CN103479933B - Chinese medicine composition of a kind of Therapeutic cancer and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of Chinese medicine composition of Therapeutic cancer, be made up of following raw materials in parts by weight proportioning: Herba Sarcandrae 15 parts, 20 parts, the Radix Rehmanniae, burnt Concha Arcae 15 parts, Concha Meretricis seu Cyclinae 30 parts, Haematitum 10 parts, Radix Clematidis 10 parts, the Radix Pulsatillae 12 parts, Fructus Fici 12 parts, Fructus Hordei Germinatus (parched to brown) 20 parts, Herba Schizonepetae 10 parts, Semen Coicis 30 parts, willow root 60 parts, process Endothelium corneum 15 parts, Spica Prunellae 12 parts, Herba Equiseti Hiemalis 40 parts, Fructus Corni 20 parts, Radix Saposhnikoviae 10 parts, Semen Vaccariae 15 parts.Vivo and vitro experimental studies results shows, and the growth of this Chinese medicine composition to typeⅡ pneumocyte system and people pulmonary carcinoma NCI-H460 cell has obvious inhibitory action.The present invention can be used for Therapeutic cancer, especially for treatment pulmonary carcinoma.

Description

Chinese medicine composition of a kind of Therapeutic cancer and preparation method thereof

Technical field

The present invention relates to the field of Chinese medicines, be specifically related to Chinese medicine composition of a kind of Therapeutic cancer and preparation method thereof.

Background technology

Malignant tumor, also claims cancer, is that phalangeal cell is out of hand, abnormality proliferation, and continued propagation and then infringement close on normal structure, and intravascular, lymphatic vessel and body cavity can transfer to other positions of health, finally jeopardizes a class serious disease of human life.In the various cause of the death, come second in China's malignant tumor, be then discharged to first place in urban; And nearly 20 years, the sickness rate of tumor and mortality rate were obvious ascendant trend, and wherein lung cancer morbidity rate ranks first.At present, the clinical treatment of tumor mainly applies the methods such as excision, radiotherapy, chemotherapy, Biotherapeutics and treatment by Chinese herbs.Because the grade malignancy of tumor is high, current treatment means is difficult to obtain good efficacy, leads oncogenic recurrence and transfer, and there is the deficiency of some treatment meanss, and the toxic and side effects as chemotherapy usually causes bone marrow depression.Therefore, new antitumor drug is developed significant to treatment tumor.The traditional Chinese medicine of China has very important value on oncotherapy, have accumulated the experience of many preciousnesses, for performing the operation and postoperative patient, and has auxiliary therapeutic action in toxic and side effects etc. in minimizing radiotherapy, chemotherapy and Biotherapeutics.Therefore, effective Chinese medicine compound has played important effect in clinical treatment tumour.

Strengthening vital QI to eliminate pathogenic factors is the universal rule of Chinese medicine disease therapy, can be used for the prevention and therapy of tumor.Chinese medicine composition of the present invention is made up of 18 medicines, and mainly for the loss of pulmonary carcinoma lung yin, spleen the moon is depressed, accumulation and obstruction of sputum, qi depression to blood stasis, and native weak gold such as to decline at the side's of establishing legislation, is used for the treatment of cancer, especially for treatment pulmonary carcinoma.

Summary of the invention

For the deficiencies in the prior art, the invention provides a kind of Chinese medicine composition.The growth of this Chinese medicine composition to typeⅡ pneumocyte system and people pulmonary carcinoma NCI-H460 cell has obvious inhibitory action, can be used for Therapeutic cancer, especially for treatment pulmonary carcinoma.

The present invention relates to a kind of Chinese medicine composition, it is made up of by corresponding weight following raw material: Herba Sarcandrae 15 parts, 20 parts, the Radix Rehmanniae, burnt Concha Arcae 15 parts, Concha Meretricis seu Cyclinae 30 parts, Haematitum 10 parts, Radix Clematidis 10 parts, the Radix Pulsatillae 12 parts, Fructus Fici 12 parts, Fructus Hordei Germinatus (parched to brown) 20 parts, Herba Schizonepetae 10 parts, Semen Coicis 30 parts, willow root 60 parts, process Endothelium corneum 15 parts, Spica Prunellae 12 parts, Herba Equiseti Hiemalis 40 parts, Fructus Corni 20 parts, Radix Saposhnikoviae 10 parts, Semen Vaccariae 15 parts.Preferably, this Chinese medicine composition can be made up of the raw material of following weight: Herba Sarcandrae 15 grams, 20 grams, the Radix Rehmanniae, burnt Concha Arcae 15 grams, Concha Meretricis seu Cyclinae 30 grams, Haematitum 10 grams, Radix Clematidis 10 grams, the Radix Pulsatillae 12 grams, Fructus Fici 12 grams, Fructus Hordei Germinatus (parched to brown) 20 grams, Herba Schizonepetae 10 grams, Semen Coicis 30 grams, willow root 60 grams, process Endothelium corneum 15 grams, Spica Prunellae 12 grams, Herba Equiseti Hiemalis 40 grams, Fructus Corni 20 grams, Radix Saposhnikoviae 10 grams, Semen Vaccariae 15 grams.

