CN107670033A - Antineoplastic pharmaceutical compositions and its application - Google Patents

Abstract

The invention provides a kind of pharmaceutical composition and its application, its described pharmaceutical composition includes Oridonin and Cetuximab.When for treating laryngocarcinoma, obvious synergy is presented in the drug regimen.The present invention relates to pharmaceutical composition in vivo and experiment in vitro proves substantially to suppress laryngeal cancer cell growth, its mechanism is suppresses the activation of EGFR signal paths；Meanwhile the pharmaceutical composition can promote active oxygen to produce and then induce laryngeal cancer cell that apoptosis and cell-cycle arrest occurs.Drug regimen of the present invention has the advantages of antitumous effect is obvious and toxic side effect is small in the application for treating laryngocarcinoma.

Description

Antineoplastic pharmaceutical compositions and its application

Technical field

The present invention relates to a kind of antineoplastic pharmaceutical compositions and its application, and in particular to Cetuximab and Oridonin Composition and its anti-laryngeal cancer application, belong to pharmaceutical technology field.

Background technology

Head and neck neoplasm is the 6th big kinds of tumor in global range.Wherein, laryngocarcinoma (laryngeal cancer) is head The common malignant tumour of neck, in China, the disease accounts for the 1%~5% of general tumour, in annual 100000 tumor patient in China just There are 1.5~3.4 newly-increased laryngocarcinoma cases.Laryngocarcinoma pathological is based on squamous cell carcinoma.In recent years, adding with industrial pollution Weight, the incidence of disease of laryngocarcinoma have obvious ascendant trend (Cancer Treatment Reviews, 2006,32:504-515； Clinical Otolaryngology&Allied Sciences,2001,26:442-446).Due to early stage laryngocarcinoma symptom and body Unobvious are levied, are not aroused attention more, more than 60% patients with laryngeal carcinoma has been Locally Advanced (Experimental Cell when diagnosing Research,2014,321(2):133-141；Human Gene Therapy,2009,20:1565-1575), its carninomatosis becomes model Enclose extensively, surgery alone treatment often cuts off full larynx, hypopharynx or esophagus, hence it is evident that reduces life and the life quality of patient.It is based on Consideration in terms of Organoprotective and existence, collaboration chemicotherapy have turned into critical treatment means (the Chinese oral and maxillofacial surgery of patients with laryngeal carcinoma Surgical magazine, 2009,7 (4):290-295).

At present, 5 FU 5 fluorouracil (5-FU) and cis-platinum are the clinical chemotherapeutics for being usually used in treating laryngocarcinoma, but both Chemotherapeutics can cause sizable side effect, such as bone marrow suppression, liver damage to body tissue.Conventional drug combination side Case can increase curative effect, but also bring more side effects simultaneously, can cause the fibrosis, scar and oedema organized on a large scale, Cause the side effect such as dysphagia, expiration throat retardance.The side effect of traditional embolic chemotherapy has reached toxic limits (Journal of Clinical Oncology,2006,24(17):2603-2605), therefore, explore and find efficiently, it is less toxic Newtype drug and treatment method to anti-laryngeal cancer are always the focus of medicine drug development.

In recent years, with being deepened continuously to tumour cell signal transduction pathway research, turn for cell receptor, signal Lead, the molecular targeted therapy of cell cycle and angiogenesis etc., it has also become the new direction of oncotherapy.Wherein epidermal growth factor Sub- acceptor (Epidermal growth factor receptor, EGFR) is target spot main.Research report 80%- EGFR gene in 100% head and neck squamous cell carcinoma be present and be overexpressed (Drugs, 2000,60 (Suppl1):15-23； Cancer Research,1994,4:3153-3159).Our seminars use FISH (fluorescence in Situ hybridization, FISH) method detect people's squamous carcinoma of larynx HEp-2 cells and 11 laryngeal carcinoma tissue marks Exist in this EGFR gene amplification (Acta Genetica Sinica, 2004,31:109-113；Chinese Journal Medical Genetics,2000,17:278-280).Chrysovergis A etc. have found there is 48 in 55 laryngeal carcinoma tissue samples Example (87.2%) EGFR is overexpressed (Medical Oncology, 2011,28 (3):871-7).It can be seen that the EGFR of excessive activation can Can be cause people's squamous carcinoma of larynx occur, development the reason for one of.A large amount of clinical researches show, for the molecular targeted of EGFR The late relapse rate of head-neck malignant tumor can be reduced in head-neck malignant tumor treatment by treating, and can substitute some chemotherapy Medicine and usage amount (Journal of Clinical Oncology, 2004,22 (1) for reducing chemotherapeutic toxicity medicine:77- 85).In view of EGFR gene is overexpressed in people's squamous carcinoma of larynx, EGFR turns into laryngocarcinoma candidate therapeutic target spot the most prominent.

EGFR is a kind of multi-functional glycoprotein being distributed widely on each cell membranes in tissue of human body, is HER/ErbB families One of four members.When EGFR and acceptor EGF (Epidermal growth factor, EGF) or conversion grow After the factor-α (Transforming growth factor α, TGF- α) is combined, cause the dimerization of acceptor to act on, form homotype Or heterodimer.The acceptor of dimerization crosslinks phosphorylation, i.e. an acceptor makes specific tyrosine on another acceptor Residue phosphorylation, the EGFR-TK subprovince of intracellular region is activated, so as to excite next stage signal transduction.EGFR downstream signal transductions Path mainly has：Ras/MAPK paths, PI3K/Akt paths, PLC- γ paths, Jak/STAT paths (Biochimica et Biophysica Acta,2006,1766(1):120-139).EGFR adjusts cell propagation and differentiation, increase by these paths The invasiveness of tumour cell, promote angiogenesis and suppress tumour cell apoptosis (EMBO Journal, 2000,19:623- 630)。

Cetuximab (cetuximab, trade name Erbitux；C225) be it is currently the only through III clinical trial phase demonstrate,prove The real anti-EGFR targeted drugs for having obvious curative effects to Locally Advanced G. cephalantha.Cetuximab is a kind of anti-EGFR people mouse Chimeric mAb, extracellular section of EGFR is specifically bound to, can effectively block ligands, EGF, TGF- α and EGFR knot Close, suppress receptor phosphorylation, so as to block downstream signal passage, play antitumor action.But existing clinical test confirms Antitumor action is very limited when EGFR inhibitor is used alone, and the effective percentage of single medicine is less than 15% (European Archives Oto-Rhino-Laryngology,2008,265(Suppl 1):S3-S12).Current research is concentrated mainly on itself and biography Unite treatment method or with the strategy that other antineoplastics are combined, i.e. can using the antitumor data of Cetuximab as Benchmark, head and neck squamous cell carcinoma (Clinical Cancer are treated by the way of two kinds, three kinds of combination therapies Research,2004,10(19):6487-6501).In recent years, the natural antitumor medicine of plant origin is generally to normal people Source cell is nontoxic or hypotoxicity, can reduce supporting for tumour cell by amplifying weaker dead signal from different drug combinations Anti- valve, it is ensured that effective cell death (Products in China magazine, 2011,24 (9) occur for tumour cell:1112-1115). Plant extract medicine known to some mechanism is typically used to antitumor synergy research (Journal of Cellular Physiology,2010,222(2):433-443；Cancer Research,2009,69(23):8910-8917).

