CN102068477B

CN102068477B - Effective parts of ginseng and preparation method and application thereof

Info

Publication number: CN102068477B
Application number: CN2010106203637A
Authority: CN
Inventors: 高瑞兰; 苗青; 林筱洁; 肖鲁伟; 许家鸾; 尹利明; 陈华; 王潇; 余潇苓
Original assignee: Zhejiang Provincial Hospital of Traditional Chinese Medicine
Current assignee: Zhejiang Provincial Hospital of Traditional Chinese Medicine
Priority date: 2010-12-31
Filing date: 2010-12-31
Publication date: 2012-07-25
Anticipated expiration: 2030-12-31
Also published as: WO2012088777A1; CN102068477A

Abstract

The invention discloses effective parts of ginseng and a preparation method and an application thereof, and the preparation of the effective parts of the ginseng comprises the following steps: (1) extraction: taking the ginseng, cutting into small sections, extracting with water-containing ethanol or ethanol-containing water, concentrating obtained extraction solution to 80 DEG C till the relative density is 0.8-1.3, and recording as A solution; (2) performing alcohol precipitation or water precipitation treatment on the A solution, concentrating supernatant fluid obtained by separation till the ratio of concentrated solution to raw herb achieves 1: 0.25-2.5, and recording as B solution; and (3) chromatography: performing macroporous resin column chromatography twice, separating and getting the effective parts of the ginseng. The effective parts of the ginseng are effective for resisting leukemia and solid tumors without obvious toxic and side effects.

Description

Radix Ginseng effective site

(1) technical field

The present invention relates to the preparation technology and the application in preparation treatment leukemia and tumour medicine of a kind of Chinese medicine Radix Ginseng effective site and extraction separation thereof.

(2) background technology

Leukemia belongs to the hemopoietic system common malignancy, and one of domestic ten big malignant tumor occurred frequently are serious threat human life's dangerous property diseases, occupies the first place of the tumor invasion of crowd below 35 years old, and is the trend that rises year by year.It is characterized in that hematopoietic stem malignant clone property hypertrophy and extensively soak into bone marrow, liver, spleen and lymph node that final failure whole body organ-tissue causes symptoms such as anemia, hemorrhage, infection.Though Therapeutic Method has HSCT, induces differentiation, immunization therapy etc., the specific aim targeted therapy of newly opening up in addition just under study for action, chemotherapy is still the main means of treatment.When killing cancerous cell, chemotherapeutics makes the patient be difficult to bear very greatly because of its toxic and side effects, and be prone to produce drug resistance simultaneously and cause treatment failure and recurrence, all are the difficult problems that press for solution.The New Policy of current domestic and international oncologist lay special stress on development anticarcinogen is an attenuation synergistic; And raising patients ' life quality; And be different from traditional chemotherapeutic; Promptly suppress malignant cell propagation, induce differentiation, promote apoptosis, strengthen the chemotherapy susceptibility, and the pernicious signal transmission of blocking-up cancerous cell, but low to normal cytotoxicity.

The 42nd brainstrust that American Society of Clinical Oncology attends a meeting fully stresses to improve the quality of life of tumor patient; Particularly point out treatment and must abandon in the past the combat model of " treating the poisonous disease with poisonous drugs "; Change traditional chemotherapy radiotherapy and kill the therapy of tumor cell; And come control cancer cell with molecular targeted agents, become the leukemic New Policy of treatment.The emphasis of foreign study leukemia new drug is the inducing leukemia cell differentiation, promotes apoptosis, to reduce the toxic and side effects of antitumor drug to human body.Switzerland Novartis Co.,Ltd releases the new drug imatinib mesylate of treatment chronic myelocytic leukemia (CML); It is exactly the Bcr-Abl fusion gene product-EGFR-TK " target spot " that directly is directed against chromosome translocation; The signal pipeline of blocking-up cancerous cell, treatment CML chronic phase, can reach hematologic response.But shortcoming is can recurrence after the drug withdrawal, and drug resistance occurs, and also needs more long-term prescription to estimate to the influence of life cycle.Also have the present imatinib mesylate dependence on import of still needing, it costs an arm and a leg, treat needed 20 in 1 year surplus ten thousand yuan.Chinese medicine is the valuable source of exploitation natural drug and leading combination drug; Received domestic and international great attention; Because some Chinese medicine has leukemia resisting action, and the advantage little to normal cell injury, a lot of researcheres both at home and abroad are devoted to develop the effective and low toxicity new Chinese medicine of leukemia.

Domestic from the isolating harringtonine of Folium et Ramulus Cephalotaxi plant extract, be first antitumor drug (chemosynthesis) that Chinese succeed in developing voluntarily, become a line medicine of treatment acute myelocytic leukemia; Become the effective medicine of treatment acute promyelocytic leukemia from the arsenic trioxide of arsenicum extraction separation, but above-mentioned two medicines still belong to chemotherapeutic." joining a capsule " (Rg from Radix Ginseng ₃Monomer) share with chemotherapeutics, help the curative effect of primary lung cancer, hepatocarcinoma, but a little less than the effect of this medicine anti-tumor in vivo.Other research such as Cordyceps, Radix Sophorae Flavescentis, Herba Hedyotidis Diffusae, Rabdosia rubescens, Radix Puerariae, white hellebore, Radix Et Rhizoma Rhei, Radix Tripterygii Wilfordii, Radix Trichosanthis etc.; All report has leukemia resisting action; But majority still rests on the experimental stage, and its mechanism of action and approach are all not clear and definite.Therefore, extract and isolate the effective site or the effective ingredient that play therapeutical effect in the Chinese medicine, with its effect of method research and the mechanism of modern medicine, and to push international medical community to be a current difficult problem that presses for solution.

Though it is limited that Chinese medicine is directly killed the effect of tumor cell, have the effect of many target spots, for suppressing tumor cell, improve patients ' life quality etc. and have important value.The main application point of treatment by Chinese herbs leukemia is to promote apoptosis of leukemia, induces differentiation and ripe, and collaborative chemotherapy and the toxicity that alleviates chemotherapeutic promote the recovery of normal hematopoiesis function, enhancing human body immunity power, and prolong life cycle.Discover that some Chinese medicine has the unique advantage of two-ways regulation; Radix Ginseng is exactly the strengthening the body resistance medicine, increases the nonspecific resistance of body, to various being harmful to protective effect stress be arranged all; On the one hand through the tonification human righteousness; Promote to suppress Leukemia Cell Proliferation on the other hand by normal hematopoiesis, and induce its apoptosis and play the leukemic effect of treatment.Radix Ginseng is obligato medicine in the leukemia tcm syndrome differentiation and treatment, has reinforcing body resistance, human body immunity improving power, and regulating functions of ZANG FU-organs alleviates the infringement of chemotherapeutics to body, strengthens and consolidate effects such as curative effect.

The main effective ingredient of Radix Ginseng is a Radix Ginseng total saponins; Confirmed structure have 40 surplus a saponin monomer, be divided into three types of panoxadiol's saponins (Panaxadiol), panaxatriol's saponins (Panaxatriol) and oleanolic acid by the contained hydroxyl quantity of sapogenin difference.Wherein glycol, triol saponins are in the great majority, and are topmost active component in the Radix Ginseng.Glycol saponins is mainly Ra, Rb ₁, Rb ₂, Rb ₃, Rc, Rd, F ₄, Rg ₃, Rg ₅, Rh ₂, Rh ₄, Rk ₁, Rk ₃With monomers such as Rs; The triol saponins is mainly Re, Rf, Rg ₁, Rg ₂And Rh ₁Deng monomer.

The inventor has reported voluntarily 6 pieces of the antileukemie papers of Radix Ginseng total saponins that extract, uses the test of leukemia clonal expansion and shows that it is that HL-60, macronucleus are the propagation of Meg-01 cell colony that Radix Ginseng total saponins 50～100mg/L suppresses the leukemia grain significantly.Strengthen the effect of chemotherapy susceptibility; Make high Folium et Ramulus Cephalotaxi, cytosine arabinoside, amycin and etoposide strengthen 1.84～2.23 times, and drug-fast leukaemia is also had the effect that suppresses and strengthen the chemotherapy susceptibility the lethal effect of acute myeloid leukemia patient CFU-GM colony of former generation.Radix Ginseng total saponins also has the effect of the HL-60 of inducing apoptosis; Annexin V positive cell and dna degradation trapezoid-shaped strips appear; The visible G0/G1 phase cell of flow cytometry dna content reduces, S phase and G2/M phase cytosis, the hypodiploid apoptotic peak of expression characteristics property on the rectangular histogram simultaneously.

Other authors are more to the research of Radix Ginseng extract antineoplastic, but all rest on the experimental stage.Have the report Radix Ginseng total saponins to handle oncoprotein Bcl-2 behind the K562 cell, the c-myc protein expression obviously reduces, Fas expresses and increases, and is relevant with induced apoptosis in leukemia cell lines.Mtt assay is observed ginsenoside monomer Rb1 and is suppressed HL-60 cell proliferation, Rb ₁, Rb ₂With Rc cytochrome C is discharged in mitochondrion, induce the HL-60 apoptosis through activating apoptosis effect enzyme Caspase-3.P-glycoprotein (P-gp, ATP binding film glycoprotein) can pump the chemicals in the leukaemia born of the same parents and make have the cell drug resistance report Rb1 can suppress the expression of P-gp, and improve the sensitivity of tumor cell to chemotherapeutics outward.Report Rh in addition ₂Suppress slow grain K562 cell, leukaemias' such as children's grain HL-60 cell, T lymph Jurkat cell propagation early, induce its apoptosis, and be concentration and time-dependent effect etc.In sum, Chinese medicine extract has become an important directions of current anti-cancer agent research.

