glue is matched The 0.6%Agar that method processed is 3mL be added 3 μ L DMSO solution or 3 μ L 0.3, the Acetylshikonin of 0.6,1.2mM, make second Final concentration of 0,0.3,0.6,1.2 μM of acyl alkannin, the 0.3%Agar that the preparation method of upper layer glue is 1mL are added 3 μ L's DMSO solution or 3 μ L 0.3, the Acetylshikonin of 0.6,1.2mM, make Acetylshikonin final concentration of corresponding 0,0.3, 0.6,1.2 μM, and take pictures respectively in the liang, three weeks, and then calculate suppression of the Acetylshikonin to the non-anchor growth of cell Efficiency processed, concrete outcome are detailed in Fig. 4 C.

The foundation of 2.2 Human colorectal carcinoma Immune deficient mices plantation tumor model

2.2.1 fresh tumor tissue is taken out into necrotic tissue, being cut into diameter is 3 cubic millimeters or so, fills in 6 week old, 18g Mouse is put into the raising of mouse IVC system by the dorsal sc of the female mice of left and right.Tumour is waited to form about 15mm or so, nothing Bacterium cuts off subcutaneous tumor, selects Space-occupying lesions, and materials are cut into 3 cubic millimeters of fritters, transplants in other mouse.Cervical dislocation is put to death Mouse, 75% alcohol disinfecting of tumour surrounding skin open an osculum with molten medicine needle bundle, tumour are taken out after being strutted with tweezers and is put into Mouse is subcutaneous.Tumour passage is carried out in aforementioned manners, until third generation mouse, mouse subcutaneous transplanting tumor occurs and tumor size is poor It is different little.It is subcutaneous that immunodeficient mouse is migrated to again, passes three generations.Subcutaneous transplantation tumor mice tumors grew feelings each time are recorded respectively Condition and tumor formation rate take forth generation mouse subcutaneous transplanting tumor sample when subcutaneous transplantation tumor waits tumour to grow to 1.5mm, and tissue is used 10% formalin is fixed, paraffin embedding, slice HE dyeing.

3. the Therapy study of experimental animal

3.1 inoculation after after a week, back of mice tumor nodule starts to be grouped when growing to 200 cubic millimeters or so, i.e., according to Gross tumor volume size evenly distributes mouse to every group, HJG17 12 mouse of every component.3 groups of mouse respectively according to following dosage from By drinking water.When control group mice gross tumor volume reaches 1500 cubic millimeters or so, experiment is terminated, removes dorsal sc tumour simultaneously Weighing, and tumor specimen is fixed using 10% formalin, paraffin embedding, it is sliced, while being detected using immunohistochemical experiment The albumen such as ki-67, p-TOPK, p-ERK1/2 and p-c-Jun in control group and high dose Acetylshikonin processing group tumor sample Expression.

A. DMSO, PEG400 and H in control group₂The volume ratio of O three: 400 45%+50%H of DMSO 5%+PEG₂O, Contain 50 μ L DMSO, the water of the PEG400 of 450 μ L and 500 μ L i.e. in 1mL control group.

B.40mg/kg DMSO, PEG400 and H in Acetylshikonin₂The volume ratio of O three: contain Acetylshikonin 400 45%+50%H of DMSO 5%+PEG₂O, i.e. 1mL 40mg/kg Acetylshikonin by 33.5 μ L 120mg/mL acetyl The water of alkannin, the DMSO of 16.5 μ L, the PEG400 of 450 μ L and 500 μ L form.

C.60mg/kg DMSO, PEG400 and H in Acetylshikonin₂The volume ratio of O three: contain Acetylshikonin 400 45%+50%H of DMSO 5%+PEG₂O, i.e. 1mL 60mg/kg Acetylshikonin are purple by the acetyl of 50 μ L, 120mg/mL The water composition of careless element, the PEG400 of 450 μ L and 500 μ L.

D. daily with 10 μ L/g mouse weight stomach-filling Acetylshikonins, mouse weight and tumour body are recorded twice a week Product.When control group mice gross tumor volume is grown to 15 cubic millimeters or so (32 days), tumor tissues are taken out in termination experiment, weigh swollen It tumor weight and takes pictures.

Experimental result

As shown in Figure 3A, living to TOPK kinases after handling TOPK kinases with 0.6,1.25,2.5 μM of Acetylshikonin group The inhibiting effect of property is apparently higher than control group, and with the increase of Acetylshikonin concentration, inhibiting effect is also gradually increased.Such as figure Shown in 3B, sepharose 4B Binding experiment illustrates that Acetylshikonin can effectively be combined with kinases TOPK.Computer simulation in Fig. 3 C As a result as can be seen that Acetylshikonin can with TOPK in specific site in conjunction with.

