CN109354629A - It is a kind of neutrality notoginseng polysaccharide method for extraction and purification and application - Google Patents

Abstract

The present invention relates to a kind of method for extraction and purification of neutral notoginseng polysaccharide and applications, using Radix Notoginseng waste residue as raw material, using water extraction and alcohol precipitation method, obtain Radix Notoginseng Thick many candies, purify Radix Notoginseng Thick many candies, it obtains neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V eluent, freeze-drying obtains 5 samples.It studies neutrality notoginseng polysaccharide PNPS I and the therapeutic effect of bone marrow suppression and the protective effect of hypoimmunity mice and combined chemotherapy to tumor-bearing mice is caused to cyclophosphamide.As a result, it has been found that only neutrality notoginseng polysaccharide PNPS I has inhibiting effect to the proliferation of 7 kinds of tumour cells；Neutral notoginseng polysaccharide PNPS I causes bone marrow suppression and the protective effect of hypoimmunity mice to cyclophosphamide；Neutral notoginseng polysaccharide PNPS I and the tumour inhibiting rate of cyclophosphamide combined medication group are higher than cyclophosphamide independent medication group.Neutral notoginseng polysaccharide PNPS I can be used as the new drug for the treatment of bone marrow suppression, hypoimmunity and combination chemotherapy tumour and the natural material of functional health-care food.

Description

It is a kind of neutrality notoginseng polysaccharide method for extraction and purification and application

Technical field

The invention belongs to field of medicaments, it is related to method for extraction and purification and the application of a kind of neutral notoginseng polysaccharide.

Background technique

Radix Notoginseng is Araliaceae Panax herbaceos perennial also known as radix notoginseng, pseudo-ginseng etc., and the genuine place of production is Yunnan text Mountain.Three seventh is that China's valuable ingredients of traditional Chinese medicine, effective component include saponin(e, dencichine, flavones, polysaccharide, carbene alcohol, amino acid, rouge Fat acid and cyclic peptide etc..Saponin constituent is concentrated mainly on to the research and utilization of compound components of panax notoginseng at present, after saponin constituent extracts, Other effective components are taken as waste residue and liquid to be discarded.

The utilization and use of saponin active ingredient are concentrated mainly on to the research of Radix Notoginseng at present, to other effective components Research is insufficient.The long-term cultivated area of Radix Notoginseng of the genuine place of production Yunnan mountain of papers of Radix Notoginseng has reached 120,000 mu or so, 9000 tons of yield.Often Year for extract the Radix Notoginseng of arasaponin to be about 1400 tons, studies have shown that after saponin extraction, notoginseng polysaccharide in pseudo-ginseng slag, Measuring content of the notoginseng polysaccharide in Radix Notoginseng is about 2%, discards nearly 30 tons of notoginseng polysaccharide every year.At present to the research of notoginseng polysaccharide Report is concentrated mainly on raising studies on immune function.Therefore, it is very must that exploitation, which can give full play to the product of notoginseng polysaccharide effect, It wants.

The patent of invention that number of patent application is 201210058942.6 discloses a kind of system of the notoginseng polysaccharide of physiological activity Preparation Method and its application.This method application water mentions, resin column chromatography (purifying absorption resin and ion exchange resin on Aqueous extracts Including LSA-8, LSA-21, LSA-30, LSA-10, LSA-AS, LSA-100, LX-20, LX-68, OU-29, LAR-714, D- The resin of 101 models), purified water be eluent, eluent crosses the ultrafiltration membrane of molecular weight 300-500, after concentration plus ethanol precipitation, Notoginseng polysaccharide will be spray-dried to obtain after precipitating dissolution.Experimental study: notoginseng polysaccharide enhance trauma in rat immune function effect and Facilitation of the notoginseng polysaccharide to immune function of mice；The influence that notoginseng polysaccharide grows animal-transplanted tumor；But in the invention The content of notoginseng polysaccharide is low, respectively only 68.76%, 67.34% and 70.24%, does not use DEAE Sepharose Fast Flow anion exchange chromatography, step of not dialysing do not use Freeze Drying Technique.Immunization is adjusted to be directed to Trauma in rat and normal mouse, anti-tumour phological activity only use notoginseng polysaccharide, thin for tumour without reference to notoginseng polysaccharide The influence of born of the same parents' in-vitro multiplication；The protection for causing immunocompromised and bone marrow suppression mouse for chemical drug without reference to notoginseng polysaccharide, does not have It is related to therapeutic effect of the joint chemical drug to tumour.