The present invention provides a kind of method preparing above-mentioned Chinese medicine composition simultaneously, the method comprises following steps: mixed by corresponding weight proportion by aforesaid raw material, comprises Herba Sarcandrae 15 parts, 20 parts, the Radix Rehmanniae, burnt Concha Arcae 15 parts, Concha Meretricis seu Cyclinae 30 parts, Haematitum 10 parts, Radix Clematidis 10 parts, the Radix Pulsatillae 12 parts, Fructus Fici 12 parts, Fructus Hordei Germinatus (parched to brown) 20 parts, Herba Schizonepetae 10 parts, Semen Coicis 30 parts, willow root 60 parts, processs Endothelium corneum 15 parts, Spica Prunellae 12 parts, Herba Equiseti Hiemalis 40 parts, Fructus Corni 20 parts, Radix Saposhnikoviae 10 parts and Semen Vaccariae 15 parts; First rinse twice, to remove dust, pesticide, fertilizer residue with clear water; Then used warm water soaking, decocted two times for 100 DEG C, become flowing soaking paste, then dilute three times, shake up to obtain extractum; Finally by described extractum cryogenic seal or storage.This extractum can be done medicinal, also can be used for as animal inhibiting tumor assay.

Particularly, this Chinese medicine composition can be mixed by corresponding weight by following raw materials according, comprises Herba Sarcandrae 15 grams, 20 grams, the Radix Rehmanniae, burnt Concha Arcae 15 grams, Concha Meretricis seu Cyclinae 30 grams, Haematitum 10 grams, Radix Clematidis 10 grams, the Radix Pulsatillae 12 grams, Fructus Fici 12 grams, Fructus Hordei Germinatus (parched to brown) 20 grams, Herba Schizonepetae 10 grams, Semen Coicis 30 grams, willow root 60 grams, processs Endothelium corneum 15 grams, Spica Prunellae 12 grams, Herba Equiseti Hiemalis 40 grams, Fructus Corni 20 grams, Radix Saposhnikoviae 10 grams and Semen Vaccariae 15 grams; First rinse twice, to remove dust, pesticide, fertilizer residue with clear water; Then used warm water soaking, decocted two times for 100 DEG C, become flowing soaking paste, then dilute three times, shake up to obtain extractum; Finally by described extractum cryogenic seal or storage.This extractum can be done medicinal, also can be used for as animal inhibiting tumor assay.

Further, the present invention also provides the working solution be made into by aforementioned extractum.Aforementioned extractum is added the distilled water of certain volume, be mixed with the mother solution that concentration is 100mg/ml, after autoclaving, by this mother solution frit 2-3 time, and dilution can be made into certain gradient concentration working solution.This working solution can cryogenic seal or storage, can do medicinal, also can be used for research, as the research of cellular level.

Specifically, described Chinese medicine composition provided by the invention can technique add adjuvant and makes tablet, capsule, granule, drop pill, micropill, dispersible tablet, oral cavity disintegration tablet, pill, oral liquid routinely; Described adjuvant comprises one or more in solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent, lubricant and substrate.

In addition, the invention still further relates to described Chinese medicine composition and prepare the application in Therapeutic cancer medicine.More specifically, the present invention relates to the application of described Chinese medicine composition in preparation treatment lung-cancer medicament.

Chinese medicine constituent in the present invention is made up of 18 taste Chinese medicines, and mainly for the loss of pulmonary carcinoma lung yin, spleen the moon is depressed, accumulation and obstruction of sputum, qi depression to blood stasis, native weak gold such as to decline at the side's of establishing legislation, and its side separates as follows: " lung governing qi; towards hundred arteries and veins ", and energy and blood of human body body fluid normally runs the promotion of complete bad gas.Patients with lung cancer stagnation of QI-blood, must affect the normal defeated cloth in lung Tianjin, lung not cloth Tianjin then body fluid stop gathering, smoulder not all right, and then it is turbid to be converted into expectorant.Because we have no report, only in the other side, the therapeutic efficiency of each Chinese medicine is expressed as follows:

(1) Herba Sarcandrae: nature and flavor toil is put down.Enter heart channel, Liver Channel.Can clearing away heat and cooling blood, speckle removing of invigorating blood circulation, dispelling wind and removing obstruction in the collateral, has antitumaous effect;

(2) Radix Rehmanniae: sweet in flavor and cold in property.Enter heart channel, Liver Channel, kidney channel.Heat clearing and blood cooling, YIN nourishing and the production of body fluid promoting, removing blood stasis to promote tissue regeneration, moisturize laxation, pain relieving.Be used for treatment yin asthenia generating intrinsic heat, hectic fever due to YIN-deficiency consumptive fever, crimson tongue excessive thirst, send out speckle dermexanthesis etc.;

(3) burnt Concha Arcae: nature and flavor are salty flat.Return lung meridian, stomach warp, Liver Channel.Expectorant blood stasis dispelling, hard masses softening and resolving, relieving gastric hyperacidity to alleviate stomachache.For the long-pending knot of pertinacious phlegm, thickness difficulty is coughed up, goiter, scrofula, abdominal mass mass in the abdomen, stomachache pantothenic acid etc.;

(4) Concha Meretricis seu Cyclinae: nature and flavor are salty flat.Enter heart channel, kidney channel.Can clearing away heat and promoting diuresis, softening and eliminating sputum.Be used for the treatment of heat-phlegm to breathe heavily and cough, edema, goiter is gathered, blood agglomeration chest pain etc.;