As shown in Figure 4 A, 2.5,5,10,20 μM of Acetylshikonin group handles people's normal colon epithelial cells CCD841CoN Non-toxic influence is grown on cell.As shown in Figure 4 B, the inhibition colon that 2.5,5,10 μM of Acetylshikonin group can be extremely significant Proliferation p < 0.01 of cancer cell.In Fig. 4 C as can be seen that 0.3,0.6,1.25 μM of Acetylshikonin group can be extremely significant suppression Non-anchor growth p < 0.01 of colon cancer cell processed.

It follows that Acetylshikonin targeting and TOPK and can inhibit the growth of colon cancer cell, have external The effect of neoplasm growth.

As shown in Figure 5A, 40mg/kg, 60mg/kg Acetylshikonin group have no significant effect mouse weight.Such as Fig. 5 B institute Show, high dose (60mg/kg) Acetylshikonin group after administration the 32nd day mouse tumor volume compared with control group than there were significant differences p <0.05.As shown in Figure 5 C, Acetylshikonin group (40,60mg/kg) mouse tumor and control group picture after administration the 32nd day.

As shown in fig. 6, Ki-67, p-TOPK, p-ERK1/2 and p-c- in Acetylshikonin high dose processing tumor tissues The expression of Jun is significantly higher than control group.

Claims (9)

1. Acetylshikonin prevents and treats the application in colon cancer drug in preparation, which is characterized in that have (1) or (2) institute The function of stating: (1) being used as kinases TOPK inhibitor, and (2) inhibit cancer cell multiplication.

2. application according to claim 1, which is characterized in that kinases TOPK mass is 2 μ g, Acetylshikonin dose concentration It is 0.6~1.25 μM.

3. application according to claim 1, which is characterized in that colon cancer cell quantity is 1000, Acetylshikonin suppression The dosage range of Growth of Colon Cancer Cells processed is 1.25~10 μM, and in non-anchor experiment, tumour cell quantity is 8000, every hole, Acetylshikonin measures range is 0.6~1.25 μM.

4. application according to any one of claims 1 to 3, which is characterized in that the Acetylshikonin is obtained by following methods :

(1) gained extracting solution is carried out concentration and obtained by the extracting solution for being obtained alkanet to alkanet raw material refluxing extraction using ethyl alcohol Obtain crude extract；

(2) crude extract obtained by step (1) is diluted with water, is successively extracted, is obtained with petroleum ether, ethyl acetate, n-butanol Obtain effective component；

(3) it is that 200-300nm large pore resin absorption column separates by aperture by step (2) resulting effective component, elutes Liquid is positive hexane-chloroform-methanol three's mixed solution, and n-hexane, chloroform and methanol volume ratio are 25:6:1, utilizes thin layer color Spectrum is identified, is merged the eluent for collecting same ingredient and is concentrated, obtains 7 kinds of extract component, 1,2,3,4 He of ingredient 5 be the crude product of effective component, is the plant residue in alkanet in ingredient 6 and 7.

5. application according to claim 4, which is characterized in that the detailed process of the step (1) are as follows: to 10kg alkanet 90% or more the ethanol solution of 60L is added in raw material, extracts 2~4 times, flow back 1.5~3h every time, and liquid is collected in filtering, steams Except solvent to get；The detailed process of the step (2) are as follows: by crude extract obtained by step (1) after the dilution of 2L distilled water, first It with petroleum ether extraction 2~4 times, then is extracted with ethyl acetate 2~4 times, finally carries out extraction 2~4 times through n-butanol again, the stone The dosage of oily ether, ethyl acetate and n-butanol is 3L, merges petroleum ether, ethyl acetate and n-butanol layer, solvent is evaporated off, To obtain the final product.

6. application according to claim 4, which is characterized in that by extract component 3 through sephadex-LH20 Sephadex-LH20 column is separated, and eluent is the petroleum ether and ethyl acetate mixtures of volume ratio 20:1, obtains two kinds of changes Close object beta, beta-dimethyl acry-lalkannin and Acetylshikonin.

7. application according to claim 4, which is characterized in that extract component 4 is flowed through the macropore that aperture is 200-300nm Adsorption resin column, eluent are that volume ratio is 25:6:1 n-hexane-chloroform-methanol mixed solution, obtain Acetylshikonin.

8. a kind of method using PDX mouse model detection Acetylshikonin effective dose in preparation treatment colon cancer drug, It is characterized in that, by the colon cancer sample taken plantation in will be obtained in immunodeficient mouse body, after 2-5 week be immunized mouse Colon Tumor takes resulting colon cancer tumours and passes in immunodeficient mouse, until obtaining colon cancer PDX mouse mould after 3-4 generation Type, and PDX model mice Acetylshikonin is fed using the method for stomach-filling, detect the growth feelings of colon cancer mouse interior tumor Condition.

9. according to the method described in claim 8, it is characterized in that, by Acetylshikonin be dissolved in PEG400 and water be made 40~ The Acetylshikonin solution of 60mg/kg, daily with 10 μ L/g mouse weight stomach-filling Acetylshikonin solution.