The advanced method that the inventor adopts the BA screening to combine with preparation technology; At first from the plurality of Chinese extract, filter out Radix Ginseng total saponins; Further isolate a plurality of extracts again from Radix Ginseng total saponins; Warp is biological activity research repeatedly, and from a plurality of Radix Ginseng extracts, filter out the inhibition leukaemia and act on the best, and to the little effective site of normal hemopoietic progenitor cell infringement.Adopt the liquid chromatograph mass spectrography analytical method, this Radix Ginseng effective site master contains ginsenoside Rd, Rg ₆, F ₄, Rk ₃, Rh ₄, F ₂, Rg ₃, Rk ₁, Rg ₅This content in the ginseng raw material of these monomers is not high, is a large amount of enrichments in extraction separation preparation back.Different with traditional chemotherapeutic mechanism of action, have the advantage that leukemia does not effectively have obvious toxic-side effects.Imagination is in the leukemia chemotherapy phase, with the cell toxicity medicament Combined application, brings into play its collaborative chemotherapy effect and improves curative effect; The chemotherapy intermission, single with and bring into play the effect that it suppresses Leukemia Cell Proliferation, promotes apoptosis and induce differentiation, to keep and to consolidate curative effect etc.

(3) summary of the invention

First technical problem that the present invention will solve provides the Radix Ginseng effective site that a kind of leukemia and entity tumor effectively do not have obvious toxic-side effects.

For solving the problems of the technologies described above, the present invention adopts following technical scheme:

A kind of Radix Ginseng effective site, the preparation of described Radix Ginseng effective site comprises the steps:

(1) extract: get Radix Ginseng and cut into segment, with aquiferous ethanol or contain ethanol water and extract, relative density was 0.8～1.3 when the gained extracting solution was concentrated into 80 ℃, was designated as A liquid;

(2) A liquid is handled through precipitate with ethanol or water precipitating, separates supernatant concentration to the concentrated solution crude drug ratio that obtains to reach 1: 0.25～2.5 (be to contain approximately in the per kilogram concentrated solution to be equivalent to ginseng crude drug 0.25～2.5kg), be designated as B liquid;

(3) chromatography

3.1 separate for the first time: get B liquid, it is mixed with crude drug content is 25%～150% medicinal liquid, adds sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 0.1～2.0molL ^-1, fully stir, to leave standstill 0.5～3.0 hour, centrifugal or filtration gets transparent liquid, is designated as C liquid; Get C liquid, last macroporous adsorptive resins, described macroporous adsorbent resin are low pole or nonpolar, use alkali liquor, water, 5%～45% washing with alcohol successively, use 45%～80% ethanol elution then, collect post liquid, are designated as D liquid; Get D liquid, decompression recycling ethanol, residual liquid is concentrated into flowing soaking paste, and drying obtains the dry thing of first separation, is designated as first;

3.2 separate for the second time: get first, its concentration by 0.01～0.1g/ml be dissolved in 5%～35% the ethanol, abundant stirring and dissolving, centrifugal or filter, transparent liquid, be designated as E liquid; Get E liquid, last macroporous adsorptive resins, described macroporous adsorbent resin are low pole or nonpolar, use 10%～50% washing with alcohol then, and reuse 50%～80% ethanol elution was collected post liquid, was designated as F liquid; Get F liquid, decompression recycling ethanol, residual liquid is concentrated into flowing soaking paste, and drying obtains secondary separation dry thing, i.e. Radix Ginseng effective site.

The Radix Ginseng effective site that the present invention makes mainly contains the chemical compound of following weight portion:

3～43 parts of protopanoxadiol type saponin Rd

Protopanoxadiol type saponin Rg ₆4～11 parts

Protopanoxadiol type saponin F ₄6～17 parts

Protopanaxatriol ginsenoside Rk

₃2～9 parts

Protopanaxatriol ginsenoside Rh

₄5～16 parts

Protopanaxatriol ginsenoside F ₂13～18 parts

Protopanaxatriol ginsenoside Rg ₃0.5～4 parts

Panoxadiol's type saponin RK ₁2～20 parts

Panoxadiol's type saponin Rg ₅1～30 part

Second technical problem that the present invention will solve provides a kind of method of the concrete above-mentioned Radix Ginseng effective site of preparation, and the technical scheme of employing is following:

A kind of method for preparing of described Radix Ginseng effective site comprises the steps:

(1) extracts: get Radix Ginseng and cut into segment (as being cut into 3～5mm segment); With aquiferous ethanol or contain ethanol water and extract (decoction, hot reflux or percolation), it is centrifugal or leave standstill fully to extract the back, centrifugal liquid or supernatant; Relative density is 0.8～1.3 when being concentrated into 80 ℃, is designated as A liquid;

(2) A liquid adopts 2.1 or 2.2 described methods to handle:

2.1 precipitate with ethanol: get A liquid, add an amount of ethanol, make it contain the alcohol amount and reach 40%～80%, leave standstill precipitate with ethanol, draw the precipitate with ethanol supernatant then; Residue is with an amount of 40%～80% washing with alcohol; The centrifugal back of washing liquid merges with supernatant; The decompression recycling ethanol continued is concentrated into concentrated solution crude drug ratio and reaches 1: 0.25～and 2.5 (be to contain approximately in the per kilogram concentrated solution to be equivalent to ginseng crude drug 0.25～2.5kg), be designated as B liquid;

2.2 water precipitating: get A liquid, add the water be equivalent to long-pending 1～5 times of A liquid (cold water or hot water all can), leave standstill water precipitating, draw the water precipitating supernatant then; Residue washs with suitable quantity of water, and the centrifugal back of washing liquid merges with supernatant, be concentrated into concentrated solution crude drug ratio to reach 1: 0.25～2.5, be designated as B liquid;

(3) chromatography

3.1 separate for the first time

3.1.1 preparation upper prop liquid: get B liquid, it is mixed with crude drug content is 25%～150% medicinal liquid, adds sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 0.1～2.0molL ^-1, fully stir, to leave standstill, centrifugal or filtration gets transparent liquid, is designated as C liquid;

3.1.2 upper prop: get C liquid, last macroporous adsorptive resins, described macroporous adsorbent resin are low pole or nonpolar, all get in the macroporous resin column until C liquid;

3.1.3 neutralizing treatment: with 0.1～1.25molL ^-1The sodium hydroxide solution washing is crossed post liquid and is discarded;

3.1.4 water washing: be washed with water to post liquid and be only neutral, and crossed post liquid and discard;

3.1.5 alcohol washing: the washing with alcohol with 5%～45%, cross post liquid and discard;

3.1.6 eluting: with 45%～80% ethanol elution, collect post liquid, be designated as D liquid, for use;

3.1.7 concentrate: get D liquid, decompression recycling ethanol, residual liquid is concentrated into flowing soaking paste, and drying obtains the dry thing of first separation, is designated as first;

3.2 separate for the second time

3.2.1 preparation upper prop liquid: get first, its concentration by 0.01～0.1g/ml be dissolved in 5%～35% the ethanol, abundant stirring and dissolving, centrifugal or filter, transparent liquid, be designated as E liquid;

3.2.2 upper prop: get E liquid, last macroporous adsorptive resins, described macroporous adsorbent resin are low pole or nonpolar, all get in the macroporous adsorptive resins until E liquid;

3.2.3 alcohol washing: the washing with alcohol with 10%～50%, cross post liquid and discard;

3.2.4 eluting: with 50%～80% ethanol elution, collected post liquid, and be designated as F liquid;

3.2.5 concentrate: get F liquid, decompression recycling ethanol, residual liquid concentrate (normal pressure or concentrating under reduced pressure all can) to flowing soaking paste, dry (normal pressure, decompression, spraying or lyophilization all can), obtain secondary separation dry thing, i.e. Radix Ginseng effective site.

Further, in the said step (2), A liquid preferably adopts following 2.1 or 2.2 described methods to handle:

2.1 precipitate with ethanol: get A liquid, add an amount of ethanol, make it contain the alcohol amount and reach 40%～80%; Fully stir, airtight, room temperature or cold preservation were left standstill more than 12 hours; Get the precipitate with ethanol supernatant then, residue is with the washing with alcohol more than 75%, and washing liquid is centrifugal or filtration is back merges with supernatant; Decompression recycling ethanol continued decompression or normal pressure be concentrated into concentrated solution crude drug ratio and reach 1: 0.25～and 2.5, be designated as B liquid;

2.2 water precipitating: get A liquid, add the cold water or the hot water that are equivalent to long-pending 1～5 times of A liquid, fully stir; Airtight, room temperature or cold preservation were left standstill more than 12 hours, then the heavy supernatant of water intaking; Residue is used water washing, and washing liquid is centrifugal or filter the merging supernatant; Decompression or normal pressure be concentrated into concentrated solution crude drug ratio and reach 1: 0.25～and 2.5, be designated as B liquid.

Further, in the described step (2), after preferred A liquid adopts 2.1 or 2.2 described methods to handle, make concentrated solution crude drug ratio reach 1: 0.5～2.5, more preferably make concentrated solution crude drug ratio reach 1: 1～2.5.

Further, described macroporous adsorptive resins is preferably from one of following: D ₁₀₁, DA ₂₀₁, D ₁₀₁+ DA ₂₀₁, nonpolar or low pole macroporous adsorbent resin such as PHD-100, PHD-200.

Further, the blade diameter length ratio of step 3.1.2 or the described macroporous adsorptive resins of 3.2.2 independently is 1: 2～10 separately, and the flow of C liquid and E liquid independently is 0.25～2.5SV (promptly per hour the discharge of liquid is 0.25～2.5 times of amount of resin volume) separately.

Further, among said step 3.1.3,3.1.4,3.1.5,3.1.6,3.2.3 or the 3.3.4, washing or eluting flow independently are 0.25～2.5SV separately.