The patent of invention that number of patent application is 201210481377.4 discloses a kind of notoginseng polysaccharide extract and its preparation Method and preparation and application, the notoginseng polysaccharide extract be using extract the waste residue after arasaponin it is extracted as raw material, Concentration, purification, dry, crushing, checking procedure obtain, and wherein notoginseng polysaccharide content is not less than 15%.The notoginseng polysaccharide mentions The preparation method for taking object includes extracting, concentration, purification, drying, crushing, checking procedure, specifically includes: will extract arasaponin Waste residue afterwards is added purified water and extracts, and filters, and ethanol precipitation is added in concentration, filters, and purification is dried to obtain object dry product； The notoginseng polysaccharide extract is added pharmaceutic adjuvant and powder, capsule, granule, tablet or pill is made；The Radix Notoginseng Polyoses extract is used to prepare the application in anti-tumor drug and strengthen immunity drug.But the invention is only preliminary extraction, Do not carry out chromatographic purifying, in terms of concrete application also without reference to.

Number of patent application is pure for the extraction that the patent of invention of CN201710459344.2 discloses a kind of neutral notoginseng polysaccharide Change method and promotion cell Proliferation pharmacology activity research and application, the invention patent is also the inventor institute Shen of the invention patent Report, the method for extraction and purification of neutral polysaccharide is identical, but related pharmacological activity and application are different.

In terms of the application of neutrality notoginseng polysaccharide is concentrated mainly on health food at present, otherwise application study is less.

Summary of the invention

For overcome the deficiencies in the prior art, the present invention provides the method for extraction and purification of neutral notoginseng polysaccharide a kind of and answers With, it is a kind of neutrality notoginseng polysaccharide method for extraction and purification and to bone marrow suppression, immunocompromised and combination chemotherapy tumour etc. The research and application of aspect.Using the Radix Notoginseng waste residue for the notoginsenoside extracted in industrial production as raw material, using water extraction and alcohol precipitation method, Radix Notoginseng Thick many candies are obtained, Radix Notoginseng Thick many candies are purified using DEAE Sepharose Fast Flow anion exchange chromatography, point Not with 0,0.1,0.2,0.3,0.4mol/L NaCl solution gradient elution, 5 groups of eluents are obtained: neutral notoginseng polysaccharide PNPS I And acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V eluent, by being concentrated in vacuo, dialysing, freezing is dry It is dry to obtain 5 samples.Investigate influence of the above-mentioned 5 kinds of polysaccharide to 7 kinds of Proliferation of Tumor Cells In Vitro；Investigate neutral notoginseng polysaccharide PNPS I causes bone marrow suppression and the protective effect of hypoimmunity mice and combined chemotherapy to make the treatment of tumor-bearing mice cyclophosphamide With.As a result, it has been found that only neutrality notoginseng polysaccharide PNPS I has inhibiting effect to the proliferation of 7 kinds of tumour cells；Neutral notoginseng polysaccharide PNPS I causes bone marrow suppression and the protective effect of hypoimmunity mice to cyclophosphamide；Neutral notoginseng polysaccharide PNPS I and ring phosphinylidyne The tumour inhibiting rate of amine drug combination group is higher than cyclophosphamide independent medication group.Neutral notoginseng polysaccharide PNPS I can be used as treatment marrow suppression The natural material of system, the new drug of hypoimmunity and combination chemotherapy tumour and functional health-care food.The present invention provides Neutral notoginseng polysaccharide preparation method, it is easy to operate, it is reproducible, can be used for industrial production.Three described in the application Seven polysaccharide include Radix Notoginseng Thick many candies, notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, At least one of PNPS V or several.