Oridonin (Oridonin) is a kind of shellfish isolated from Labiatae Rabdosia (Rabdosia) plant Shell China fir alkene Diterpenes natural organic-compound.Great mass of data proves that Oridonin is to human colon carcinoma SW1116 cells, human pancreas The kinds of tumors such as cancer PaCa cells and human gastric cancer SNU-5 cells be respectively provided with obvious inhibitory action (BMC Cancer, 2010,10: 610；International Journal of Oncology 2012,41:949-958；Tumor Biology,2014,35: 9139-9146), its therapeutic action to solid tumor has been widely used in clinic at present, and it is a variety of to be usually used in cancer of the esophagus, liver cancer etc. The treatment of solid tumor, and achieve obvious clinical efficacy (cancer, 1984,8 (1):50；Treatment and prevention of tumour is studied, and 1984, 11(2):86).We have found that Oridonin can suppress the increasing of people squamous carcinoma of larynx HEp-2 cells first in previous work Grow, mechanism of action may be to suppress the activation of EGFR signal paths.When Oridonin and AG1478 (EGFR specific inhibitors) Further suppress EGFR activation after combination, and cooperate with increase HEp-2 Apoptosis (Cancer Letters, 2010,294: 147-158), prompt, by Oridonin and EGFR clinic target therapeutic agents --- it is probably a kind of that Cetuximab, which is combined, The method of effective treatment laryngocarcinoma.

Apoptosis (apoptosis) is cell active, the orderly suicide carried out under specific gene regulation Journey.It is that cell stimulates the responsing reaction made to internal/external signal, is one of important mechanisms for maintaining homeostasis.Apoptosis Morphological change is mainly reflected in nucleus, shows as chromatin pyknosis and aggegation, is the principal mode of cell death.Apoptosis Two approach is respectively death receptor pathway * and mitochondria pathway.Fas signal pathways are most representational death receptor pathway *s. After corresponding part is combined with Fas acceptors, dimerization will occur for the acceptor on film, and the domain being present in endochylema will Change, raise intracellular adaptor protein FADD (Fas-associated death domain) afterwards, and then make FADD Other end Death Effector Domain (death effector domain, DED) structure change, then raise Procaspase-8, be collectively forming dead inducement signal compound (death inducing signaling complex, DISC).After DISC is formed, procaspase-8 itself activation is can induce, produces the caspase-8 of activity, further under activation The caspase-3 of trip, so as to apoptosis-induced.

Cell cycle (cell cycle) is the essential characteristic of cell activities.Generation, the development of most of malignant tumour It is relevant with cell cycle regulating dysfunction, so, regulation cell cycle progression is one of approach for treating tumour.Cell week Interim main DNA damage check point has G1/S phases check point, S phases check point and G2/M phase check points.Adjust cell cycle prison The key protein of measuring point has three major types：Cell cycle dependent protein (Cyclins), cell cycle protein dependent kinase (Cyclin-dependent kinases, CDKs), cell cycle protein dependent kinase inhibitor (Cyclin-dependent Kinase inhibitors, CDKIs), they collectively form the regulated and control network of cell cycle.Wherein CDKs is regulated and control network Core, Cyclins have positivity regulating and controlling effect to CDKs, and CKIs has negativity regulating and controlling effect (Science, 1996,272 (5263): 877).Cyclins of the Cyclin B as the G2 phases, with cell cycle protein dependent kinase CDK1 (also known as Cdc2) knot Synthesizing complex promotes the cell cycle to carry out G2/M phase transformations.CDK1 activity is adjusted by cyclin CyclinB forward direction Control, also need the regulation and control of catalytic subunit phosphorylation.Cdc25C is a protein phosphatase, and it can be responsible for making CDK1 dephosphorylations And activate.After Cdc25C phosphorylations, its own activity inhibited, so as to suppress CDK1 activation, and then CDK1- is prevented The activation of cyclinB1 compounds and it is mitotic carry out (Oncogene, 2004,23:905-913).

Active oxygen (reactive oxygen species, ROS) is a general designation, refers to a kind of short-lived and has height The small molecule of activity, is mainly formed by the incomplete one-electron reduction of oxygen molecule.ROS includes oxygen anion, free radical and mistake Oxide etc., such as superoxide anion, hydroxy radical and hydrogen peroxide etc..A small amount of ROS cell proliferations, maintain redox Balance is played an important role, and cellular protein kinases, the activity of transcription factor and early stage gene expression can be adjusted as courier (Journal of Biological Chemistry,2005,280(47):39485-39492).When various external sources or endogenous When ROS caused by factor exceedes internal Scavenging activity, the horizontal rises of ROS are may result in, cause oxidative stress, in oxidative cell The macromolecular structures such as how unsaturated fat, protein and DNA, so as to destroy their biological function, further result in cell week Phase blocks and Apoptosis (Free Radical Biology and Medicine, 1996,20 (5):675-705； Biochemical Pharmacology,2010,79:825-841；Trends in Biochemical Sciences,1996, 21:83-86)。

MAPK (mitogen-activated protein kinase, MAPK) is a kind of silk ammonia The protein kinase of acid/threonine residues, it is important channel (the J Biol Chem.2006 of cell signalling:281,5734- 5740).MAPK signal path is a three-level phosphorylation cascade reaction.Research shows that each member of MAPK families has Respective substrate, different effects is played for cell living or death under different cells and unlike signal stimulation. (Cell.2000,103:239-252)

So far, on be used in combination in antineoplaston Cetuximab and Oridonin and the two anti- There is not been reported for synergy in tumour.

The content of the invention

The invention aims to increase treatment of laryngeal carcinoma effect, there is provided a kind of pharmaceutical composition is in laryngocarcinoma is treated Using the specially application of Oridonin and Cetuximab in people's squamous carcinoma of larynx.