The present invention is concrete to recommend said method for preparing to carry out according to following steps:

(1) extract: get Radix Ginseng and cut into segment, with aquiferous ethanol or contain that pure water decocts, hot reflux or percolation extract, it is centrifugal or leave standstill fully to extract the back, centrifugal liquid or supernatant, relative density is 0.8～1.3 when being concentrated into 80 ℃, is designated as A liquid;

(2) A liquid adopts 2.1 or 2.2 described methods to handle:

2.1 precipitate with ethanol: get A liquid, add an amount of ethanol, make it contain the alcohol amount and reach 40%～80%, leave standstill precipitate with ethanol, draw the precipitate with ethanol supernatant then; Residue is with an amount of 40%～80% washing with alcohol, and the centrifugal back of washing liquid merges with supernatant, and the decompression recycling ethanol continued is concentrated into concentrated solution crude drug ratio and reaches 1: 0.25～and 2.5, be designated as B liquid;

2.2 water precipitating: get A liquid, add the cold water or the hot water that are equivalent to long-pending 1～5 times of A liquid, leave standstill water precipitating, draw the water precipitating supernatant then; Residue washs with suitable quantity of water, and the centrifugal back of washing liquid merges with supernatant, be concentrated into concentrated solution crude drug ratio to reach 1: 0.25～2.5, be designated as B liquid;

(3) chromatography

3.1 separate for the first time

3.1.1 preparation upper prop liquid: get B liquid, it is mixed with crude drug content is 25%～150% medicinal liquid, adds sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 0.1～2.0molL ^-1, fully stir, to leave standstill 0.5～3.0 hour, centrifugal or filtration gets transparent liquid, is designated as C liquid;

3.1.2 upper prop: get C liquid, last blade diameter length ratio is 1: 1～10 macroporous adsorptive resins, and flow 0.25～2.5SV all gets in the macroporous resin column until C liquid, leaves standstill 1～60 minute or does not leave standstill; Said macroporous adsorptive resins is a low pole or nonpolar;

3.1.3 neutralizing treatment: with 0.1～1.25molL ^-1The sodium hydroxide solution washing, flow is 0.25～2.5SV, crosses post liquid and discards;

3.1.4 water washing: be washed with water to post liquid and be only neutral, flow 0.25～2.5SV crosses post liquid and discards;

3.1.5 the alcohol washing: with 5%～45% washing with alcohol, flow is 0.25～2.5SV, crosses post liquid and discards;

3.1.6 eluting: with 45%～80% ethanol elution, flow is 0.25～2.5SV, collects post liquid, is designated as D liquid, and is for use;

3.2 separate for the second time

3.2.2 upper prop: get E liquid, last blade diameter length ratio is 1: 1～10 macroporous adsorptive resins, and flow 0.25～2.5SV all gets in the macroporous resin column until E liquid, leaves standstill 1～60 minute or does not leave standstill, and is for use; Said macroporous adsorptive resins is a low pole or nonpolar;

3.2.3 the alcohol washing: with 10%～50% washing with alcohol, flow is 0.25～2.5SV, crosses post liquid and discards;

3.2.4 eluting: with 50%～80% ethanol elution, flow is 0.25～2.5SV, collects post liquid, is designated as F liquid;

3.2.5 concentrate: get F liquid, decompression recycling ethanol, residual liquid is concentrated into flowing soaking paste, and drying obtains secondary separation dry thing, i.e. Radix Ginseng effective site.

In the technique scheme, concentration of alcohol is volumetric concentration.

The 3rd technical problem that the present invention will solve is the application of described Radix Ginseng effective site, and concrete is the application in the preparation anti-leukemia medicine; Suppress the application in the solid tumor cell medicine in preparation, concrete, described solid tumor cell is that L929 becomes fibrous tumours cell or ECV-304 tumor endothelial cell.

The present invention adopts animal model, cytology and Protocols in Molecular Biology research; Prove the subcutaneous lotus tumor of Radix Ginseng effective site treatment leukemia animal model of the present invention nude mouse; And the monokaryon leukemia is soaked into the evident in efficacy of nude mouse; Suppress the leukemia progenitor cell proliferation, promote apoptosis, induce differentiation, collaborative chemotherapeutic, and the pernicious signal conduction of retardance Leukemia Cell Proliferation and reach antileukemie effect, it is effective and do not have an advantage of obvious toxic-side effects to have a leukemia.Therefore, Radix Ginseng effective site of the present invention can be used for preparing anti-leukemia medicine.

Radix Ginseng effective site of the present invention also has significant inhibitory effect to other solid tumor cell, comprises that L929 becomes fibrous tumours cell, ECV-304 tumor endothelial cell etc., so it can be used for preparing the medicine that suppresses solid tumor cell.

Compared with prior art, the beneficial effect of Radix Ginseng effective site of the present invention is:

1. the interior curative effect of 2 kinds of leukemia animal models of research proof Radix Ginseng effective site treatment is remarkable; Can prolong life cycle significantly; Suppress the growth of tumor body in the subcutaneous lotus tumor of the K562 leukaemia nude mouse body significantly, the tumor tissue pathologic finding has degeneration necrosis to some extent.Treatment monokaryon SHI-1 leukemiacell infiltration nude mouse model can suppress leukaemia's growth of bone marrow effectively, and significantly reduce the infiltration of leukaemia at cerebral tissue and a plurality of internal organs.

2. suppress the propagation of acute myeloid leukemia patient leukemia hemopoietic progenitor cell of former generation, colony forms number average and obviously descends, and the colony suppression ratio of 20～100mg/L reaches 25.6～68.6%.Suppress the red K562 of being simultaneously, grain is that HL-60 and monokaryon are the propagation of SHI-1 leukemia cell line, makes colony form number average and obviously descends, the colony suppression ratio of 20～100mg/L reaches 26.7～83.5%.Suppress proliferation function and be dose dependent, one of its mechanism of action is a downward modulation leukemia K 562 cell proliferation specific b CR/ABL Expression of Fusion Protein.

3. low dose of can strengthen the sensitivity of leukaemia, show the concertedness of itself and chemotherapeutics chemotherapeutics.Increase K562, the HL-60 leukaemia sensitivity to chemotherapeutic homoharringtonine and cytosine arabinoside, effect obviously strengthens to the leukemia cell inhibiting to make low dose of chemotherapeutics.

4. the effect of induced apoptosis in leukemia cell lines is obvious; It is that HL-60 and monokaryon are apoptosis of leukemia such as SHI-1 that Radix Ginseng effective site is induced the red K562 of being, grain; The male apoptotic cell rate of Annexin V obviously increases, and the visible typical dna degradation specificity of apoptosis trapezoid-shaped strips.

5. the effect of inducing leukemia germinal cell differentiation is remarkable, and the positive cell rate that flow cytometry demonstration leukaemia breaks up specific marker obviously increases.Simultaneously, confirm to promote that through several different methods such as laser scanning confocal microscopy, Western immunoblotting and the dyeing of cellular immunization group the mechanism of action of differentiation is to break up relevant proteic expression through raising multiple leukaemia.

6. the relevant kinase whose expression of MAPK signal pathway multiple protein of downward modulation Leukemia Cell Proliferation; The Western immunoblotting assay shows the expression that can suppress propagation related protein kinases such as AKT-1, AKT-2, ERK-2, MEK-1 and MEK-2 in human leukemia stem cell KG-1a cell, the red K562 of the being cell to some extent; Explain that it passes through the kinase whose expression of downward modulation propagation coherent signal pathway protein; The conduction of blocking pernicious signal, and inhibition leukaemia's abnormality proliferation.

7. other solid tumor cell is also had significant inhibitory effect, comprise that L929 becomes fibrous tumours cell, ECV-304 tumor endothelial cell etc.In view of becoming the fibrous tumours cell is that the growth of tumor cell provides support, and endotheliocyte then provides tumor growth required nutrition through forming blood vessel, therefore points out Radix Ginseng effective site except antileukemie effect, also has the effect of anti entity tumour simultaneously.

(4) description of drawings

Nude mouse interior tumor tissue in Radix Ginseng effective site treatment back has in various degree degeneration and necrosis (HE dyeing, 200 times) among Fig. 1: the embodiment 4, wherein, and A: model control group, B:50mg/Kg group, C:100mg/Kg group, D:200mg/Kg group.100, after the 200mg/Kg treatment, pathologic finding tumor tissue cell has swelling, breaks, and phenomenons such as karyolysis explain in lotus leukemia tumor nude mouse to have stronger leukemia resisting action.

Radix Ginseng effective site treatment back nude mice cerebral tissue HCD45 among Fig. 2: the embodiment 5 ⁺Leukaemia's infiltration obviously reduces (200 times), wherein: A: model control group, B:50mg/Kg group, C:100mg/Kg group, D:200mg/Kg group.A large amount of people CD45 in the brain essence of immunohistochemical staining display model matched group ⁺The leukaemia piles up, and 100mg/Kg group brain essence and dura mater stranger CD45 ⁺The quantity of cellular infiltration obviously reduces, and does not see in the brain essence infiltration is arranged, and only the brain adventitia is coated with people CD45 ⁺Cell; The leukemiacell infiltration of 200mg/Kg group cerebral tissue does not still less find to have the infiltration of brain essence, only is covered in fracture surface between brain, XIAONAO and brain on a small quantity.

Radix Ginseng effective site downward modulation Leukemia Cell Proliferation specific b CR/ABL Expression of Fusion Protein among Fig. 3: the embodiment 6, wherein, A: contrast K562 cell, B:20mg/L, C:50mg/L, D:100mg/L.The BCR/ABL expressing fusion protein that is the green fluorescence reaction in laser co-focusing microscopy demonstration 20, the 50mg/L group K562 cell weakens gradually; The green fluorescence of 100mg/L group a little less than, prompting can suppress leukemic propagation through downward modulation BCR/ABL Expression of Fusion Protein effectively.