(5) Haematitum: property bitter but sweet flavor is put down.Enter Liver Channel, stomach warp, heart born of the same parents warp.Can suppressing the hyperactive liver and subsiding YANG, sending down the abnormal ascending QI stops blooding.For vertigo and tinnitus, pant, eructation, vomiting, singultus, spits blood, epistaxis etc.;

(6) Radix Clematidis: the pungent salty temperature of nature and flavor is poisonous.Enter urinary bladder channel.Can expelling wind and removing dampness, removing obstruction in the collateral to relieve pain, expectorant water, the loose hypochondriac lump;

(7) Radix Pulsatillae: nature and flavor bitter cold; Return stomach warp, large intestine channel.There is heat-clearing and toxic substances removing, eliminating pathogenic heat from blood to cure dysentery, effect of removing dampness and killing parasites.Carbuncle skin ulcer can be treated, scrofula etc.;

(8) Fructus Fici: nature and flavor are sweet micro-pungent flat, slightly poisonous.Cure mainly swelling and pain of hemorrhoid etc.;

(9) Fructus Hordei Germinatus (parched to brown): nature and flavor are sweet flat.Return spleen channel, stomach warp.Can strengthening the spleen and nourishing the stomach, food stagnation removing food stagnation;

(10) Herba Schizonepetae carbon: the pungent puckery slight fever of nature and flavor.Return lung meridian, Liver Channel.Can hemostasis with astringents;

(11) Semen Coicis: nature and flavor are cool sweet light.Can spleen invigorating eliminating dampness by diuresis, eliminating impediment antidiarrheal.Can be used for treatment edema, dysuria, arthralgia chiefly caused by damp pathogen contracture, diarrhea due to hypofunction of the spleen;

(12) willow root: can expelling wind and removing dampness, reducing swelling and alleviating pain;

(13) Endothelium corneum is processed: property is hidden sweet cold.Return spleen channel, stomach warp, small intestine meridian, urinary bladder channel.Can promoting digestion and invigorating the stomach aid digestion, arresting seminal emission.Be used for the treatment of dyspepsia, shallow complexion, do not think Na Gu, the disease such as infantile malnutrition, emaciated physique, enlarged abdomen abdominal distention, weakness of the spleen and stomach, food stagnation distension;

(14) Spica Prunellae: nature and flavor are trembled with fear sweet pungent micro-hardship, have the effect of letting out clearly liver-fire, mass dissipating and swelling eliminating, heat-clearing and toxic substances removing, expelling phlegm for arresting cough, cooling blood for hemostasis, are applicable to the diseases such as lymphoid tuberculosis, goiter, acute mastitis.Modern pharmacological research shows, Spica Prunellae has the effect of anticancer;

(15) Herba Equiseti Hiemalis: nature and flavor sweetness and bitterness is put down, nontoxic, is slightly cold.Enter heart channel, Liver Channel, stomach warp, urinary bladder channel.Can stop blooding, liver heat removing and eyesight improving, inducing diuresis for treating stranguria syndrome, is used for the treatment of the diseases such as cough, conjunctival congestion and swelling pain, discharging fresh blood stool;

(16) Fructus Corni: tepor that nature and flavor are sour and astringent.As Liver Channel, kidney channel.Can liver and kidney tonifying, restrain astringent or styptic treatment for spontaneous sweating, controlling nocturnal emission with astringent drugs reducing urination, hidroschesis promotes the production of body fluid.Can be used for soreness of waist and knee joint, profuse sweating is collapsed, and interior-heat such as to be quenched one's thirst at the disease;

(17) Radix Saposhnikoviae: property acrid-sweet flavor tepor.Enter urinary bladder channel, lung meridian, spleen channel, Liver Channel.Can expelling pathogenic wind from the body surface, removing dampness to relieve pain, spasmolytic antipruritic.Be used for the treatment of affection of exogenous wind-cold, headache general pain, rheumatic arthralgia, joint is ached, and stomachache such as to be had loose bowels the disease;

(18) Semen Vaccariae: nature and flavor hardship is flat.Enter Liver Channel, stomach warp.Can promoting the circulation of blood regulating menstruation, reducing swelling and alleviating pain, lactogenesis of invigorating blood circulation, expedite the emergence of stimulating milk secretion, puerperal thymus dredging.Be used for the treatment of blood stasis amenorrhea, dysmenorrhea, difficult labour, acute mastitis such as to swell and ache at the disease.

The basis of this prescription can increase other adjuvant drugs and/or make medicine, to improve curative effect further.

Advantage of the present invention is that the Chinese medicine composition provided has significant curative effect in Therapeutic cancer, particularly treatment pulmonary carcinoma.Inventor found through experiments, and the suppression ratio of said composition to people's immortalized hepatocyte LO2 reaches 70%, can reach 75%, improve significantly and therapeutical effect in clinical trial to the symptom of patient's pulmonary carcinoma to the suppression ratio of nude mouse tumor.

Accompanying drawing explanation

Fig. 1 Chinese medicine composition acts on people's immortalized hepatocyte LO2 morphology photo after 48 hrs.

Fig. 2 Chinese medicine composition acts on human lung cancer cell A549's morphology photo after 48 hrs.

Fig. 3 Chinese medicine composition acts on human lung carcinoma cell NCI-H460 morphology photo after 48 hrs.

Fig. 4 Chinese medicine composition makes employment immortalized hepatocyte LO2 Trypan Blue result (p after 48 hrs ₁=0.0417, ^*p<0.05, ^*p<0.01, ^{* *}p<0.001).