Oridonin structural formula is in pharmaceutical composition of the present invention：

The purpose of the present invention is achieved through the following technical solutions：

The present invention is using Rabdosia rubescens medicinal material as raw material, by 95% ethanol solution refluxing extraction 3 times；Resulting extraction Thing, with petroleum ether degreasing, then isometric ethyl acetate extraction；Gained extract is separated by silica gel column chromatography, uses dichloro Methane and methanol elution gradient, Oridonin crude product is obtained, through being recrystallized to give Oridonin.

Cetuximab (Erbitux) preparation method and formulation method are described in United States Patent (USP) 6217866B1.Western appropriate former times Monoclonal antibody is purchased from Merck companies.

Oridonin and Cetuximab in pharmaceutical composition of the present invention include its pharmaceutically acceptable salt respectively, And pharmaceutically acceptable excipient or carrier.

The present invention has found that Oridonin can pass through suppression with Cetuximab combination first by external, experiment in vivo EGFR signal paths processed and suppress laryngocarcinoma growth.Wherein, Cetuximab and Oridonin concentration are respectively 1~100 μ g/mL With 12~36 μM.

The present invention have detected Oridonin with Cetuximab combination to people's squamous carcinoma of larynx apoptosis and cell cycle The influence of distribution.As a result the two combination of display can be blocked with co-induction Apoptosis and G2/M phases.ROS mediations are found simultaneously JNK signal paths participate in exogenous apoptosis and the G2/M phases block, and the effect that performance promotion apoptosis and G2/M phases block.

The present invention provides Oridonin and Cetuximab is combined the application of antitumor action in vivo, Papillary association With the growth for suppressing people's squamous carcinoma of larynx transplanted tumor in nude mice, inhibitory rate to 92.2%.In drug treatment, nude mice essence God is in good condition, grow, diet, activity it is normal, have no obvious adverse reaction, the weight of animals does not have significance difference between each group It is different.Oridonin and Cetuximab combination collaboration suppress tumor tissue growth, and apoptosis-induced generation simultaneously.

In the scheme that the present composition is made to the medicament being administered simultaneously, Oridonin and Cetuximab can be with It is contained in same pharmaceutical preparation such as injection, Oridonin can also does to piece agent or capsule, but Cetuximab It is administered intravenously (IV, the instruction of patient to specifications uses simultaneously；The present composition is being made to the medicine of consecutive administration In agent scheme, Oridonin can be made to tablet or capsule, but Cetuximab is administered intravenously (IV, patient can press The sequencing of book instruction is taken as directed.

In the present invention, Oridonin can be prepared into intestines and stomach or parenteral administration using the conventional method in this area Pharmaceutical preparation.In the pharmaceutical preparation of Oridonin and Cetuximab composition of the present invention, according to different dosage forms With preparation specification, the content of the composition in the formulation can be that quality is calculated as 1-99%, preferably 10%-90%；Preparation makes Auxiliary material can use the conventional auxiliary material in this area, reacted with the present composition of getting along well or do not influence medicine of the present invention Premised on curative effect；It is prepared by the preparation method that the preparation method of the preparation can use this area conventional.

The dosage of pharmaceutical composition in the present invention according to the dosage form of administration object, method of administration or medicine not It is same to carry out appropriate change, but to ensure that the pharmaceutical composition can reach effective blood concentration in mammalian body Premised on.

The beneficial effects of the invention are as follows：

(1) Oridonin preparation process selects dichloromethane, methanol and ethanol equal solvent system in the present invention, relative to make With safety.Recrystallization technology used can prepare steady quality, the Oridonin of high-purity in the technique.Preparation process Easy to operate, separating rate is fast, and cost is low.

(2) Oridonin and Cetuximab pharmaceutical composition of the present invention, it is thin for people's larynx squamous of the high expression of EGFR The growth of born of the same parents' cancer has obvious collaboration inhibition.

(3) Oridonin and Cetuximab pharmaceutical composition of the present invention, for the more single medicine Rabdosia rubescens of toxicity of nude mice A prime is obviously reduced, and reduces injury of the pharmaceutical composition for body.

Brief description of the drawings

Fig. 1 is that molecular docking shows Oridonin and EGFR binding site.

Fig. 2 be Oridonin (ORI) combine Cetuximab (Cet) to people's squamous carcinoma of larynx HEp-2 cells and The growth inhibition effect of Tu212 cells；Wherein：A：ORI cooperates with Cet combinations suppresses HEp-2 and Tu212 cells propagation；B：ORI With Cet associated with different modes of administration to the growth inhibition effects of HEp-2 and Tu212 cells；C：ORI joints Cet suppresses HEp-2 EGFR signal paths activation intracellular with Tu212.

Fig. 3 is that ORI combines Cet induction HEp-2 and Tu212 Apoptosis；Wherein：A：Fluorescence microscope ORI combines Cet inducing cell apoptosis；B：Apoptosis ratio caused by flow cytometry quantitative analysis ORI joints Cet；C：Western Immuno The influence that blotting detection ORI joints Cet is expressed apoptosis-related protein.

Fig. 4 is that ORI joints Cet induces HEp-2 the and Tu212 cells generation G2/M phases to block；Wherein：A：Flow cytometry point Analyse the influence of ORI joint Cet cell cycle distributions；B：ORI combines influences of the Cet to G2/M phase correlative protein expressions.

Fig. 5 is that ROS participates in HEp-2 the and Tu212 Apoptosis of ORI joint Cet inductions and the G2/M phases block；Wherein：A： ORI joint Cet inductions ROS is produced；B：Pre-process shadow of the ROS scavengers to the cell growth inhibition of ORI joint Cet inductions Ring；C：Pre-process influence of the ROS scavengers to the Apoptosis of ORI joint Cet inductions；D：ROS scavengers are pre-processed to apoptosis The influence of correlative protein expression；E：Pre-process influence of the ROS scavengers to HEp-2 cell cycle distributions；F：ROS is pre-processed to remove Influence of the agent to Tu212 cell cycle distributions.

Fig. 6 is that ROS mediates JNK signal paths to participate in HEp-2 and Tu212 Apoptosis and G2/M that ORI combines Cet inductions Phase blocks；Wherein：A：Pre-process influence of the MAPK inhibitor to the cell growth inhibition of ORI joint Cet inductions；B：ORI Influences of the joint Cet to JNK and p-JNK protein expressions；C：Pre-process jnk inhibitor and combine the apoptosis-induced shadows of Cet to ORI Ring；D：Pre-process influence of the jnk inhibitor to apoptosis-related protein；E：Pre-process the shadow of jnk inhibitor cell cycle distribution Ring；F：Pre-process the influence of jnk inhibitor cell cycle albumen；G：Pre-process shadow of the ROS scavengers to p-JNK protein expressions Ring.