Radix Ginseng effective site increases the sensitivity of leukaemia to chemotherapeutics among Fig. 4: the embodiment 7; Fig. 4 a: increase the sensitivity of leukaemia to homoharringtonine (Hom); When semisolid colonies was cultivated

low dose

10,20,50mg/L and low dose of Hom Combined application, the colony of K562, HL-60 cell generated number and is starkly lower than single Hom matched group of using.Fig. 4 b: increase the sensitivity of leukaemia to cytosine arabinoside (Ara-c), 10,20 and when 50mg/L and low dose of Ara-c Combined application, the colony of K562, HL-60 cell generates number and is starkly lower than and singly uses the Ara-c matched group.

Radix Ginseng effective site induced apoptosis in leukemia cell lines dna degradation trapezoid-shaped strips among Fig. 5: the embodiment 8, wherein: 1.Mark, 2.HL-60 control cells, 3.10mg/L, 4.25mg/L, 5.50mg/L, 6.75mg/L, 7.100mg/L.25,50,75,100mg/L effect 36 hours, the specificity trapezoid-shaped strips of apoptosis dna degradation appears in the agarose gel electrophoresis analysis.

Radix Ginseng effective site raises the expression that the leukaemia breaks up relevant γ-globin among Fig. 6: the embodiment 9, wherein: A: contrast K562 cell, B:10mg/L, C:20mg/L, D:40mg/L.10,3 weeks of 20mg/L effect K562 cell, the SABC Faxian shows the cell rate of expressing γ-globin " +++" strong positive control cells apparently higher than not dosing.

The relevant CD11b phenotype positive cell rate of Radix Ginseng effective site inducing leukemia cell differentiation increases among Fig. 7: the embodiment 9, wherein: 7a: contrast SHI-1 cell, 7b:5mg/L, 7c:10mg/L, 7d:20mg/L, 7e:40mg/L.10,20,40mg/L induces 3 weeks of SHI-1 cell, the relevant CD11b positive cell rate of differentiation is apparently higher than the control cells of not dosing.

The kinase whose expression of Radix Ginseng effective site downward modulation MAPK signal pathway major protein among Fig. 8: the embodiment 10; 50,100mg/L effect KG-1a cell is after 7 days, and the ribbon density of propagation relevant 5 kinds of albumen kinases AKT-1, AKT-2, ERK-2, MEK-1 and MEK-2 is lower than control cells to some extent.50, behind the 100mg/L effect K562 cell, the ribbon density of 5 kinds of albumen kinases AKT-1, AKT-2, ERK-2, MEK-1 and MEK-2 also is lower than control cells to some extent.

Radix Ginseng effective site suppresses the effect that L929 tumor cell colony generates, A. control cells among Fig. 9: the embodiment 11; B.5mg/L; C.10mg/L; D.20mg/L; E.50mg/L; F.100mg/L.10,20, the colony number obviously reduces during 50mg/L, cell clump increases, and the colony form changes to the evacuation type by tight type, when drug level is increased to 100mg/L, has basically no colony and generates.

(5) specific embodiment

With specific embodiment technical scheme of the present invention is further specified below, but protection scope of the present invention is not limited thereto:

The preparation of embodiment 1 Radix Ginseng effective site

1. get ginseng raw material 25kg, cut into 3～5mm segment, the alcohol heat reflux with 30% extracts three times, merges each time extracting solution, leaves standstill, and filters to such an extent that consider liquid, and decompression or normal pressure are concentrated into relative density 1.1 (80 ℃), numbering: A liquid, and for use.

2A liquid is handled

2.1 pure taking the pulse heavily A liquid, 95% ethanol of 3 times of volumes of adding fully stirs, and airtight, room temperature left standstill more than 12 hours.Get the precipitate with ethanol supernatant next day; Residue is with 71% washing with alcohol three times, and the centrifugal back of washing liquid merges with supernatant, and decompression recycling ethanol continued normal pressure is concentrated into concentrated solution crude drug ratio and reaches 1: 1.0 (being to contain approximately in the per kilogram concentrated solution to be equivalent to ginseng crude drug 1.0kg); Numbering: B liquid, for use.

3. chromatography

3.1 separate for the first time

3.1.1 preparation upper prop liquid is got B liquid, adds water to 50L, adds an amount of sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 1.0molL ^-1, fully stir, left standstill 0.5 hour, 300 mesh sieves filter, and get transparent liquid, numbering: C liquid, for use.

3.1.2 upper prop is got C liquid, last blade diameter length ratio is 1: 5 D ₁₀₁Macroporous adsorptive resins, flow 1.0SV all gets in the macroporous resin column until C liquid, leaves standstill 30 minutes, and is for use.

3.1.3 neutralizing treatment is used 0.1molL ^-1The caustic lye of soda washing, flow is 1.0SV, crosses post liquid and discards.

Be only neutral 3.1.4 water washing is washed with water to post liquid, flow 1.0SV crosses post liquid and discards.

3.1.5 30% washing with alcohol is used in the alcohol washing, flow is 0.1SV, crosses post liquid and discards.

3.1.6 eluting is used 80% ethanol elution, flow is 1.0SV, collects post liquid, numbering: D liquid, and for use.

3.1.7 concentrate and get D liquid, decompression recycling ethanol, the residual liquid normal pressure is concentrated into flowing soaking paste, and vacuum drying obtains the dry thing of first separation, numbering: first, for use.

3.2 separate for the second time

3.2.1 preparation upper prop liquid is got first, with it by 0.02g/ml) concentration be dissolved in 20% the ethanol and join, abundant stirring and dissolving, centrifugal or filter, transparent liquid, number: E liquid, for use.

3.2.2 upper prop is got E liquid, last blade diameter length ratio is 1: 5 a D101 macroporous resin column, and flow 1.0SV all gets in the macroporous resin column until E liquid, leaves standstill 30 minutes, and is for use.

3.2.3 the alcohol washing is with 45% washing with alcohol, flow is 1.0SV, crosses post liquid and discards.

3.2.4 eluting is used 80% ethanol elution, flow is 1.0SV, collects post liquid, numbering: F liquid, and for use.

3.2.5 concentrate and get F liquid, decompression recycling ethanol, the residual liquid normal pressure is concentrated into flowing soaking paste, and vacuum drying obtains secondary separation dry thing, i.e. Radix Ginseng effective site.

4. the evaluation of Radix Ginseng effective site chemical constituent

4.1 instrument and chromatographic condition liquid-matter coupling detector adopts Agilent 1200 series of high efficiency liquid chromatograph-TOFQ 125 combined instruments, ion source is the ESI source.

Chromatographic column: Agilent ZORBAX 80AExtend-C ₁₈(4.6x250mm, 5 μ m);

Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 205nm;

Eluent gradient is seen table 1.

4.2 each 10mg of test agent in the sample treatment peek batch Radix Ginseng effective site, respectively with 1ml methanol ultrasonic dissolution 2min, the centrifugal 10min of 3000rmp gets settled solution and supplies test usefulness, sample size 2 μ l.

4.3 analysis result and conclusion

4.3.1 through analyze to find content in the Radix Ginseng effective site higher 9 chemical compounds are arranged, according to the cleavage of mass spectrum situation of these 9 chemical compounds, combine the cleavage of mass spectrum rule of ginsenoside's constituents simultaneously, infer that they are respectively: protopanoxadiol type saponin: Rd, F ₂With Rg ₃, protopanaxatriol ginsenoside: Rg ₆, F ₄, Rk ₃, Rh ₄And panoxadiol's type saponin: RK ₁, Rg ₅

4.3.2 find through analyzing: the content ratio of 9 main compound in the total ginsenoside of Radix Ginseng effective site is as shown in table 2.

Table 1 eluent gradient table

Annotate the A:0.1% aqueous formic acid; B:0.1% formic acid acetonitrile solution

9 main ginsenosides' content ratio in the table 2 embodiment 1 Radix Ginseng effective site

The preparation of embodiment 2 Radix Ginseng effective sites

1. get ginseng raw material 37.5kg, cut into 3～5mm segment, the alcohol heat reflux with 50% extracts three times, merges each time extracting solution, leaves standstill, and filters, and filtrate decompression is concentrated into relative density 1.1 (80 ℃), numbering: A liquid, and for use.

2. concentrated solution is handled

2.1 water precipitating is got A liquid, adds the cold water that is equivalent to long-pending 4 times of A liquid, fully stirs, airtight, cold preservation was left standstill more than 12 hours.The heavy supernatant of water intaking next day, residue are with water washing three times, and the centrifugal back of washing liquid merges with supernatant, is evaporated to concentrated solution crude drug ratio and reaches 1: 1.0 (being to contain approximately in the per kilogram concentrated solution to be equivalent to ginseng crude drug 1.0kg), numbering: B liquid, and for use.

3. chromatography

3.1 separate for the first time

3.1.1 preparation upper prop liquid is got B liquid, adds water to 50L, adds an amount of sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 1.0molL ^-1, fully stir, left standstill 0.5 hour, filter, get transparent liquid, numbering: C liquid, for use.

3.1.2 upper prop is got C liquid, last blade diameter length ratio is 1: 5 D ₁₀₁+ DA ₂₀₁Macroporous resin column, flow 0.5SV all gets in the macroporous resin column until C liquid, leaves standstill 30 minutes or does not leave standstill, and is for use.

3.1.3 neutralizing treatment is used 1.0molL ^-1The caustic lye of soda washing, flow is 1.0SV, crosses post liquid and discards.

3.1.5 the alcohol washing is with 20% washing with alcohol, flow is 1.0SV, crosses post liquid and discards.

3.1.6 eluting is used 70% ethanol elution, flow is 1.0SV, collects post liquid, numbering: D liquid, and for use.