Fig. 5 Chinese medicine composition effect human lung carcinoma cell trypan blue coloration result (p after 48 hours ₁=0.0053, p ₂=0.0013; p ₃=0.0048).

Fig. 6 Chinese medicine composition effect human lung carcinoma cell NCI-H460 Trypan Blue (p after 48 hrs ₁=0.0075, p ₂=0.0158, p ₃=0.0167).

Fig. 7 Chinese medicine composition effect immortalized hepatocyte LO2 MTT testing result (p after 48 hrs ₁=0.0163).

Fig. 8 Chinese medicine composition effect human lung cancer cell A549 MTT testing result (p after 48 hrs ₁=0.017, p ₂=0.0070, p ₃=0.0061).

Fig. 9 Chinese medicine composition effect human lung carcinoma cell NCI-H460 MTT result (p after 48 hrs ₁=0.0125, p ₂=0.0358, p ₃=0.0167).

Figure 10 Chinese medicine composition makes employment immortalized hepatocyte LO2 streaming result after 48 hrs.

Figure 11 Chinese medicine composition effect lung cell A549 streaming result after 48 hrs.

Figure 12 BALB/C nude mice weight relation over time.

Figure 13 BALB/C nude mouse tumor volume relation over time.

The tumor mass photo of Figure 14 BALB/C nude mice gastric infusion after 45 days (often organizing 6).

The tumor mass block diagram of Figure 15 BALB/C nude mice gastric infusion after 45 days.

Figure 16 is used for the RNA quality analysis results of genechip detection.

Detailed description of the invention

Embodiment 1 Chinese medicine composition and preparation thereof

[1] pharmaceutical compositions and preparation thereof

Chinese medicine composition by following raw materials in parts by weight proportioning is: Herba Sarcandrae 15 parts, 20 parts, the Radix Rehmanniae, burnt Concha Arcae 15 parts, Concha Meretricis seu Cyclinae 30 parts, Haematitum 10 parts, Radix Clematidis 10 parts, the Radix Pulsatillae 12 parts, Fructus Fici 12 parts, Fructus Hordei Germinatus (parched to brown) 20 parts, Herba Schizonepetae 10 parts, Semen Coicis 30 parts, willow root 60 parts, process Endothelium corneum 15 parts, Spica Prunellae 12 parts, Herba Equiseti Hiemalis 40 parts, Fructus Corni 20 parts, Radix Saposhnikoviae 10 parts, Semen Vaccariae 15 parts.

In the present embodiment, described Chinese medicine composition by Herba Sarcandrae 15 grams, 20 grams, the Radix Rehmanniae, burnt Concha Arcae 15 grams, Concha Meretricis seu Cyclinae 30 grams, Haematitum 10 grams, Radix Clematidis 10 grams, the Radix Pulsatillae 12 grams, Fructus Fici 12 grams, Fructus Hordei Germinatus (parched to brown) 20 grams, Herba Schizonepetae 10 grams, Semen Coicis 30 grams, willow root 60 grams, processs Endothelium corneum 15 grams, Spica Prunellae 12 grams, Herba Equiseti Hiemalis 40 grams, Fructus Corni 20 grams, Radix Saposhnikoviae 10 grams and Semen Vaccariae 15 grams composition.

The preparation of this Chinese medicine composition, comprises the following steps:

A) raw material of aforesaid kind and component first rinses twice, to remove dust, pesticide, fertilizer residue with clear water;

B) with warm water soaking, decoct two times for 100 DEG C, become flowing soaking paste, dilution three times, shake up to obtain extractum;

C) by described extractum cryogenic seal or storage.

This extractum has therapeutic effect, may be used for animal inhibiting tumor assay, also can do medicinal.

Aforementioned extractum is added the distilled water of certain volume, be mixed with mother solution, concentration is 100mg/ml, after autoclaving, with frit 2-3 time, can be diluted to certain gradient concentration working solution.This work also can cryogenic seal or storage, can be used for research, the research of such as cellular level.

[2] process for preparing raw material

1. press prescription and medical material base supply business please supply medicine.After medicine is bought, first check into the formality of medicine channel, understand the place of production; Whether next side's of checking medicine conforms to; Again screen, determine whether medicine has impurity, the adulterated and medicine that goes mouldy, and could put processing in storage after errorless.

2. according to quantity medicine is dropped into reactor, add water cleaning two times, object removes ash, keep medicine clean, reduce agriculture chemical to remain simultaneously, stir in fine laundering process in addition and also can reduce content of beary metal, make medicine process the various content of beary metal such as rear lead, arsenic and meet enterprise, national standard completely, ensure drug quality.

3. medicine-feeding ready after, adopts 100 DEG C of high temperature to decoct secondaries, each decoctions two hours, first time adds 10 times of soak by water, and second time adds six times of soak by water, then extracts decoction liquor and pours in cylinder, be powder by liquid spray drying again, load in sealing bag after finally xeraphium being harvested and stored.

[3] tablet machining process

1., by dry semi-finished product powder, add adjuvant pelletize, the tablettings such as corn starch, stearic acid U.S., microcrystalline Cellulose, silicon dioxide.

2. after medicine processing, every sheet drug weight is 550 milligrams, and wherein crude drug powder is 420 milligrams, corn starch is 90 milligrams, U.S. 5 milligrams of silica 15 milligrams, stearic acid.