Fig. 7 is that ORI combines Cet to people's squamous carcinoma of larynx HEp-2 cells transplanted tumor in nude mice propagation, apoptosis and GAP-associated protein GAP The influence of expression；Wherein：A：ORI combines influences of the Cet to transplanted tumor in nude mice volume and body weight；B：ORI combines Cet to HEp-2 The influence of the EGFR signal proteins of cell transplanted tumor in nude mice；C：ORI combines ROS contents of the Cet to HEp-2 cell transplanted tumor in nude mice Influence；D：ORI combines Cet to nude mice model tumor tissue EGFR signaling pathway proteins, PCNA expression and the influence of apoptosis.

Embodiment

The present invention can be illustrated by following embodiment.

Embodiment 1：The preparation method of Oridonin

By Rabdosia rubescens dry aerial parts (9kg), with 95% ethanol solution refluxing extractions 3 times, each 2h of 10 times of amounts, Medicinal extract can be obtained after the ethanol extract of gained is recycled into no alcohol taste.Medicinal extract is dissolved in the water, respectively with isometric oil Ether degreasing, then isometric ethyl acetate extraction, obtains ethyl acetate layer extract 180g.Ethyl acetate layer extract is passed through afterwards Silica gel column chromatography separation is crossed, with dichloromethane and methanol with 100:0,100:1,100:2,100:3,100:5,100:7,100:10 Volume ratio continuously elute, Oridonin crude product is obtained, through being recrystallized to give white, needle-shaped crystals (3g).

The white, needle-shaped crystals (MeOH), vanillic aldehyde-strong sulfuric acid response are positive.Speculate the chemical combination with reference to 1H, 13C-NMR The molecular formula of thing is C20H28O6, degree of unsaturation 7.Speculate that the compound is shellfish by above physicochemical property and Wave Spectrum feature Shell China fir alkane type diterpene.

Terminal double bond proton signal δ 6.31 (1H, br s), 5.51 (1H, br s) are provided in 1H-NMR spectrums；Three company's oxygen carbon Proton signal δ 5.35 (1H, s), 4.30 (1H, dd, J=10.6,6.9Hz), 3.63 (1H, m)；Even Oxymethylene proton signal δ 4.80 (1H, d, J=9.9Hz), 4.42 (1H, d, J=9.9Hz)；Pair of horns methyl proton signal δ 1.20 (3H, s), 1.13 (3H,s)。

13C-NMR composes the carbon signal δ 209.7,153.8,119.5 for providing alpha, beta-unsaturated ketone；Even dioxy carbon signal δ 98.9；Four company oxygen carbon signal δ 75.3,74.0,73.6,64.5.

In summary, and by the hydrocarbon modal data of the compound and known compound Oridonin (Chemical＆ Pharmaceutical Bulletin,2000,48(1):148-149) it is compared, it is basically identical, therefore identify that the compound is Oridonin.Nuclear magnetic data ownership in detail is shown in Table one.

The 1H-NMR of table one (600MHz), 13C-NMR (75MHz) Data for oridonin

Embodiment 2：Molecular docking

1 experimental method

Oridonin and EGFR are subjected to molecular docking using Autodock4.23 molecular dockings software, obtain Rabdosia rubescens A prime and EGFR Conjugated free energy (binding free energy).

2 experimental results

As seen from Figure 1, Oridonin is combined with EGFR Lys721 and Asp831 in the form of hydrogen bond, and Val702, Leu694 and Leu820 is combined in the form of hydrophobic bond.Oridonin and EGFR Conjugated free energy are -8.34kcal/mol, Its binding site be EGFR intracellulars portion ATP-binding site (Cell, 2006,125:1137-1149).Under normal circumstances, The Conjugated free energy of Autodock predictions reaches -9kcal/mol, that is, thinks that affinity can reach nM ranks.Molecular Docking Study As a result combine previous experiments result of study to prompt, Oridonin has very strong binding ability with EGFR.

Embodiment 3：Cetuximab combines Oridonin to people's squamous carcinoma of larynx HEp-2 cells and Tu212 cells Growth inhibition effect

1 experimental method

(1) mtt assay detection cell propagation：HEp-2 and Tu212 cells are inoculated in blue or green containing 10% hyclone, 1 × 105U/L In the RPMI-1640 culture mediums of mycin and 100g/L streptomysins, 5%CO2 incubators, cultivate at 37 DEG C.By exponential phase HEp-2 and Tu212 cells, 96 orifice plates are inoculated in every hole 3 × 104 and 5 × 104 respectively.After cultivating 24h, adding concentration is 0th, 1,10,100 μ g/mL Cetuximab and 0,12,24,36 μM of Oridonin.3 multiple holes of every group of setting, set simultaneously Put negative control.5%CO2 incubators are placed in, 37 DEG C are continued to cultivate 24 and 48h.100 μ L 5mg/mL MTT, culture are added per hole After 2.5h, nutrient solution in hole is carefully discarded, the μ L of DMSO 150 are added per hole, micro oscillator vibration 10min Shi formazans are completely molten Solution, each hole light absorption value (A) value is determined using ELIASA under 490nm wavelength, inhibiting rate is calculated according to formula below：

Survival rate (%)=[(A492, Sample-A492, Blank)/(A492, Control-A492, Blank)] × 100%

(2) drug interaction is evaluated：Combination index (CI) is the multi-medicament effect equation according to Chou-Talalay The drug synergism index of quantization.The various concentrations in cell proliferation experiment are calculated respectively using Calcusyn biological softwares The united CI values of Oridonin and Cetuximab of combination.Calculating can obtain when obtaining the equation under different effect levels To different CI values, work as CI<When 1, two medicines show as acting synergistically；During CI=1, two medicines show as summation action；CI>When 1, two Medicine shows as antagonism.

(3) compared with Oridonin shares different dosing order with Cetuximab：By the HEp-2 of exponential phase and Tu212 cells are inoculated in 96 orifice plates, and 24h is cultivated in 37 DEG C of 5%CO2 incubators.Then use at three kinds of drug combination modes Manage two kinds of laryngeal cancer cell strains, respectively Oridonin and Cetuximab act on simultaneously, after Oridonin pretreatment 1h again Oridonin is given again after giving Cetuximab and Cetuximab pretreatment 1h, and different dosing is detected using mtt assay The inhibitory rate of cell growth of mode.