3.2 separate for the second time

3.2.1 preparation upper prop liquid is got first, its concentration by 0.04g/ml is dissolved in 20% the ethanol, fully stirring and dissolving is centrifugal, transparent liquid, numbering: E liquid, for use.

3.2.2 upper prop is got E liquid, last blade diameter length ratio is 1: 5 D ₁₀₁+ DA ₂₀₁Macroporous resin column, flow 1.0SV all gets in the macroporous resin column until E liquid, leaves standstill 30 minutes, and is for use.

3.2.3 the alcohol washing is with 40% washing with alcohol, flow is 1.0SV, crosses post liquid and discards.

3.2.4 eluting is used 70% ethanol elution, flow is 1.0SV, collects post liquid, numbering: F liquid, and for use.

3.2.7 concentrate and get F liquid, decompression recycling ethanol, the residual liquid normal pressure is concentrated into flowing soaking paste, and vacuum drying obtains secondary separation dry thing, i.e. Radix Ginseng effective site.

4. the evaluation of Radix Ginseng effective site chemical constituent

Detection method is with embodiment 1, and testing result shows: the contained chemical constituent of Radix Ginseng effective site sample is as shown in table 3 with the content ratio of 1,9 main compound of embodiment in the total ginsenoside of Radix Ginseng effective site:

9 main ginsenosides' content ratio in the table 3 embodiment 2 Radix Ginseng effective sites

The preparation of embodiment 3 Radix Ginseng effective sites

1. get ginseng raw material 50kg, cut into 3～5mm segment, the alcohol heat reflux with 30% extracts three times, merges each time extracting solution, and is centrifugal, gets centrifugal liquid, is evaporated to relative density 1.1 (80 ℃), numbering: A liquid, and for use.

2. concentrated solution is handled

2.1 pure taking the pulse heavily A liquid, 95% ethanol of 4 times of amounts of adding fully stirs, and airtight, cold preservation was left standstill more than 12 hours.Get the precipitate with ethanol supernatant next day; Residue is with 76% washing with alcohol three times, and the centrifugal back of washing liquid merges with supernatant, and decompression recycling ethanol continued normal pressure is concentrated into concentrated solution crude drug ratio and reaches 1: 2.0 (being to contain approximately in the per kilogram concentrated solution to be equivalent to ginseng crude drug 2.0kg); Numbering: B liquid, for use.

3. chromatography

3.1 separate for the first time

3.1.1 preparation upper prop liquid is got B liquid, adds water to 50kg, adds an amount of sodium hydroxide, makes the naoh concentration in the medicinal liquid reach 1.0molL ^-1, fully stir, leave standstill 0.5 hour, centrifugal, get transparent liquid, numbering: C liquid, for use.

3.1.2 upper prop is got C liquid, last blade diameter length ratio is 1: 5 D ₁₀₁Macroporous resin column, flow 1.0SV all gets in the macroporous resin column until C liquid, leaves standstill 30 minutes or does not leave standstill, and is for use.

3.1.3 neutralizing treatment is used 0.5molL ^-1The caustic lye of soda washing, flow is 1.0SV, crosses post liquid and discards.

3.1.5 the alcohol washing is with 30% washing with alcohol, flow is 1.0SV, crosses post liquid and discards.

3.1.6 eluting is used 60% ethanol elution, flow is 1.0SV, collects post liquid, numbering: D liquid, and for use.

3.2 separate for the second time

3.2.1 preparation upper prop liquid is got first, its concentration by 0.06g/ml is dissolved in 20% the ethanol, fully stirring and dissolving is centrifugal, transparent liquid, numbering: E liquid, for use.

3.2.2 upper prop is got E liquid, last blade diameter length ratio is 1: 5 D ₁₀₁Macroporous resin column, flow 1.0SV all gets in the macroporous resin column until E liquid, leaves standstill 30 minutes or does not leave standstill, and is for use.

3.2.3 the alcohol washing is with 35% ethanol gradient washing, flow is 1.0SV, crosses post liquid and discards.

3.2.4 eluting is used 60% ethanol elution, flow is 1.0SV, collects post liquid, numbering: F liquid, and for use.

4. the evaluation of Radix Ginseng effective site chemical constituent

Detection method is with embodiment 1, and testing result shows: the contained chemical constituent of Radix Ginseng effective site sample is as shown in table 4 with the content ratio of 1,9 main compound of embodiment in the total ginsenoside of Radix Ginseng effective site:

9 main ginsenosides' content ratio in the table 4 embodiment 3 Radix Ginseng effective sites

Embodiment 4 treatments are red to be the evident in efficacy of the subcutaneous lotus tumor of K562 leukaemia nude mouse model

1. material and method

1.1 preparation model: 4～5 all immunodeficiency in age (Nude) nude mices are produced by Shanghai Si Laike laboratory animal company, with the high tumorigenesis K562 cell 1 * 10 of exponential phase ⁷It is subcutaneous that/0.2ml is inoculated in the outside, nude mice forelimb oxter, and the inoculation back can become tumor in 1 week, the tumor growth stability of characteristics, and the maximum tumor body weight in inoculation 3 week back can reach 2.0g.

1.2 experiment is divided into groups and Therapeutic Method: the nude mice that will inoculate the K562 cell is divided into 5 groups at random; Every group 10; Model group, Radix Ginseng effective site (embodiment 3 makes) low dosage 50mg/Kg/d, middle dose groups 100mg/Kg/d, high dose group 200mg/Kg/d all irritate the stomach treatment and connect 21 days.Model group normal saline every day is irritated stomach, positive drug matched group homoharringtonine (Hom) 1mg/Kg/d lumbar injection.

2. result

2.1 Radix Ginseng effective site suppresses growth of tumor in the nude mouse significantly: low, in and the tumor stereometry of high-dose therapy group be respectively 4.88 ± 3.91cm ³, 2.89 ± 1.49cm ³, 2.03 ± 1.71cm ³, all be significantly less than the 7.52 ± 4.05cm that does not treat model group ³(P difference＜0.05,0.01) pressed the tumour inhibiting rate formula and calculated, and tumour inhibiting rate is respectively 35.1 ± 28.1%, 61.6 ± 19.8% and 72.9 ± 22.8%.Explanation has the effect of stronger inhibition leukaemia growth in lotus leukemia tumor nude mouse.

2.2 tumor tissue has in various degree degeneration and necrosis: Fig. 1 to show that treatment back tumor tissue has in various degree degeneration and necrosis in the nude mouse of Radix Ginseng effective site treatment back; 100,200mg/Kg treatment group pathologic finding shows that the tumor tissue cell has swelling, breaks; Phenomenons such as karyolysis explain in lotus leukemia tumor nude mouse to have stronger leukemia resisting action.

3. brief summary

Research proof Radix Ginseng effective site is treated the evident in efficacy of erythroleukemia tumor bearing nude mice; Not only in vitro inhibition K562 leukaemia's propagation; And also can suppress the growth of tumor body in vivo effectively; And make the leukemia tumor tissue that in various degree degeneration and necrosis arranged, thereby bring into play its antileukemie effect.

Embodiment 5 treats the evident in efficacy of monokaryon SHI-1 leukemiacell infiltration nude mouse models

1. material and method

1.1 preparation model: 4～5 all immunodeficiency in age (Nude) nude mices are produced by Shanghai Si Laike laboratory animal company, with the acute monokaryon SHI-1 leukaemia (1 * 10 of garbled high oncogenicity people ⁷Cell, people CD45 ⁺Cell) through tail vein injection in nude mouse.Pathologic finding shows that the SHI-1 cell can soak into multiple internal organs, all can see expressing human CD45 in various degree in tissues such as liver, lung, kidney, spleen, bone marrow, lump and brain after the molding ⁺The leukaemia shows that this model preparation is successful.

1.2 experiment is divided into groups and Therapeutic Method: experiment divides 6 groups; Every group 12; Comprise the nude mice matched group of not inoculating the leukaemia; Inoculation leukaemia's model nude mice is divided into 5 groups at random, and Radix Ginseng effective site (embodiment 3 makes) low dose group 50mg/Kg/d, middle dose groups 100mg/Kg/d, high dose group 200mg/Kg/d all irritate stomach treatment 30 days.Do not inoculate the nude mice matched group and do not treat model group normal saline every day filling stomach, positive drug matched group homoharringtonine (Hom) 1mg/Kg/d lumbar injection.

2. result and analysis

2.1 Radix Ginseng effective site obviously prolongs the life cycle that the monokaryon leukemia is soaked into nude mice model: after the middle and high dosage treatment; Be 50.92 ± 10.42 days, 54.75 ± 9.11 days the life cycle of leukemia nude mice; All obviously be longer than 43.83 ± 7.17 days (P all＜0.01) not treating model group, explain that it has antileukemie effect in vivo.

2.2 reduce bone marrow and peripheral blood people CD45 ⁺Leukaemia's quantity: the middle and high dosage treatment group of Flow cytometry bone marrow is expressed CD45 ⁺Cell rate is respectively 2.98 ± 1.02% and 2.37 ± 0.89%, is starkly lower than 3.94 ± 1.35 (P＜0.05 and 0.01) of not treating model group; And the high dose group peripheral blood is expressed CD45 ⁺Cell rate 24.44 ± 6.22% also is starkly lower than 33.19 ± 5.35% (P＜0.01) of not treating model group.Prompting Radix Ginseng effective site can suppress leukaemia's propagation in vivo significantly.

2.3 obviously reduce people CD45 ⁺The leukaemia shows Radix Ginseng effective site treatment back nude mice cerebral tissue people CD45 at the infiltration of cental system: Fig. 2 ⁺Leukaemia's infiltration obviously reduces, and immunohistochemical staining shows a large amount of leukemia CD45 in the brain essence of not treating model control group ⁺Cell accumulation, and middle dosage 100mg/Kg/d treatment group brain essence and epidural CD45 ⁺The quantity of cellular infiltration obviously reduces, and does not see in the brain essence infiltration is arranged, and only the brain adventitia is coated with CD45 ⁺Cell; The leukemiacell infiltration of high dose 200mg/Kg/d treatment group cerebral tissue subtracts then still less, does not find to have the infiltration of brain essence, only covers to be placed on brain on a small quantity, XIAONAO, fracture surface between brain.