[4] dry capsule prilling process

1. get spray drying powder extract powder 300 milligrams, microcrystalline Cellulose 60 milligrams, silica 15 milligrams, U.S. 5 milligrams of stearic acid, add up to 400 milligrams, load No. 0 capsule after pelletize, by extract powder and microcrystalline Cellulose mix homogeneously; Add 80% ethanol and make suitable soft material, 16 order nylon mesh are granulated; 50 ~ 60 DEG C of dryings, 18 order nylon mesh granulate, add methylol ingot powder sodium, silicon dioxide during granulate, stearic acid is beautiful, mix homogeneously; Load No. 0 capsule, to refill and airtight.

[5] suspension and grain ball processing method

The method of suspension processing is, after secondary decoction liquor is concentrated, then heats in after heat loading sealing Packaging Bottle, then heating, vacuum process

[6] drop pill processing method

Drop pill is by spraying dry after centrifuge process, and removed by the impurity such as two oxycarbides, remaining micropowder adds adjuvant, can be processed into drop pill.

Embodiment 2: pharmacodynamic study

[1] experiment material and instrument and equipment

100% ethanol (Tianjin chemical reagent wholesaling firm), hyclone (Invitrogen, USA), RPMI1640(Invitrogen, USA), pancreatin (Sangon Biotech (Shanghai) Co., Ltd.), trypan blue (prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state), MTT(full name is 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide, 3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide bromine salt, trade name tetrazolium bromide, Thermo, USA), DMSO(dimethyl sulfoxide, prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state), propidium iodide (Sigma, USA), blood counting chamber (Shanghai refinement biochemical reagents company limited), low speed desk centrifuge (Changsha Xiang Yi centrifuge Instrument Ltd.), enzyme-linked immunosorbent assay instrument (BIO-RAD, USA), flow cytometer (BD FACSDiVa, USA), decolorization swinging table (Beijing De Tianyou development in science and technology company limited), CO2 incubator (sanyo, Japan), inverted microscope (OLYMPUS, Japan).

[2] cell culture

Non-small cell lung carcinoma cell line A549, National People's Congress sclc cell line NCI-H460 and people's immortalized hepatocyte strain LO2 are purchased from Nanjing KaiJi Biology Science Development Co., Ltd.The application of above-mentioned cell strain containing the RPMI1640 culture medium (Gibco, USA) of 10% hyclone (HyClone, Australia) at the 5%CO of 37 DEG C ₂cultivate in incubator.

[3] statistical method

Adopt Student ' s t inspection to carry out statistical analysis to data, P<0.05 and P<0.01 is significant difference.

[4] morphological observation

1. prepare the Chinese medicine mother solution of certain density Chinese medicine composition, autoclaving, filtering and impurity removing 2 ~ 3 times, be diluted to a series of gradient concentration (0.01,0.1,1.0,5.0 and 10.0mg/ml) for subsequent use; Cell strain adopts typeⅡ pneumocyte system, people pulmonary carcinoma NCI-H460 cell and people's immortalized hepatocyte strain LO2;

2. growth is spread 6 orifice plates at the cell of logarithmic (log) phase, put into incubator and be cultured to cell when growing to 80% ~ 90%, add the Chinese medicine 200 μ l of variable concentrations respectively, wherein matched group adds 200 μ l PBS;

3. incubator cultivates 48 hours, the impact of basis of microscopic observation variable concentrations medicine (0.01,0.1,1.0,5.0 and 10.0mg/ml) on cell, and takes pictures;

4. observed result:

After the Chinese medicine composition of 1mg/ml concentration acts on immortalized hepatocyte LO2 cell, form, observation of cell is without significant change, and when drug level is increased to 5mg/ml, LO2 cell death also comes off; TypeⅡ pneumocyte is about 1/3 cell survival under the drug level of 1mg/ml, and under the drug level of 5mg/ml and 10mg/ml, almost have little cell survival; People pulmonary carcinoma NCI-H460 cell and A549 cell situation similar, even more responsive than A549 cell when low concentration.

[5] Trypan Blue

1. normal living cells, after birth structural integrity, can repel trypan blue, makes it can not enter in born of the same parents; And loss of activity or the incomplete cell of cell membrane, the permeability of after birth increases, and can be dyed blueness by trypan blue.It has been generally acknowledged that cell membrane integrity is lost, can think that cell is dead, this is contrary with dimethyl diaminophenazine chloride effect.Therefore, by Trypan Blue can very easy, distinguish living cells and dead cell rapidly;

2. growth is spread 6 orifice plates at the cell of logarithmic (log) phase, puts into incubator and be cultured to cell when growing to 80% ~ 90%, add the Chinese medicine 200 μ l(0.01 of variable concentrations respectively, 0.1,1.0,5.0 and 10.0mg/ml), wherein matched group adds 200 μ l PBS;

3. incubator cultivates 48 hours, and basis of microscopic observation variable concentrations medicine is on the impact of cell.Collect supernatant respectively, pancreatin digests the cell of different pharmaceutical concentration process respectively, the cell that in merging, cleer and peaceful digestion is got off; Centrifugal 10 minutes of 800rpm, abandons supernatant; Add 5ml PBS resuspended, centrifugal 10 minutes of 800rpm, abandons supernatant; Add 1ml PBS resuspended;