(4) Oridonin is combined the influence to the expression of EGFR pathway proteins with Cetuximab：By HEp-2 and Tu212 Cell is inoculated in blake bottle with 8 × 105 and 9 × 105/mL density respectively, and it is 10 μ g/ that concentration is added after adherent 24h ML Cetuximabs and 24 μM (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonin effect 48h.Cell is collected, is made 30min is cracked with protein lysate ice bath, centrifugation, collects supernatant.Determined with Bio-Rad protein assay reagent Protein content.Sample is separated with 10%SDS- polyacrylamide gel electrophoresises, and gone on nitrocellulose filter.Primary antibody is incubated Overnight, TBST is washed 3 times, and secondary antibody is incubated 2h, and TBST is washed 3 times, the colour developing of ECL luminous substrates, film for medical X-ray radiography exposure.

2 experimental results

From Fig. 2A, Oridonin is capable of induction HEp-2 and the Tu212 cell death of time, dose dependent.Winter The careless A prime list medicine of IC50 values when insulting to(for) HEp-2 and Tu212 cell 48h are respectively 20 and 24 μM.And Cetuximab list medicine Little for two kinds of human laryngeal cancer cell growth inhibitions, highest inhibiting rate is only 19%.Drug combination group for HEp-2 and The growth inhibition effect of Tu212 cells is then the increase of extension and drug concentration over time and increases, and to tumour cell Inhibitory action be higher than corresponding single medicine treatment group.

As shown in table two and three, the CI values of various concentrations Cetuximab and Oridonin combination are respectively less than 1, prompt the winter Insult careless A prime joint Cetuximab and can cooperate with and promote HEp-2 and Tu212 cell deaths.

The Cetuximab of table two and Oridonin combine the growth inhibition effect Cetuximab winter to HEp-2 cells Insult careless A prime inhibiting rate (%) CI

The Cetuximab of table three and Oridonin combine the growth inhibition effect Cetuximab winter to Tu212 cells Insult careless A prime inhibiting rate (%) CI

As shown in Figure 2 B, three kinds of different Oridonins and the growth inhibition ratio of Cetuximab combination administration group are bright It is aobvious to be better than single medicine group.In three kinds of administering modes, first handle 1h with Oridonin and give Cetuximab (ORI → Cet) group pair again It is most strong in HEp-2 and Tu212 inhibitory action.And first with Cetuximab handle 1h after Oridonin (Cet → ORI) group with Treatment group (ORI+Cet) is compared simultaneously, in the absence of significant difference.These results suggest that, Oridonin first handle 1h again to Give the order of administration effect of Cetuximab the most obvious, later stage experiment in vitro will use this pattern.

As shown in Figure 2 C, in HEp-2 and Tu212 cells, Cetuximab list medicine can substantially suppress p-EGFR expression. Oridonin list medicine can also lower p-EGFR expression to a certain degree.After two medicines are combined, p-EGFR expression quantity enters one Step is reduced.Simultaneously the two combine after for EGFR downstream signaling pathways (p-Erk and p-Akt) influence with p-EGFR expressions Change it is consistent.Illustrate that Cetuximab promotes cell death with Oridonin combination by suppressing EGFR signal paths.

Embodiment 4：Cetuximab and Oridonin combination induction people's squamous carcinoma of larynx HEp-2 and Tu212 cell Apoptosis

1 experimental method

(1) fluorescence microscope apoptosis：By HEp-2 the and Tu212 cells of exponential phase, with every hole 2 × 105 and 3 The density of × 105 cells is inoculated in 6 orifice plates, and addition concentration is 10 μ g/mL Cetuximabs and 24 μM after cultivating 24h (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonin effect 48h, add 20 μ g/mL AO and dye l in 37 DEG C of lucifuges H, then in the form of fluorescence microscopy Microscopic observation nucleus.

(2) the double dyes of Annexin V-PE/7-AAD combine flow cytomery apoptosis ratio：HEp-2 and Tu212 is thin Born of the same parents are inoculated in 6 orifice plates with the density in every hole 2 × 105 and 3 × 105 cells respectively, and addition concentration is 10 μ g/ after cultivating 24h ML Cetuximabs and 24 μM (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonin effect 48h, after pancreatin digestion Single cell suspension is made, is adjusted cell density to 5 × 104/mL left sides with the RPMI-1640 culture mediums containing 10% hyclone The right side, 1mL cell suspension is measured after 4 DEG C of centrifugation 10min of 1000rpm, supernatant is carefully discarded, collects cell.Add PBS Wash, 1000rpm, supernatant is abandoned after 4 DEG C of centrifugation 10min, then add PBS washings, centrifugation, the cell of 1 × 105 concentration of collection.Will Suspension cell is added in 500 μ L Binding Buffer, after adding 5 μ L Annexin V-PE and suspension cell mixing, then is added The 7-AAD for entering 5 μ L is mixed.At room temperature, pay attention to lucifuge, stand reaction 15min.Above-mentioned cell mixture up flow type cell immediately Instrument detects apoptosis result.

(3) expression of Western blot methods detection apoptosis-related protein：Western blot methods are (the same as implementation The method of example 3)

2 experimental results

From Fig. 3 A, using fluorescence microscope, the cell of Oridonin list medicine group and drug combination group can be observed There occurs obvious morphological change.Cellular control unit nuclear staining is uniform, and cell membrane is complete.Oridonin list medicine and joint are used After medicine processing, the visible obvious change of form of cell, cell size, the inhomogeneity of form are gradually obvious, and nucleus is in cause Close dense dye or formation apoptotic body, apoptotic cell substantially increase.

From Fig. 3 B, compared with cellular control unit, Oridonin list medicine group and drug combination group have in various degree Apoptotic cell produces.The apoptosis ratio of drug combination is added merely apparently higher than the apoptosis rate after two kinds of medicines individually processing.On State result to show, Cetuximab and Oridonin are combined that to produce co-induction on two plants of cells of HEp-2 and Tu212 thin The effect of born of the same parents' apoptosis.

As shown in Figure 3 C, Oridonin list medicine can substantially raise Fas, FADD and FasL protein expression, and increase and live Property form caspase-8, caspase-3, ICAD and activity form PARP substantially increases.But Cetuximab list medicine for Caspase-3 and its downstream albumen PARP and ICAD effect unobvious.Oridonin can be obvious with Cetuximab combination The influence that increase Oridonin list medicine is expressed for apoptosis-related protein.As a result show that Oridonin joins with Cetuximab With the activation of caspase families by activating external source apoptosis pathway, is promoted so as to inducing HEp-2 and Tu212 Apoptosis.