3. brief summary

Research proof Radix Ginseng effective site demonstrates stronger leukemia resisting action in acute monocyte infiltration nude mice model body; The life cycle of leukemia nude mice can be prolonged effectively; Suppress SHI-1 leukaemia and breed in vivo, reduce bone marrow and peripheral blood people CD45 ⁺Leukaemia's quantity, and suppress transfer and the infiltration of this leukaemia effectively at cerebral tissue.

The effect of the multiple leukemia hemopoietic progenitor cell propagation of embodiment 6 vitro inhibition

1. material and method

1.1 the proliferation test of acute myeloid leukemia patient bone marrow leukemia hemopoietic progenitor cell of former generation: acute myeloid leukemia patient 10 examples (n=10), just control 3 examples, recur 7 examples, be diagnosed as acute myeloblastic leukemia through morphocytology and histochemical stain.Get patient's bone marrow prepare; Isolate mononuclearcell, adopt phytohaemagglutinin-leukocyte conditioned medium two step method cultivating system, the leukaemia is inoculated in semi-solid cultivating system; All do 3 multiple holes, with the support of agar as the formation of leukemia CFU-GM colony.Experimental group adds Radix Ginseng effective site and is respectively 5,10,20,50 and 100mg/L, is matched group with what do not add medicine.Cultivated 7 days, inverted microscope is observed down and counting leukemia CFU-GM colony (>=40 cells), and the colony number is got the mean in 3 multiple holes.

1.2 leukemia cell line is semi-solid and the proliferation test of 2 kinds of culture methods of liquid: selecting the red K562 of being, monokaryon is that SHI-1, grain are that the HL-60 cell is as target cell; The leukaemia is inoculated in semi-solid cultivating system; All do 3 multiple holes;, cultivated 7 days as the support that leukemia CFU-GM colony forms with agar, inverted microscope is observed down and counting leukemia CFU-GM colony (>=40 cells).In the liquid culture system,, all do 3 multiple holes, adopt the MTT method to measure the result then the drug incubation of leukaemia and variable concentrations 3 days.2 kinds of culture methods add Radix Ginseng effective site (embodiment 3 makes) 5,10,20,50 and 100mg/L respectively, are matched group with what do not add medicine.Calculate the suppression ratio of medicine, identical experiment repetition 6 times (n=6).

1.3 laser confocal microscope is observed the fluorescence intensity of BCR/ABL protein expression: Radix Ginseng

effective site

20,50 and 100mg/L were hatched the K562 cell 7 days; Get cell with the film-making of centrifugation smearing machine; Adopt BCR/ABL fusion rotein antibody to carry out association reaction; Reuse fluorescent labeling, observed result then.

2. result

2.1 suppress the effect of acute myeloid leukemia patient leukemia hemopoietic progenitor cell of former generation propagation: the effect of Radix Ginseng effective site vitro inhibition propagation is dose dependent; The effect that 20mg/L inhibition colony generates is obvious; The colony number obviously reduces, and the colony suppression ratio is 25.6 ± 14.5% (P＜0.01); 50 is more remarkable to the inhibitory action of leukemia colony with 100mg/L, and the colony suppression ratio is respectively 54.3 ± 21.8% and 68.6 ± 22.3% (P all＜0.01).

2.2 suppress the effect of the red K562 of being Leukemia Cell Proliferation

Be dose dependent 2.2.1 suppress the K562 Leukemia Cell Proliferation effectively: liquid shows that all Radix Ginseng effective site can suppress Leukemia Cell Proliferation effectively, low concentration 20mg/L onset with semi-solid the cultivation.The liquid culture mtt assay detects the suppression ratio that shows Radix Ginseng

effective site

20,50 and 100mg/L and is respectively 25.5 ± 17.7%, 38.4 ± 26.1% and 66.1 ± 13.3% (P all＜0.01).Equally, the test of semi-solid multiplication by culture also show 20,50 and the colony suppression ratio of 100mg/L be respectively 23.7 ± 2.8%, 51.6 ± 3.8% and 88.6 ± 4.6% (P equal＜0.01).

Show Radix Ginseng effective site downward modulation K562 cell proliferation specific b CR/ABL Expression of Fusion Protein 2.2.2 suppress K562 Leukemia Cell Proliferation relative specific BCR/ABL Expression of Fusion Protein: Fig. 3; The fluorescent labeling laser confocal microscope is observed increasing along with Radix Ginseng effective site dosage; The BCR/ABL expressing fusion protein that is the green fluorescence reaction in the leukaemia weakens gradually; 20, the green fluorescence reaction is lower than control cells in the 50mg/L group cell; The green fluorescence of 100mg/L group a little less than, prompting can suppress leukaemia's propagation effectively through downward modulation BCR/ABL Expression of Fusion Protein.

2.3 suppress monokaryon effectively is the effect of SHI-1 Leukemia Cell Proliferation

Be dose dependent 2.3.1 suppress monokaryon leukaemia's propagation: liquid culture MTT detects and shows Radix Ginseng effective site 20,50mg/L group; Leukemia cell inhibiting rate is respectively 16.2 ± 1.8% and 38.6 ± 5.5% (P＜0.05 and 0.01), and the 100mg/L suppression ratio reaches 72.6 ± 9.8% (P＜0.01).

Be dose dependent 2.3.2 suppress the generation of monokaryon SHI-1 leukemia CFU-GM colony: the semi-solid colony of cultivating generates test demonstration Radix Ginseng effective site low concentration 10mg/L onset, and the suppression ratio that colony generates is 15.6 ± 2.5% (P＜0.05); 20,50 and the suppression ratio of 100mg/L be respectively 36.6 ± 3.5%, 56.1 ± 12.4% and 82.3 ± 10.6% (P all＜0.01).

2.4 suppressing grain effectively is the effect of HL-60 Leukemia Cell Proliferation

Be dose dependent 2.4.1 suppress a propagation that is the leukaemia: the liquid culture mtt assay analyzes Radix Ginseng

effective site

20,50 and 100mg/L organizes the suppression ratio difference 24.6 ± 2.1%, 45.6 ± 2.5% and 61.4 ± 2.4% (P all＜0.01) to HL-60 leukaemia.

2.4.2 suppress grain is that the generation of leukemia CFU-GM colony is dose dependent: in the external semisolid colonies cultivating system; Radix Ginseng effective site obviously reduces the formation of leukemia CFU-GM colony, 20,50 and 100mg/L group to the suppression ratio of HL-60 leukemia colony respectively 19.9 ± 2.2%, 42.2 ± 2.1% and 79.5 ± 3.4% (P equal＜0.01).

3. brief summary

Semi-solid and 2 kinds of culture methods of liquid prove that all Radix Ginseng effective site not only can suppress the propagation of acute myeloid leukemia patient leukemia hemopoietic progenitor cell of former generation; And be that SHI-1, grain are that multiple leukemia cell line such as HL-60 all has and suppresses proliferation function significantly to the red K562 of being, monokaryon, its depression effect is dose dependent.The dependent interaction Study on Mechanism shows through downward modulation leukemia K 562 cell proliferation specific b CR/ABL Expression of Fusion Protein level, and brings into play its antileukemie effect.

Increase the sensitivity of leukaemia in embodiment 7 In vitro culture to chemotherapeutics

1. material and method

1.1 target cell: the red K562 of being, grain are HL-60 leukaemia.

1.2 the concertedness test with chemotherapeutics homoharringtonine (Hom) and cytosine arabinoside (Ara-c): Radix Ginseng effective site (embodiment 3 makes) and low dose of Hom or Ara-c Combined application.It is that 5 μ g/L, Ara-c final concentration are 2 μ g/L that cell culture adds the Hom final concentration, hatches 1 hour for 37 ℃, with culture fluid flush away chemotherapeutics; Counting cells is seeded in the agar semisolid colonies cultivating system, adds that Radix Ginseng effective site is respectively 0,10,20,50mg/L; All do three multiple holes, cultivated 7 days, counting leukemia colony number (>=40 cells); Get the mean in three multiple holes respectively, and calculate medicine suppression ratio, identical experiment repetition 6 times (n=6).The drug susceptibility standard is according to results reported before this laboratory, with medicine suppression ratio >=30% as standard to this susceptibility sense.

2. result

Show the sensitivity of increase leukaemia to homoharringtonine 2.1 semisolid colonies is cultivated: Fig. 4 a shows that Radix Ginseng effective site increases the inhibitory action of Hom to the leukemia cell colony; When low dose of Radix Ginseng

effective site

10,20,50mg/L and low dose of Hom Combined application; The suppression ratio that the K562 cell colony is generated is respectively 40.7 ± 5.6%, 75.3 ± 5.7%, 92.8 ± 5.0%, apparently higher than single 20.3 ± 6.5% (P all＜0.01) with the Hom matched group.

Show the sensitivity of increase leukaemia to cytosine arabinoside 2.2 semisolid colonies is cultivated: Fig. 4 b shows that Radix Ginseng effective site increases the inhibitory action of Ara-c to the leukemia cell colony; When low dose of Radix Ginseng

effective site

10,20 and 50mg/L and low dose of Ara-c Combined application; Suppression ratio to the HL-60 cell colony is respectively 42.0 ± 3.6%, 51.6 ± 4.4% and 94.7 ± 12.4%, apparently higher than single 18.5 ± 5.6% (P all＜0.01) with the Ara-c matched group.