4. get the trypan blue solution mixing dyeing 3 ~ 6 minutes of 10 μ l sample solutions and 0.4% respectively, then carry out blood counting chamber living cell counting and dead cell number, painted is dead cell, and not having painted is living cells; Every strain cell line at least repeats 3 times;

5. experimental result:

Table 1 people immortalized hepatocyte system LO2 trypan blue count results

Concentration mg/ml	0	0.01	0.1	1	5	10
							Cell number 10 4	48.5	52	53	53.5	12	3

Table 2 typeⅡ pneumocyte system trypan blue count results

Concentration mg/ml	0	0.01	0.1	1	5	10
							Cell number 10 4	55.4	47.8	43.1	14.7	9.3	6.3

Table 3 people pulmonary carcinoma NCI-H460 cell line trypan blue count results

Concentration mg/ml	0	0.01	0.1	1	5	10
							Cell number 10 4	103.5	100.5	58.5	26	6.5	3.5

6. adopt Logit method to calculate Chinese medicine to the medium effective concentration IC of A549 cell ₅₀value, IC ₅₀=0.369mg/ml.

[6] mtt assay detects

1.MTT method, also known as MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to cell number;

2. exponential phase cell is spread 6 orifice plates, puts into incubator and be cultured to cell when growing to 80% ~ 90%, add the Chinese medicine 200 μ l(0.01 of variable concentrations respectively, 0.1,1.0,5.0 and 10.0mg/ml), wherein matched group adds 200 μ l PBS;

3. incubator cultivates 48 hours, and basis of microscopic observation variable concentrations medicine is on the impact of cell; Collect supernatant respectively, pancreatin digests the cell of different pharmaceutical concentration process respectively, the cell that in merging, cleer and peaceful digestion is got off; Centrifugal 10 minutes of 800rpm, abandons supernatant; Add 5ml PBS resuspended, centrifugal 10 minutes of 800rpm, abandons supernatant; Repeat do not have color to re-suspension liquid 2 ~ 3 times, it is resuspended to add 1ml PBS;

4. spread 96 orifice plates, every hole adds 100 μ l culture medium, each concentration 6 ~ 8 repetition; 15 μ l MTT(5mg/ml are added again in every hole), put incubator and cultivate 3.5 ~ 4 hours; Suck supernatant with 1ml syringe, in every hole, add 150 μ l DMSO, on shaking table, 150rpm vibrates 15 minutes, surveys OD _490-620;

5. experimental result:

The MTT testing result of table 4 people immortalized hepatocyte system LO2

Concentration mg/ml	0	0.01	0.1	1	5	10
							OD 490-620	0.252	0.270	0.269	0.274	0.071	-0.104

Table 5 typeⅡ pneumocyte system MTT testing result

Concentration mg/ml	0	0.01	0.1	1	5	10
							OD 490-620	1.078	1.047	0.857	0.210	0.147	0.079

Table 6 people pulmonary carcinoma NCI-H460 cell line MTT result

Concentration mg/ml	0	0.01	0.1	1	5	10
							OD 490-620	1.073	0.832	0.722	0.594	0.399	0.038

[7] flow cyctometry detects

1. synchronization process cell, uses the Chinese medicine composition process A549 cell of 0mg/ml, 1mg/ml concentration respectively; With the Chinese medicine composition process NCI-H460 cell of 0mg/ml, 0.1mg/ml concentration, drug effect 48 hours;

2. trypsinization attached cell, PBS dispels separation, collects (15ml centrifuge tube) in centrifuge tube; Centrifugal 10 minutes of 800rpm, abandons supernatant; Add 5ml PBS resuspended, centrifugal 5 minutes of 800rpm, abandons supernatant; Add 5ml PBS resuspended, centrifugal 5 minutes of 800rpm, abandons supernatant; Add rapidly the ethanol of 1ml pre-cooling (-20 DEG C) 75%, piping and druming cell dispersion (when Stirring prevents conglomeration, slowly will add ethanol); 4 DEG C are fixedly spent the night, the resuspended precipitation of 900 μ l PBS, and adding 100 μ l RNase A(can not add), hatch 30 minutes to 1 hour for 37 DEG C; Add 10 μ l propidium iodides (concentration is 5mg/ml), 20 minutes on ice, lucifuge, room temperature 5-10 minute, flow cytometer showed (filtering with 400 order nylon membranes);

3. experimental result:

With 0mg/ml drug treating immortalized hepatocyte (LO2), S phase LO2 cell proportion is 31.14%; By the drug treating of 1mg/ml, S phase LO2 cell proportion is 24.52%, and S phase cell proportion reduces compared with the control, and the medicine of 1mg/ml has no significant effect thin LO2 born of the same parents; With 0mg/ml drug treating human lung cancer cell line A549, S phase cell proportion is 29.94%; By the drug treating of 0.1mg/ml, S phase cell proportion is 8.95%, and S phase cell proportion reduces a lot compared with the control; Therefore, the propagation of medicine to A549 cell of 0.1mg/ml has obvious inhibitory action.