Embodiment 5：Oridonin and Cetuximab combination induction people's squamous carcinoma of larynx HEp-2 and Tu212 cell The generation G2/M phases block

1 experimental method

(1) PI dyeing combines flow cytomery cell cycle distribution ratio：By HEp-2 and Tu212 cells respectively with The density of every hole 2 × 105 and 3 × 105 cells is inoculated in blake bottle, and addition concentration is 10 μ g/mL western appropriate former times after cultivating 24h Monoclonal antibody and 24 μM (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonin effect 48h, then collect cell and use PBS Washed once, then with 70% ethanol in -20 DEG C of fixed at least 12h.Cell cake after centrifugation, which is used, contains PBS, 5 μ g/mL PI And 1mg/mL RNaseA dye solution is resuspended, 4 DEG C of incubation 30min of lucifuge, when each in the flow cytometry analysis cell cycle The cells ratio of phase.

(2) the Western blot methods detection G2/M phases block the expression of associated adjustment albumen：Western blot Method (with the method for embodiment 3).

2 experimental results

From Fig. 4 A, compared with the cells ratio of control group G2/M phases, after Cetuximab list medicine processing cell 48h, Two plants of laryngeal cancer cell period profiles are without significantly changing, and Oridonin list medicine can substantially raise G2/M phase ratios.Rabdosia rubescens A prime further increases G2/M phase cells ratios with Cetuximab combination.Prompting Oridonin lures with Cetuximab combination People squamous carcinoma of larynx HEp-2 and Tu212 cell the generation G2/M phases are led to block.

As shown in Figure 4 B, Oridonin can suppress Cyclin B1 and Cdc2 protein expressions.Cdc25c nonactive shape Formula phospho-Cdc25c expression quantity increase, and active Cdc25c is then reduced.In addition, Oridonin increases P21 and p-Cdc2 expression.With Oridonin list medicine phases ratio, drug combination can substantially increase Cdc2, Cyclin B1 and The inhibitory action of Cdc25c expression, and further increase p21 and p-Cdc2 up-regulation.This result and flow cytometric analysis results phase Unanimously.

Embodiment 6：ROS participate in regulation and control Oridonin and Cetuximab combination induction human laryngeal cancer Apoptosis and The G2/M phases block

1 experimental method

(1) it is horizontal to combine flow cytomery ROS for DCFH-DA dyeing：HEp-2 and Tu212 cells are inoculated in 6 orifice plates In, cultivate and concentration is separately added into after 24h (Tu212 is thin for 10 μ g/mL Cetuximabs and 24 μM (HEp-2 cells) or 36 μM Born of the same parents) after Oridonin effect 48h, cell is collected, 10 μM of DCFH-DA is added and is incubated 30min, fluidic cell in 37 DEG C of lucifuges Instrument fluorescence intensity.

(2) effects of the mtt assay detection ROS in cell death：HEp-2 and Tu212 cells are inoculated in 96 orifice plates, added After entering ROS scavenger NAC and Cat pretreatment cells 1h, it is that (HEp-2 is thin for 10 μ g/mL Cetuximabs and 24 μM to add concentration Born of the same parents) or 36 μM of (Tu212 cells) Oridonin function cells 48h, MTT detection drug combinations and pretreatment ROS scavengers The influence of cell proliferation (with the method for embodiment 3).

(3) the double dyes of Annexin V-PE/7-AAD investigate effects of the ROS in apoptosis：HEp-2 and Tu212 cells are inoculated with In 6 orifice plates, after adding ROS scavenger NAC and Cat pretreatment cells 1h, it is 10 μ g/mL Cetuximabs and 24 to add concentration μM (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonins effect 48h, utilize the double dyes of Annexin V-PE/7-AAD With reference to after flow cytomery drug combination and pretreatment ROS scavengers to the Apoptosis of cell (with the method for embodiment 3).

(4) PI combinations flow cytometer investigates the effect during ROS blocks in the G2/M phases：HEp-2 and Tu212 cells are inoculated with In 6 orifice plates, after adding ROS scavengers NAC and Cat pretreatment 1h, it is 10 μ g/mL Cetuximabs and 24 μM to add concentration (HEp-2 cells) or 36 μM of (Tu212 cells) Oridonin effect 48h, are combined using PI combinations flow cytomery The influence that cell cycle is distributed after medication and pretreatment ROS scavengers (with the method for embodiment 5).

2 experimental results

As shown in Figure 5A, can be obvious when Oridonin is combined with Cetuximab in HEp-2 and Tu212 cells Increase intracellular ROS to produce, ROS yields are most during 3h, gradually reduce afterwards.

As shown in Figure 5 B, MTT results are shown, with drug combination group ratio, NAC and Cat pretreated group inhibitory rate of cell growth Significantly reduce, show that ROS participates in Oridonin and the cell death of Cetuximab combination induction.

As seen in figs. 5c and 5d, pretreatment ROS scavengers can substantially reduce Oridonin and are combined with Cetuximab The ratio and Fas apoptosis pathway of caused Apoptosis, show that ROS participates in Oridonin and Cetuximab combination induction Apoptosis, and ROS plays apoptosis-promoting effect.

As shown in Fig. 5 E and 5F, pretreatment ROS scavengers can reverse the G2/M phases caused by drug combination to block, and show ROS participates in Oridonin and the G2/M phases of Cetuximab combination induction block, and ROS is played and promoted G2/M phase retardations.

Embodiment 7：The human laryngeal cancer that the JNK paths participation Oridonin of ROS mediations shares induction with Cetuximab is thin Born of the same parents' apoptosis and G2/M phases block.

1 experimental method

(1) mtt assay investigates effects of the MAPK in cell death：HEp-2 and Tu212 cells are inoculated in 96 orifice plates, After adding MAPK inhibitor pretreatment 1h, it is 10 μ g/mL Cetuximabs and 24 μM (HEp-2 cells) or 36 μM to add concentration Cell is increased after (Tu212 cells) Oridonin function cells 48h, MTT detection drug combination and pretreatment MAPK inhibitor The influence grown.

(2) effects of the Annexin V-PE/7-AAD dyeing detection JNK in apoptosis：HEp-2 and Tu212 cells are inoculated with In 6 orifice plates, after SP600125 pretreatment cells 1h, it is that (HEp-2 is thin for 10 μ g/mL Cetuximabs and 24 μM to add concentration Born of the same parents) or 36 μM of (Tu212 cells) Oridonin effect 48h, it is thin to combine streaming using the double dyes of Annexin V-PE/7-AAD Born of the same parents' instrument influences after detecting drug combination and pretreatment jnk inhibitor on Apoptosis.