The present invention provides the following technical solutions:

A kind of method for extraction and purification of neutrality notoginseng polysaccharide, uses the Radix Notoginseng waste residue for extracting notoginsenoside for raw material, It is characterized in that: the following steps are included:

(1) it takes Radix Notoginseng to be ground into coarse powder, is extracted, filtered with 70% ethyl alcohol, extracted, filtered 4 times, obtain Radix Notoginseng waste residue, done It is dry；

(2) water extraction and alcohol precipitation method extracts Radix Notoginseng Thick many candies；

(3) DEAE Sepharose Fast Flow anion-exchange chromatography and dialysis purify Thick many candies, freeze-drying Method obtains neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acid Radix Notoginseng Polysaccharide PNPS IV, acid notoginseng polysaccharide PNPS V freeze-dried powder.

The present invention also provides the neutral notoginseng polysaccharides of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide to control in preparation It treats, the application in inhibition Proliferation of Tumor Cells In Vitro drug.Neutral notoginseng polysaccharide is neutrality notoginseng polysaccharide PNPS I.

The present invention also provides above-mentioned neutral notoginseng polysaccharides in preparation treatment, inhibits or promote Proliferation of Tumor Cells In Vitro drug In application, the tumour cell include at least liver cancer cells, sarcoma cell, lymphoma cell, humanized's breast cancer cell, cream Adenocarcinoma cell, melanoma cells, colon cancer cell.

Preferably, neutral notoginseng polysaccharide is neutrality notoginseng polysaccharide PNPS I.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in preparation treatment, inhibition Proliferation of Tumor Cells In Vitro drug In application, the tumour cell include at least liver cancer cells H22, sarcoma cell S180, lymphoma cell YAC-1, humanized Breast cancer cell MCF-7, breast cancer cell 4T1, melanoma cells B16, colon cancer cell CT26.WT.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in preparation treatment, inhibition liver cancer cells H22 in-vitro multiplication medicine When application in object.Here neutral notoginseng polysaccharide include PNPS, PNPS I, PNPS IV, PNPS administration concentration be greater than 0.07mg/mL, and/or, it is in inhibiting effect to the growth of H22 cell when the administration concentration of PNPS I is greater than 0.56mg/mL, and/ Or, the administration concentration of PNPS IV in 0.07~0.28mg/mL, has a slight proliferation, concentration 1.12~ It is in inhibiting effect when 2.24mg/mL.

Any of the above-described scheme is preferably, application of the neutral notoginseng polysaccharide in terms of promoting liver cancer cells H22 proliferation, in turn For scientific research.Here neutral notoginseng polysaccharide refers to PNPS IV, and the administration concentration of PNPS IV is in 0.07~0.28mg/mL When, there is slight proliferation.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in preparation treatment, inhibition sarcoma cell S180 in-vitro multiplication When application in drug.Here neutral notoginseng polysaccharide includes PNPS, PNPS I, PNPS II and PNPS I-A, and PNPS is to S180 Cell growth is in inhibiting effect；The administration concentration of PNPS I is given in 0.035~2.24mg/mL, PNPS II and PNPS I-A Concentration is in inhibiting effect to the growth of S180 cell in 0.28~2.24mg/mL.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide increases in vitro in preparation treatment, inhibition lymphoma cell YAC-1 When growing the application in drug.Here neutral notoginseng polysaccharide includes PNPS, PNPS I, PNPS II, PNPS III, PNPS IV, When concrete application, PNPS administration concentration grows YAC-1 cell inhibited in 0.28~2.24mg/mL；PNPS I Administration concentration in 0.56~2.24mg/mL, the administration concentration of PNPS II is in 1.12~2.24mg/mL, PNPS III Administration concentration in 0.28~2.24mg/mL, to YAC-1 cell growth be in inhibiting effect；；The administration concentration of PNPS IV exists It is in inhibiting effect to the growth of YAC-1 cell when 0.56~2.24mg/mL.