2.3 showing, liquid culture MTT detection method increases the sensitivity of leukaemia: when low dose of Radix Ginseng effective site 20mg/L and low dose of Hom Combined application to homoharringtonine; Suppression ratio to K562, HL-60 cell proliferation is respectively 58.7 ± 7.6%, 55.5 ± 6.7%, apparently higher than single 19.5 ± 8.4%, 19.2 ± 7.4% (P all＜0.01) with the Hom matched group.

2.4 showing, liquid culture MTT detection method increases the sensitivity of leukaemia: when low dose of Radix Ginseng effective site 20mg/L and low dose of Ara-c Combined application to cytosine arabinoside; Suppression ratio to K562, HL-60 cell proliferation is respectively 58.5 ± 8.5%, 62.7 ± 10.0%, apparently higher than single 19.5 ± 6.2%, 18.4 ± 8.5% (P all＜0.01) with the Ara-c matched group.

3. brief summary

Radix Ginseng effective site not only can suppress the acute myeloid leukemia patient former generation the leukemia hemopoietic progenitor cell propagation, and be that SHI-1, grain are that multiple leukemia cell line such as HL-60 all has significant inhibition proliferation function to the red K562 of being, monokaryon.Simultaneously, low dose of Radix Ginseng effective site and low dose of chemotherapy drugs in combination are used, and can show its antileukemie concertedness to the sensitivity of chemotherapeutics through strengthening the leukaemia.

The effect of embodiment 8 induced apoptosis in leukemia cell lines

1. material and method

1.1 leukemia target cell: selecting the red K562 of being, grain is that HL-60, monokaryon are SHI-1 leukaemia.

1.2 method: Annexin V detects early stage apoptotic cell; Adopt liquid culture; Radix Ginseng effective site (embodiment 3 makes) was handled the leukaemia 36 hours, carried out fluorescently-labeled Annexin dyeing, added PI simultaneously and made double labelling (differentiating apoptosis and dead cell); Flow cytometer detects the male apoptotic cell percentage ratio of Annexin V, identical experiment repetition 5 times (n=5).The detection of non-viable apoptotic cell then adopts agarose gel electrophoresis to analyze the method for apoptosis-specific dna degradation trapezoid-shaped strips; Behind quadrat method drug treating cell; Cell lysis; Extract DNA, electrophoresis on 1.2% agarose gel, apoptosis-specific dna degradation trapezoid-shaped strips is taken by the ultraviolet light imaging system.

2. result

2.1 induce the effect of the red K562 of being apoptosis of leukemia: Radix Ginseng

effective site

50,75 and 100mg/L handled cell 36 hours; The male apoptotic cell rate of Annexin V obviously increases; Be respectively 5.27 ± 0.44%, 9.42 ± 0.70% and 15.72 ± 1.87%, be significantly higher than not 3.23 ± 0.47% (P all＜0.01) of dosing matched group.75 handle cell with 100mg/L after, the specificity trapezoid-shaped strips of the visible typical dna degradation of agarose gel electrophoresis.

2.2 inducing grain is the effect of HL-60 apoptosis of leukemia: Radix Ginseng

effective site

50,75 and 100mg/L handled cell 36 hours; The male apoptotic cell rate of Annexin V obviously increases; Be respectively 13.72 ± 1.44%, 21.6 ± 2.14% and 29.5 ± 3.01%, be significantly higher than not 1.23 ± 0.40% (P all＜0.01) of dosing matched group.Simultaneously; The typical characteristic of the visible apoptotic cell of microscopically morphological observation; Fig. 5 shows Radix Ginseng effective site induced apoptosis in leukemia cell lines dna degradation trapezoid-shaped strips, 25,50,75 and after 100mg/L handles cell, and the specificity trapezoid-shaped strips of the visible typical dna degradation of agarose gel electrophoresis.

2.3 inducing monokaryon is the effect of SHI-1 apoptosis of leukemia: Radix Ginseng

effective site

50,75 and 100mg/L treatment S HI-1 cell 36 hours; The male apoptotic cell rate of Annexin V obviously increases; Be respectively 8.61 ± 0.45%, 12.60 ± 1.08% and 18.40 ± 0.56%, all apparently higher than 3.24 ± 0.82% (P all＜0.01) of dosing matched group not.

3. brief summary

Radix Ginseng effective site is external, and can to induce the red K562 of being cell, grain effectively be that HL-60 and monokaryon are the apoptosis of SHI-1 leukemia germinal cell; Flow cytometry shows that the male apoptotic cell rate of Annexin V obviously increases; The typical dna degradation specificity of apoptosis trapezoid-shaped strips appears in agarose gel electrophoresis, proves that all Radix Ginseng effective site induced apoptosis in leukemia cell lines is one of its antileukemie important effect of performance.

The effect of embodiment 9 inducing leukemia germinal cells differentiation

1. material and method

1.1 target cell: selecting the red K562 of being, monokaryon is SHI-1 leukaemia.

1.2 cell culture and drug treating: liquid culture adds the Radix Ginseng effective site (embodiment 3 makes) 10,20 and 3 weeks of 40mg/L incubated cell of variable concentrations, is contrast with the cell of not dosing.Cultivate the variation (n=6) of back Flow cytometry SHI-1 cell differentiation phenotype.Get and cultivate the back cell, immunocytochemical stain and the fluorescent labeling laser co-focusing microscopy observation relevant γ-globin of differentiation and β-catenin albumen, and the expression of PU.1 and GATA-1 transcription factor with the film-making of centrifugation smearing machine.Identical experiment repeats 3 times.

1.3 the criterion of immunocytochemical stain cell positive degree: the positive cell of brown granular shape thing occurs with endochylema, "-" is no positive reactant; "+" is light brown cloud is the weak positive; " +++" is dark brown grumeleuse shape is strong positive; " ++ " is between "+" and " +++".Identical experiment repeats 3 times, and positive percentage is got average.

2. result

2.1 induce the effect of the red K562 of being leukaemia differentiation

2.1.1 induce the expression of the relevant γ-globin of differentiation to increase: Fig. 6 shows that Radix Ginseng effective site raises the expression of the relevant γ-globin of differentiation;

Low concentration

10,3 weeks of 20mg/L function cells; Immunohistochemical staining shows that it is 41.1 ± 3.7% that the relevant γ of differentiation-globin is expressed " +++" strong positive cell; 62.0 ± 5.6%, apparently higher than 21.3 ± 7.3% (P is all＜0.01) of dosing matched group not.Simultaneously, the laser co-focusing microscopy also obtains equifinality, and K562 cell itself is expressed γ-globin; But a little less than the fluorescence, and behind drug effect, strengthening to some extent appears in the green fluorescence reaction; Compare with not dosing control cells, 10,20 and the fluorescence reaction of 40mg/L group obviously strengthen.

2.1.2 induce the expression of the relevant GATA-1 transcription factor of differentiation to increase: low dose of Radix Ginseng effective site 10,20mg/L handle cell after 3 weeks; Immunohistochemical staining shows that the relevant GATA-1 transcription factor expression of differentiation " +++" strong positive cell is 42.5 ± 4.7%, 40.8 ± 10.5%, apparently higher than 23.6 ± 3.2% (P all＜0.01) of matched group.Simultaneously; The laser co-focusing microscopy also obtains equifinality, and K562 cell itself is expressed the GATA-1 transcription factor protein, but a little less than the fluorescence; And behind drug effect; Strengthening to some extent appears in red fluorescence reaction, compares 10, the red fluorescence significant reaction of 20mg/L group strengthens with not dosing control cells.

2.2 induce the effect of monokaryon SHI-1 leukaemia differentiation

2.2.1 the relevant phenotype CD11b positive cell rate of external evoked mononuclear cell differentiation increases: Fig. 7 shows that Radix Ginseng effective site induces the relevant CD11b phenotype positive cell rate of SHI-1 cell differentiation to increase;

Low concentration medicine

10,3 weeks of 20mg/L function cells; Flow cytometry shows that the relevant phenotype CD11b positive cell rate of mononuclear cell differentiation is respectively 8.1 ± 0.6% and 11.1 ± 2.9%, apparently higher than 3.4 ± 0.5% (P＜0.05 and 0.01) of dosing matched group not.The positive cell rate of 40mg/L group is 14.6 ± 3.7%, also apparently higher than matched group (P＜0.01).

2.2.2 the relevant PU.1 of external evoked differentiation and β-catenin protein expression increase:

immunocytochemical stain demonstration

10,3 weeks of 20mg/L function cells; The cell rate of the relevant PU.1 protein expression of differentiation " +++" strong positive is 44.2 ± 5.6%, 60.8 ± 8.5%, apparently higher than 21.6 ± 3.5% (P all＜0.01) of dosing matched group not; The cell rate of the relevant β of differentiation-catenin protein expression " +++" strong positive is 41.9 ± 8.6%, 56.0 ± 4.5% simultaneously, also apparently higher than 20.2 ± 6.3% (P all＜0.01) of dosing matched group not.

3. brief summary

It is the differentiation of SHI-1 leukemia germinal cell that Radix Ginseng effective site can be induced the red K562 of being cell, monokaryon, and Flow cytometry phenotypic differentiation positive cell rate all obviously raises; Immunocytochemical stain shows that with the laser co-focusing microscopy the relevant γ-globin of differentiation and β-catenin protein expression level increase; The expression of same GATA-1 and PU.1 transcription factor also increases, and proves all that Radix Ginseng effective site can be broken up through the inducing leukemia germinal cell bring into play its antileukemie effect.

The relevant kinase whose expression of MAPK signal pathway multiple protein of embodiment 10 downward modulation Leukemia Cell Proliferation

1. material and method

1.1 target cell: get the red K562 of being cell and KG-1a leukemic stem cells; The KG-1a cell is so kind as to give by Australian professor B.H.Chong; This cell itself is very original; 99.4% cell is the CD34 positive, and GM-CSF is insensitive to hemopoietic growth factor, and also insensitive to the effect of inducing differentiation agent TPA by force.