(3) effect during JNK blocks in the G2/M phases is investigated in PI dyeing：HEp-2 and Tu212 cells are inoculated in 6 orifice plates, After SP600125 pretreatment cells 1h, it is 10 μ g/mL Cetuximabs and 24 μM (HEp-2 cells) or 36 μM to add concentration (Tu212 cells) Oridonin acts on 48h, using PI dyeing with reference to flow cytomery drug combination and pretreatment JNK To the influence of cell distribution after inhibitor.

(4) Western blot methods detection JNK expression：Western blot methods (with the method for embodiment 3)

2 experimental results

As shown in Figure 6 A and 6B, MTT results show, compared to Oridonin and Cetuximab drug combination group, add The inhibitory rate of cell growth for entering jnk inhibitor SP600125 pretreated groups significantly reduces, and add ERK inhibitor PD98059 and P38 inhibitor SB203580 promotes cell death.In addition, Oridonin list medicine can increase p-JNK expression, and with it is western appropriate Former times monoclonal antibody combination p-JNK expression quantity further increases.Show JNK in Oridonin and Cetuximab combination inducing cell The effect for promoting cell death is played in death process, ERK and p38 play the effect for promoting cell survival.

As shown in figures 6 c and 6d, pretreatment SP600125 can be relieved apoptosis ratio and apoptosis caused by drug combination GAP-associated protein GAP, show that JNK promotes Oridonin and the Apoptosis of Cetuximab combination induction.

As shown in figs. 6e and 6f, pretreatment SP600125 can substantially suppress G2/M retardances and correlation caused by drug combination Albumen, show that JNK promotes Oridonin and the G2/M phases of Cetuximab combination induction to block.

As shown in Figure 6 G, compared to Oridonin and Cetuximab drug combination group, the cell p- of NAC pretreated groups The horizontal conspicuousnesses of JNK reduce, and illustrate that ROS participates in Oridonin and the p-JNK up-regulations of Cetuximab combination induction act on.

Embodiment 8：Oridonin combines Cetuximab to people's squamous carcinoma of larynx HEp-2 cell transplanted tumor in nude mice Influence

1 experimental method

(1) Oridonin is combined the influence to HEp-2 cell Nude Mouse Models with Cetuximab：HEp-2 is thin Born of the same parents are cultivated in RPMI-1640 culture mediums to exponential phase, are centrifuged after pancreatin digestion, are adjusted with PBS cushioning liquid to cell Concentration is 1 × 108/mL.40 nude mices are taken, in the right side oxter subcutaneous vaccination oncocyte liquid 0.2mL of every mouse, are raised in SPF In clean room.When tumour length is to 100-150mm3, animal is randomly divided into four groups, every group 10.Specific packet and application side Method is control group：Intraperitoneal injection PBS 100 μ L daily；Cetuximab list medicine group：It is injected intraperitoneally Cetuximab 1mg, 2 times/ 5 days, totally 4 times；Oridonin list medicine group：Oridonin is injected intraperitoneally 20mg/kg/ days, altogether 10 times；Drug combination group： Oridonin 20mg/kg+ Cetuximabs 1mg is injected intraperitoneally.Tumour growth situation is dynamically observed during experiment and is weighed, often It after medication 10 days, is put to death mice with tumor, knurl body is peeled off, by what is stripped out with vernier caliper measurement tumour major diameter (a) and minor axis (b) Tumour removes blood stains, the non-tumor tissue such as fat, respectively at preserving tumor tissue in liquid nitrogen and 4% paraformaldehyde.Calculated according to formula Gross tumor volume and gross tumor volume inhibiting rate：Mean tumor volume (mm3)=ab2/2；Gross tumor volume inhibiting rate (%)=(1- experimental groups Mean tumor volume/negative control group mean tumor volume) × 100%.

(2) Western blot detect each group transplantable tumor EGFR signal path correlative protein expression situations：Western Blot methods (with the method for embodiment 3).

(3) ELISA method measure tumor tissues ROS contents：Tumor tissue is added into appropriate PBS to smash to pieces.3000rpm is centrifuged 20min takes supernatant；Lath needed for being taken out from aluminium foil bag, sets standard sample wells and sample aperture, standard sample wells respectively to add various concentrations The μ L of standard items 50.Sample aperture first adds sample to be tested l0 μ L, then adds the μ L of Sample dilution 40, and blank well is not added with；In addition to blank well, The μ L of detection antibody 100 of horseradish peroxidase (HRP) mark are added in standard sample wells and sample aperture per hole, are sealed with shrouding film Reacting hole, 37 DEG C of water-baths or insulating box incubate 60min；Liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up with per hole, stands 1min, cleaning solution is discarded, patted dry, so repeat board-washing 5 times；Substrate A, each 50 μ L of B are added per hole, 37 DEG C of lucifuges are incubated 15min. Added per hole in terminate liquid 50 μ L, 15min, the OD values in each hole are determined at 450nm wavelength.

(4) the expression shadow of SABC detection different disposal group tumor tissues PCNA and EGFR signal path GAP-associated protein GAP Ring：Fixer fixes 24h, and 70% is alcohol-pickled overnight, 80% alcohol-pickled 2h, 95% alcohol-pickled 2 times, each 1.5h；It is pure Alcohol-pickled 2 times, each 1.5h；Dimethylbenzene I soaks 30min, and dimethylbenzene II soaks 40min；The waxdip 30min of paraffin I, paraffin II Waxdip 90min；Sample embeds, section, and roasting piece 2h, to be dyed in 60 DEG C of baking boxs.

5 μm of paraffin section, conventional dewaxing to water, 3% hydrogen peroxide at room temperature immersion 30min；Antigen retrieval micro-wave oven height Fiery 5min, the middle fiery 4min of height, closed with 10% cow's serum, room temperature 30min, incline serum deprivation；Primary antibody (potency 1 is added dropwise：100) it is placed in 4 DEG C of refrigerator overnights in wet box, TBST vibration washings, 3 times × 5min, secondary antibody is added dropwise, is incubated at room temperature 30min；TBS vibration washings, 3 times × 5min；Colour developing：DAB substrates are added dropwise at tissue, develop the color 1min；Distillation washing 5 times, haematoxylin redyes 2min, dehydration, dimethylbenzene It is transparent, neutral gum mounting, light Microscopic observation result.