Any of the above-described scheme is preferably, application of the neutral notoginseng polysaccharide in terms of promoting lymphoma cell YAC-1 proliferation, And then it is used for scientific research.Here neutral notoginseng polysaccharide includes PNPS, PNPS IV, PNPS I-A, when concrete application, PNPS Administration concentration has proliferation, PNPS IV and PNPS I- in 0.0175~0.14mg/mL, to the growth of YAC-1 cell The administration concentration of A has slight proliferation in 0.0175~0.28mg/mL, to the growth of YAC-1 cell.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in preparation treatment, inhibition humanized's breast cancer cell MCF-7 Application in in-vitro multiplication drug, neutral notoginseng polysaccharide here include PNPS I, PNPS I-A.When concrete application, PNPS I Administration concentration in 0.035~2.24mg/mL, to MCF-7 cell grow it is inhibited；The administration of PNPS I-A is dense Degree is in inhibiting effect to the growth of MCF-7 cell in 0.035~2.24mg/mL.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is promoting humanized's breast cancer cell MCF-7 in-vitro multiplication The application of aspect, and then it is used for scientific research.Here neutral notoginseng polysaccharide includes PNPS, PNPS II, PNPS III, PNPS IV, when concrete application, PNPS administration concentration has the growth of MCF-7 cell and promotees proliferation and make in 0.0175~1.12mg/mL With；PNPS II administration concentration is in 0.28~2.24mg/mL, or, PNPS III administration concentration is in 0.56~2.24mg/mL When, or, PNPS IV administration concentration has proliferation in 0.070~2.24mg/mL, to the growth of MCF-7 cell.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in preparation treatment, inhibition breast cancer cell 4T1 in-vitro multiplication Application in drug.Here neutral notoginseng polysaccharide includes PNPS, PNPS I, PNPS II, PNPS IV, when concrete application, The administration concentration of PNPS and PNPS II grows 4T1 cell inhibited in 0.56~2.24mg/mL；PNPS I 4T1 cell is grown with PNPS III inhibited；PNPS IV administration concentration is in 0.56~2.24mg/mL, to 4T1 Cell growth is inhibited.

Any of the above-described scheme is preferably, neutral notoginseng polysaccharide answering in terms of promoting breast cancer cell 4T1 in-vitro multiplication With, and then it is used for scientific research.Here neutral notoginseng polysaccharide includes PNPS, PNPS II, PNPS IV, PNPS I-A, specifically Make in application, the administration concentration of PNPS and PNPS II in 0.0175~0.28mg/mL, has the growth of 4T1 cell to promote to be proliferated With；PNPS IV administration concentration has proliferation in 0.0175~0.14 mg/mL, to the growth of 4T1 cell；PNPS I-A Administration concentration in 0.0175~0.56mg/mL, to 4T1 cell growth have proliferation.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide increases in vitro in preparation treatment, inhibition melanoma cells B16 Grow the application in drug.Here neutral notoginseng polysaccharide include PNPS, PNPS I, PNPS II, PNPS III, PNPS IV, PNPS I-A, when concrete application, for PNPS administration concentration in 0.070~2.24mg/mL, the administration concentration of PNPS I is 0.14 When~2.24mg/mL, PNPS II administration concentration in 0.56~2.24 mg/mL, PNPS III administration concentration 0.070~ When 2.24mg/mL, PNPS IV and PNPS I-A administration concentration are in 0.14~2.24mg/mL, to the growth of B16 cell in inhibition Effect.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is external in preparation treatment, inhibition colon cancer cell CT26.WT Application in hyperproliferation agent.Here neutral notoginseng polysaccharide includes PNPS, PNPS I, PNPS III, PNPS IV, concrete application When, PNPS administration concentration grows CT26.WT cell inhibited in 0.56~2.24 mg/mL；PNPS I's gives Concentration grows CT26.WT cell inhibited in 1.12~2.24mg/mL；PNPS III administration concentration exists When 2.24mg/mL, CT26.WT cell is grown inhibited；PNPS IV administration concentration in 1.12~2.24mg/mL, CT26.WT cell is grown inhibited.

Any of the above-described scheme is preferably, and neutral notoginseng polysaccharide is in terms of promoting colon cancer cell CT26.WT in-vitro multiplication Application, and then be used for scientific research.Here neutral notoginseng polysaccharide include PNPS, PNPS II, PNPS III, PNPS IV, PNPS I-A, when concrete application, PNPS administration concentration has the growth of CT26.WT cell in 0.0175~0.28mg/mL Proliferation；PNPS II administration concentration has the growth of CT26.WT cell and promotees proliferation work in 0.070~2.24 mg/mL With；PNPS III administration concentration has proliferation in 0.0175~0.56mg/mL, to the growth of CT26.WT cell； PNPS IV administration concentration has proliferation in 0.035~0.56mg/mL, to the growth of CT26.WT cell；PNPS I-A Administration concentration in 0.0175~2.24mg/mL, to MCF-7 cell growth have proliferation.