1.2 drug treating cell and extraction plasmosin: 5 kinds of albumen kinases AKT-1, AKT-2, ERK-2, MEK-1 and MEK-2 selecting the high expressed of leukaemia own; Observe the inhibitory action of Radix Ginseng effective site to these propagation related protein kinases; Handled cell 7 days through Radix Ginseng effective site (embodiment 3 makes) 20,50,100mg/L; Gather in the crops the cell of drug treating group and matched group respectively; Extract plasmosin, adopt the Western immunoblotting to analyze above-mentioned 5 kinds of kinase whose expressions of albumen, identical experiment repetition 3 times.

2. result

Show the kinase whose expression of Radix Ginseng effective site downward modulation MAPK signal pathway major protein 2.1 reduce the expression of the relevant AKT-1 of KG-1a cell proliferation, AKT-2, ERK-2, MEK-1 and MEK-2 protein kinase: Fig. 8 to some extent; 50, after the 100mg/L function cells; AKT-1 protein band density 1429.3 ± 136.5,1425.3 ± 136.1 is lower than 2692.3 ± 216.3 of matched group; AKT-2 protein band density 329.9 ± 31.4,307.7 ± 30.2 is lower than 838.0 ± 81.3 of matched group; ERK-2 protein band density 610.9 ± 61.3,563.8 ± 51.2 is lower than 1985.5 ± 189.9 of matched group; MEK-1 protein band density 1013.1 ± 102.1,997.4 ± 92.7 is lower than 2815.5 ± 282.6 of matched group; MEK-2 protein band density 1363.5 ± 125.8,1272.5 ± 124.5 is lower than 2594.5 ± 244.3 of matched group.

Show the kinase whose expression of Radix Ginseng effective site downward modulation MAPK signal pathway major protein 2.2 reduce the expression of the relevant AKT-1 of K562 cell proliferation, AKT-2, ERK-2, MEK-1 and MEK-2 protein kinase: Fig. 8 to some extent; 50, after the 100mg/L drug treating; AKT-1 protein band density 2050.4 ± 206.5,217.7 ± 26.4 is lower than 2323.2 ± 226.3 of matched group; AKT-2 protein band density 409.2 ± 41.4,12.24 ± 1.5 is lower than 652.6 ± 61.8 of matched group; ERK-2 protein band density 3015.2 ± 331.3,681.1 ± 67.3 is lower than 3478.8 ± 309.3 of matched group; MEK-1 protein band density 350.3 ± 34.1,35.0 ± 3.4 is lower than 770.0 ± 72.5 of matched group; MEK-2 protein band density 2383.4 ± 225.6,676.0 ± 64.3 is lower than 2356.8 ± 235.2 of matched group.

3. brief summary

Radix Ginseng effective site can suppress the expression of the relevant MAPK signal pathway multiple protein kinases AKT-1 of leukaemia's internal breeding, AKT-2, ERK-2, MEK-1 and MEK-2 significantly; Prove that one of its antileukemie mechanism of action is through the kinase whose expression of downward modulation propagation coherent signal approach multiple protein; The conduction of blocking pernicious signal, thereby inhibition leukaemia's abnormality proliferation.

Embodiment 11 Radix Ginseng effective sites are to other non-hematopoietic system cancer cell inhibiting effect

1. material and method

1.1 target cell: L929 becomes fibrous tumours cell, ECV-304 tumor endothelial cell.

1.2 experimental technique: external liquid culture MTT detection method; Colony generates test method(s) and observes the inhibitory action of Radix Ginseng effective site (embodiment 3 makes) to solid tumor cell; Experimental group adds Radix Ginseng effective site and is respectively 5,10,20,50 and 100mg/L; Not add medicine is matched group, all does 3 multiple holes.Cultivated 7 days, inverted microscope is observed down and counting solid tumor cell colony (>=40 cells).Identical experiment repetition 5 times (n=5).

2. result

Show that Radix Ginseng effective site suppresses the effect that the L929 cell colony generates 2.1 suppress the effect that L929 becomes the fibrous tumours cell colony to generate: Fig. 9; Dosing L929 control cells colony form is not main with tight type; And Radix Ginseng

effective site

10,20,50,100mg/L effect back colony number obviously reduces; Cell clump increases, and the colony form changes to the evacuation type by tight type, and the colony suppression ratio is respectively 25.0 ± 5.3%, 41.7 ± 1.0%, 76.6 ± 3.1% (P all＜0.01).When drug level is increased to 100mg/L, has basically no colony and form.Explain that Radix Ginseng effective site has significant inhibition proliferation function to the L929 cell, is dose dependent.

2.2 suppress the effect that L929 becomes the fibrous tumours cell proliferation: liquid culture MTT detects the increase that shows along with Radix Ginseng effective fraction medicine concentration; The OD value reduces gradually, and the suppression ratio of different

pharmaceutical concentration

10,20,50 and 100mg/L is respectively 14.1 ± 5.0%, 21.4 ± 8.1%, 37.6 ± 3.1% and 57.2 ± 3.5% (P＜0.05 or 0.01).The result of prompting MTT colorimetric determination and colony-forming test is consistent, proves that all Radix Ginseng effective site has the effect that suppresses the L929 cell proliferation, is dose dependent.In view of becoming fiber L929 cell is that growth of tumour cell provides support, and The above results prompting Radix Ginseng effective site is through being suppressed to the growth of fibrous tumours cell, and has antineoplastic action.

2.3 suppress the effect of ECV-304 tumor endothelial cell propagation: liquid culture MTT detects the increase that shows along with Radix Ginseng effective fraction medicine concentration, and the OD value reduces gradually, and inhibitory action obviously strengthens, and is dose dependent.10,20,50, the suppression ratio of 100mg/L is respectively 26.7 ± 7.1%, 36.7 ± 2.6%, 43.7 ± 5.0%, 68.6 ± 2.8% (P all＜0.01).In view of endotheliocyte passes through to form blood vessel, and provide growth of tumour cell required nutrition, The above results prompting Radix Ginseng effective site passes through to suppress the growth of tumor endothelial cell, thereby has antineoplastic action.

3. brief summary

Radix Ginseng effective site also has significant inhibitory effect to other solid tumor cell, comprises that L929 becomes fibrous tumours cell, ECV-304 tumor endothelial cell etc.In view of becoming the fibrous tumours cell is that the growth of tumor cell provides support; Endotheliocyte provides growth of tumour cell required nutrition through forming blood vessel; Therefore point out Radix Ginseng effective site except above-mentioned antileukemie effect, also have the effect of anti entity tumour simultaneously.

Claims

1. Radix Ginseng effective site is characterized in that described Radix Ginseng effective site mainly contains the chemical compound of following weight portion:

The preparation of described Radix Ginseng effective site comprises the steps:

(2) A liquid is handled through precipitate with ethanol or water precipitating, separate supernatant concentration to the concentrated solution crude drug ratio that obtains to reach 1: 0.25～2.5, be designated as B liquid;

(3) chromatography

2. the method for preparing of a Radix Ginseng effective site as claimed in claim 1 comprises the steps:

(1) extract: get Radix Ginseng and cut into segment, with aquiferous ethanol or contain pure water and extract, it is centrifugal or leave standstill fully to extract the back, centrifugal liquid or supernatant, relative density is 0.8～1.3 when being concentrated into 80 ℃, is designated as A liquid;

(2) A liquid adopts 2.1 or 2.2 described methods to handle:

2.2 water precipitating: get A liquid, add the water that is equivalent to long-pending 1～5 times of A liquid, leave standstill water precipitating, draw the water precipitating supernatant then; Residue washs with suitable quantity of water, and the centrifugal back of washing liquid merges with supernatant, be concentrated into concentrated solution crude drug ratio to reach 1: 0.25～2.5, be designated as B liquid;

(3) chromatography

3.1 separate for the first time

3.1.2 upper prop: get C liquid, last macroporous adsorptive resins all gets in the macroporous resin column until C liquid;

3.2 separate for the second time

3.2.2 upper prop: get E liquid, last macroporous adsorptive resins all gets in the macroporous adsorptive resins until E liquid;

3. the method for preparing of Radix Ginseng effective site as claimed in claim 2, it is one of following to it is characterized in that described macroporous adsorptive resins is selected from: D ₁₀₁, DA ₂₀₁, D ₁₀₁+ DA ₂₀₁, PHD-100, PHD-200.

4. the method for preparing of Radix Ginseng effective site as claimed in claim 2 is characterized in that in the described step (2), after A liquid adopts 2.1 or 2.2 described methods to handle, make concentrated solution crude drug ratio reach 1: 0.5～2.5.

5. the method for preparing of Radix Ginseng effective site as claimed in claim 2, the blade diameter length ratio that it is characterized in that step 3.1.2 or the described macroporous adsorptive resins of 3.2.2 independently is 1: 1～10 separately, the flow of C liquid and E liquid independently is 0.25～2.5SV separately; Among said step 3.1.3,3.1.4,3.1.5,3.1.6,3.2.3 or the 3.3.4, washing or eluting flow independently are 0.25～2.5SV separately.

6. the method for preparing of Radix Ginseng effective site as claimed in claim 2 is characterized in that said method for preparing carries out according to following steps:

(2) A liquid adopts 2.1 or 2.2 described methods to handle:

(3) chromatography

3.1 separate for the first time

3.2 separate for the second time

7. the application of Radix Ginseng effective site as claimed in claim 1 in the preparation anti-leukemia medicine.

8. Radix Ginseng effective site as claimed in claim 1 suppresses the application in the solid tumor cell medicine in preparation.

9. application as claimed in claim 8 is characterized in that described solid tumor cell is that L929 becomes fibrous tumours cell or ECV-304 tumor endothelial cell.