[8] animal nude mouse xenotransplant model

1. experimental technique: by 1 × 10 ⁷a549 cell inoculation nude mice, become after tumor and get tumor tissue block, tumor tissue is cut into size and is about 1.5mm ³, be inoculated in animal side with the trocar or bilateral axillary regions subcutaneous, tieback nude mice totally 12, is divided into two groups (experimental group and matched groups), often organize 6.When tumor BOB(beginning of block) increases (about 1 week after), start to apply irrigation stomach device medicine feed, daily 1 time, each 0.3ml, measure Mouse Weight and tumor block size every day and record.Administration, after 45 days, puts to death mice.Get tumor measurement volumes, weigh and take pictures.

2. experimental result

Therapeutic evaluation formula: in tumor control rate=(C-T)/C × 100% formula, T is the average tumor weight of administration group; C is the average tumor weight of matched group.Table 7 result shows, and tumour inhibiting rate is 75%.

Table 7 model in nude mice administration tumor mass after 45 days

Group	Tumor heavy (mg)	Tumour inhibiting rate
			Matched group (CG)	23.52	-
Experimental group (EG)	5.87	75%

Embodiment 3 Chinese medicine composition acts on the analysis of lung carcinoma cell gene expression profile

[1] total serum IgE sample preparation methods following (attached cell lung cancer A549 cell system):

1. cultivated by the lung cancer A549 cell of cultivation and grow to exponential phase, experimental group adds the Chinese medicine composition 10 μ l of 100mg/ml, and matched group adds PBS10 μ l; Put into incubator to continue to cultivate, drug effect 48 hours;

2. discard culture fluid, PBS cleaning 2 ~ 3 times; Respectively to adding 1.5mlTRLzol reagent in culture bottle, repeatedly beat with 1ml rifle head, the culture bottle surface making TRLzol contact all cells fully digests; Transfer in the test tube of RNase-free and repeatedly lash cell until cannot see agglomerating cell lump with disposable syringe, whole solution should be state that is refrigerant and not thickness;

3. put into ultra-low temp to preserve, then deliver to Boao Biological Co., Ltd and carry out genechip detection experiment.

[2] experimental result:

Gene chip (Gene Chip, DNA Chip), also known as DNA microarray (DNAMicroarray), refers to and to be fixed on solid phase carrier according to precalculated position the microarray array that ten million nucleic acid molecules in very small size forms.Under certain condition, the nucleic acid molecules on carrier can be hybridized with the nucleic acid fragment of the complementary from sample.If the nucleic acid fragment in sample is carried out labelling, special chip reading instrument just can detect hybridization signal.

Biochip technology mainly comprises four key steps: chip preparation, sample preparation, hybridization and signal detection and interpretation of result.Chip gene expression profile adopts cDNA or the general acid fragment of few core to make probe, is solidificated on chip; The mRNA of testing sample (processed group) and control sample is carried out labelling with two kinds of different fluorescence molecules, then hybridize with chip simultaneously, by analyzing the ratio of the fluorescence intensity of two kinds of samples and probe hybridization, carry out the change of gene expression detection level.This chip gene expression profile detects and is completed by Boao Biological Co., Ltd.

The total serum IgE sample liquid nitrogen prepared above is transported to Boao Biological Co., Ltd, and do chip gene expression profile analysis, sample quality inspection is described as follows:

Table 8 sample quality inspection result

Sample	A 260/280	A 260/230	Concentration (μ g/ μ L)	Total amount (μ g)	Sample quality
						0.1mg/ml	1.98	1.99	0.708	35.40	RNA is more complete
0mg/ml	1.97	1.58	0.884	44.19	RNA is more complete

RNA purity: RNA sample A _260/280> 1.70, meets rich chip of expression spectrum requirement of experiment difficult to understand.RNA total amount: RNA sample total amount > 8 μ g, total amount meets rich chip of expression spectrum requirement of experiment difficult to understand.RNA integrity: through electrophoresis detection, RNA sample band is more clear, and the brightness of 28S ﹕ 18SrRNA electrophoretic band is greater than 1 ﹕ 1, meets rich chip of expression spectrum requirement of experiment difficult to understand.In sum: RNA sample purity, total amount and integrity meet rich chip of expression spectrum requirement of experiment difficult to understand, can continue follow-up array experiment.According to the gene chip results that Boao Biological Co., Ltd provides, we apply the molecule annotation system MAS3.0 provided of Boao Biological Co., Ltd to data analysis.

Differential expression result shows: in administration group (0.1mg/ml) vs matched group, difference expression gene has 1103, wherein the upper gene lowered that is in harmonious proportion is respectively 490 and 613, screening criteria is Ratio(administration group/matched group) be greater than 2.5, be less than 0.5, P value is less than the gene of 0.05 is simultaneously the gene of significant difference.

The enrichment analysis result display of Pathway: in administration group (0.1mg/ml) vs matched group, the signal path of significant difference mainly comprises oxidative phosphorylation (Oxidative phosphorylation), ribosome (Ribosome), cell cycle (Cell cycle), Heparan sulfate synthesis (Heparansulfate biosynthesis), O-glycosyl synthesis (O-Glycan biosynthesis), DNApolymerase(DNA polymerase), Wnt signal path (Wnt signaling pathway), MAPK signal path (MAPK signaling pathway), TGF-signal beta Signal Transduction Pathways (TGF-beta signaling pathway), compact siro spinning technology (Tight junction), Adipocyte Factor signal transduction pathway (Adipocytokine signaling pathway), cell adhesion (Celladhesion molecules), cytokine interphase interaction (Cytokine-cytokine receptorinteraction), ECM acceptor interaction (ECM-receptor interaction), focal adhension (Focal adhesion), recessed bond ing (Gap junction), apoptosis (Apoptosis), Jak-STAT signal path (Jak-STAT signaling pathway), p53 signal path (p53signaling pathway), PPAR signal path (PPAR signaling pathway), glycosyl-phosphatidyl inositol anchor synthesizes surely (Glycosylphosphatidylinositol (GPI)-anchorbiosynthesis) etc.Related gene expression level changes, and such as: PCNA lowers 0.29 times, WNT6 lowers 0.22 times, and SKP2 lowers 0.29 times, and TNF raises 4.16 times, etc.