(5) In situ terminal labeling technology (TUNEL) detection transplantable tumor histocyte apoptosis：Histotomy routinely dewaxes extremely Water；With 20 μ g/mL protease K digestings 30min；Every section be added dropwise 50 μ L TUNEL reaction solutions (use before by TdT liquid I with it is glimmering The dUTP liquid II of light element mark mixes in proportion, the μ L of I 50 μ L+ II 450), put 60min in 37 DEG C of water baths；Rinsed with PBS, 3 times ×5min；POD liquid is added dropwise, reacts 40min at a temperature of 37 DEG C in dark wet box；PBS is rinsed 3 times, each 5min；Dripped at tissue Add DAB substrates, develop the color 10min；PBS is rinsed, 3 times × 5min；It is dehydrated after being redyed with haematoxylin with graded ethanol, then Dimethylbenzene is transparent, finally with neutral gum mounting.Light Microscopic observation, nucleus is dyed to brown color in positive cell.Use Image pro-plus 6.0 count positive apoptotic cells ratio.

2 experimental results

As shown in Figure 7 A, compared with control group, the tumor growth curve of each group is suppressed by different degrees of.Western appropriate former times Monoclonal antibody list medicine group and winter grass A prime list medicine group tumour inhibiting rate are respectively 61.7% and 83.1%.The wherein tumour life of drug combination group Long most slow, inhibitory rate to 92.2%.Compared to Oridonin list medicine group, drug combination has no influence for nude mice body weight. Illustrate that Oridonin, Cetuximab and the two drug combination have inhibitory action to transplanted tumor in nude mice, and in identical medicine Under thing concentration, drug combination group is significantly stronger than corresponding single medicine group to the inhibitory action of laryngocarcinoma transplantable tumor.In drug treatment, The nude mice state of mind is good, grow, diet, activity it is normal, have no obvious adverse reaction.Oridonin slightly reduces naked Mouse body weight, and drug combination for nude mice body weight without influence, illustrate that drug combination is non-toxic for nude mice.

As shown in Figure 7 B, compared with control group, Oridonin list medicine group, Cetuximab list medicine group and the two joint are used Medicine group p-EGFR, p-Akt and p-Erk expression are decreased obviously.The protein expression of drug combination group is substantially less than single medicine group.And the winter The shear pattern protein expressions of caspase 8 can substantially be increased by insulting careless A prime list medicine group and drug combination group.Illustrate Rabdosia rubescens first Element and Cetuximab combination suppress EGFR signal paths and activate and promote apoptosis.

As seen in figure 7 c, compared with control group, Oridonin, Cetuximab and the two combination can be with induced tumors Tissue produces ROS.Compared with Oridonin list medicine group, drug combination does not increase ROS generation further, prompts Rabdosia rubescens A prime and Cetuximab combination may suppress tumor tissue growth by promoting ROS to produce.

As illustrated in fig. 7d, proliferating cell nuclear antigen PCNA throughout expressions in reason group tumor tissue：PCNA is used as and commented One common counter of valency proliferative activity, its positive expression are located at nucleus, and brown color is positive reaction cell.Control group Laryngeal cancer cell breeds hyperactive, and its PCNA positive expression rate is very high, Oridonin list medicine group and Cetuximab list medicine The positive expression rate of group is significantly lower than control group.And drug combination group PCNA expression rate is only (9 ± 2.6) %, its tumour is thin Born of the same parents' proliferation activity is more substantially less than single medicine group.As a result illustrate, drug combination can substantially suppress transplantable tumor tissue cell proliferation.

The positive cell of p-Akt, p-EGFR and p-Erk expression is in brown color after immunohistochemical staining.With control group phase Than the obvious reduction of p-EGFR expression of Oridonin list medicine group and Cetuximab list medicine group.Drug combination can be further Reduce p-EGFR expression.Oridonin after Cetuximab combination for EGFR downstreams albumen p-Akt and p-Erk with influenceing It is consistent with the change of p-EGFR expressions.It these results suggest that, drug combination can substantially suppress EGFR signal paths.

TUNEL methods detect each administration group tumor tissue Apoptosis situation：Apoptotic cell is bright in each treatment group's transplanting tumor tissue Aobvious to be clear to, the more pyknosis of apoptotic nucleus, DAB colour developings are dark-brown, and the simultaneously unobvious in control group.Statistical result showed, winter The positive apoptosis cells for insulting careless A prime list medicine and Cetuximab list medicine group are respectively 13% and 3%, and drug combination group cell withers Rate is died for 46.3%, hence it is evident that higher than single medicine group.Prompting Oridonin can substantially induce laryngocarcinoma naked with Cetuximab combination Mouse transplants apoptosis of tumor.

Claims (8)

1. a kind of antineoplastic pharmaceutical compositions, it is characterised in that the composition contains Oridonin and Cetuximab.

2. pharmaceutical composition according to claim 1, it is characterised in that the extraction process of the Oridonin is with the winter It is raw material to insult careless medicinal material, passes through ethanol solution refluxing extraction；Resulting extract, with petroleum ether degreasing, then isometric second Acetoacetic ester extracts；Gained extract is separated by silica gel column chromatography, with dichloromethane and methanol elution gradient, obtains Rabdosia rubescens A prime crude product, through being recrystallized to give Oridonin.

3. antineoplastic pharmaceutical compositions according to claim 1, it is characterised in that the Oridonin and the western appropriate former times are single It is anti-to include its pharmaceutically acceptable salt, and pharmaceutically acceptable excipient or carrier respectively.

4. the application of antineoplastic pharmaceutical compositions according to claim 1, it is characterised in that described tumour behaviour larynx squama Shape cell cancer.

5. antineoplastic pharmaceutical compositions according to claim 1, it is characterised in that：The Cetuximab concentration be 1~ 100 μ g/mL, Oridonin concentration are 12~36 μM.

6. antineoplastic pharmaceutical compositions according to claim 4, it is characterised in that Oridonin and Cetuximab combination are logical Cross increase reactive oxygen species to produce and then activate death receptor pathway * and mitochondria pathway, and then activate downstream caspase man Apoptosis occurs for race's albumen, induction laryngeal cancer cell；Oridonin can induce laryngocarcinoma with Cetuximab combination by ROS simultaneously The cell generation G2/M phases block.

7. antineoplastic pharmaceutical compositions according to claim 1, it is characterised in that Oridonin is combined with Cetuximab By suppressing EGFR signal paths and promoting active oxygen to produce to suppress tumour growth in transplanted tumor in nude mice.

A kind of 8. application of antineoplastic pharmaceutical compositions according to claims 1, it is characterised in that the drug regimen Oridonin and Cetuximab in thing are used or used with the order of any priority simultaneously.