Embodiment 4 patients with lung cancer model case

In the present invention, select the case meeting pulmonary cancer diagnosis standard, take pharmaceutical composition of the present invention three times by every day.

Now enumerate some model cases as follows:

Zhang, man, 65 years old.Find pulmonary shadow during health check-up in May, 2009, the prompting of check on March 3rd, 2010 breast CT, makes a definite diagnosis pulmonary carcinoma, and under general anesthesia on March 22 in 2010, carry out thoracoscope, lower inferior lobe of right lung excision, hilus pulumonis, mediastinal lymphadenectomy art, postoperatively do scorching anti symptom treatment, recover smoothly.Ordinary circumstance when leaving hospital, without main complaint, without heating, otch is taken out stitches, and II/ first heals, and check hemogram is normal, and rabat shows Postoperative changes, and postoperative do not have chemotherapy.On April 7th, 2010 starts to take medicine, and takes medicine continuously 10 months, every day three times, significantly improves body constitution after taking medicine, and to JIUYUE in 2011 1 day, checks and is showed no recurrence and transfer for 5 times.

Hu, man, 60 years old.There is cough in June, 2012, chest pain, tells a large amount of brown expectorant, dyspnea, without appetite, and the symptoms such as sleep difference.CT is in hospital, and imaging diagnosis is reported: inferior lobe of right lung is not opened, right lung inflammation, right pleural effusion.Make a definite diagnosis pulmonary carcinoma.After reaffirm as pulmonary carcinoma, because of health and economic cause abandoning cure.On July 21st, 2012 starts to take medicine.Medication after 20 days expectorant color transfer yellow sticky expectorant to, medication 40 days, without any discomfort, defecation is normal, and sleep, appetite is good, appetite is normal, does not substantially cough, and amount of expectoration minimizing, expectorant yellow skin is sticky, and apoplexy due to phlegm is once in a while with any scarlet blood streak.

Experimental data shows: take Chinese medicine composition of the present invention, has alleviation or therapeutical effect to the state of an illness of patient.Therefore, these experiments further illustrate the present invention has good pharmacodynamic action to Therapeutic cancer, particularly pulmonary carcinoma, is Therapeutic cancer, particularly treats the good Chinese medicine preparation of pulmonary carcinoma.

Claims (6)

1. a Chinese medicine composition for Therapeutic cancer, is made up by corresponding weight of following raw material: Herba Sarcandrae 15 parts, 20 parts, the Radix Rehmanniae, burnt Concha Arcae 15 parts, Concha Meretricis seu Cyclinae 30 parts, Haematitum 10 parts, Radix Clematidis 10 parts, the Radix Pulsatillae 12 parts, Fructus Fici 12 parts, Fructus Hordei Germinatus (parched to brown) 20 parts, Herba Schizonepetae 10 parts, Semen Coicis 30 parts, willow root 60 parts, process Endothelium corneum 15 parts, Spica Prunellae 12 parts, Herba Equiseti Hiemalis 40 parts, Fructus Corni 20 parts, Radix Saposhnikoviae 10 parts, Semen Vaccariae 15 parts.

2. Chinese medicine composition according to claim 1, described Chinese medicine composition is made up by corresponding weight of following raw material: Herba Sarcandrae 15 grams, 20 grams, the Radix Rehmanniae, burnt Concha Arcae 15 grams, Concha Meretricis seu Cyclinae 30 grams, Haematitum 10 grams, Radix Clematidis 10 grams, the Radix Pulsatillae 12 grams, Fructus Fici 12 grams, Fructus Hordei Germinatus (parched to brown) 20 grams, Herba Schizonepetae 10 grams, Semen Coicis 30 grams, willow root 60 grams, process Endothelium corneum 15 grams, Spica Prunellae 12 grams, Herba Equiseti Hiemalis 40 grams, Fructus Corni 20 grams, Radix Saposhnikoviae 10 grams, Semen Vaccariae 15 grams.

3. the Chinese medicine composition described in claim 1 or 2, described pharmaceutical composition routinely technique adds adjuvant and makes tablet, capsule, granule, pill or oral liquid, and described adjuvant comprises one or more in solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent, lubricant and substrate.

4. prepare a method for the Chinese medicine composition described in claim 1 or 2, comprise the following steps:

A) raw material of kind described in claim 1 or 2 and component is rinsed twice, to remove dust, pesticide, fertilizer residue with clear water;

B) with warm water soaking, decoct two times for 100 DEG C, become flowing soaking paste, dilution three times, shake up to obtain extractum;

C) by described extractum cryogenic seal or storage.

5. the Chinese medicine composition described in claim 1 or 2 is preparing the application in Therapeutic cancer medicine.

6. the application of the Chinese medicine composition described in claim 1 or 2 in preparation treatment lung-cancer medicament.