The present invention also provides the neutral notoginseng polysaccharides of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide to control in preparation Treat the application in bone marrow suppression drug.

The present invention also provides the neutral notoginseng polysaccharides of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide to mention in preparation Application in high immunity and marrow DNA drug.

The present invention also provides the neutral notoginseng polysaccharides of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide to mention in preparation Application in high tumour inhibiting rate, raising immunity and routine blood indexes drug.

The present invention also provides above-mentioned neutral notoginseng polysaccharides to merge bone marrow suppression thymus gland, spleen system to preparation treatment immunocompromised Application in number, promotion bone marrow suppression hematopoiesis function drug.

Quantity drug, promotion marrow the present invention also provides notoginseng polysaccharide and Radix Notoginseng Thick many candies in preparation raising immunocyte Inhibit the application in hematopoiesis function drug.

The present invention also provides neutral notoginseng polysaccharides in the drug that preparation improves immunosupress macrophage phagocytic index Using.

Immunocompromised, which is improved, in preparation the present invention also provides neutral notoginseng polysaccharide merges bone marrow suppression serum hemolysis cellulose content Drug in application.

The present invention also provides Radix Notoginseng Thick many candies to improve the drug that immunocompromised merges bone marrow suppression NK cell activity in preparation In application.

Immunocompromised, which is improved, in preparation the present invention also provides neutral notoginseng polysaccharide merges bone marrow suppression spleen lymphocyte proliferation The application of power drug.

Immunocompromised, which is improved, in preparation the present invention also provides notoginseng polysaccharide merges answering in bone marrow suppression myelosis drug With.Here notoginseng polysaccharide includes neutral notoginseng polysaccharide, Radix Notoginseng Thick many candies.

The present invention also provides PNPS I to improve the application in tumour cell thymus index value drug, PNPS I preparation in preparation It is used in combination after drug with cyclophosphamide CTX and improves thymus index value.Tumour cell includes murine hepatocarcinoma cell H22.

The present invention also provides improve tumour cell thymus index value drug in preparation after PNPS I and cyclophosphamide CTX mixing In application.

The present invention also provides improve answering in tumour tumour inhibiting rate drug in preparation after PNPS I and cyclophosphamide CTX mixing With.

The present invention also provides PNPS I to improve the application in tumour tumour inhibiting rate drug in preparation, PNPS I prepare after drug with Raising tumour inhibiting rate is used in combination in cyclophosphamide CTX.Tumour cell includes murine hepatocarcinoma cell H22.

The present invention also provides reduce in tumour phagocytic index value drug after PNPS I and cyclophosphamide CTX mixing in preparation Using.Tumour includes at least liver cancer.Here PNPS I is high dose 300mg/kg.

The present invention also provides improve in tumour phagocytic index value drug after PNPS I and cyclophosphamide CTX mixing in preparation Using.Here PNPS I is low dosage 100mg/kg.

The present invention also provides improve H22 lotus knurl monocytes/macrophages carbon in preparation after PNPS I and cyclophosphamide CTX mixing Clean up the application in ability drug.Tumour includes at least rat liver cancer.

The present invention also provides improve lotus knurl serum hemolysin in preparation after PNPS I and cyclophosphamide CTX mixing to generate drug In application.Here PNPS I is low dosage 100mg/kg.Tumour include at least rat liver cancer,

The present invention also provides PNPS I to improve the application in H22 lotus knurl serum hemolysin generation drug in preparation.Tumour is extremely It less include rat liver cancer.

The present invention also provides neutral notoginseng polysaccharides to improve tumor blood leucocyte, red blood cell, lymphocyte concentration in preparation Or the application in reduction hemoglobin concentration drug.

The present invention also provides PNPS I to improve H22 lotus knurl blood leucocyte, red blood cell, lymphocyte acute drug in preparation In application.Tumour includes at least rat liver cancer.

The present invention also provides improve H22 lotus knurl blood leucocyte, red thin in preparation after PNPS I and cyclophosphamide CTX mixing Application in born of the same parents, lymphocyte acute drug.

The present invention also provides PNPS I to reduce the application in H22 lotus knurl Blood Hemoglobin Concentration drug in preparation.

It is dense the present invention also provides H22 lotus knurl blood-hemoglobin is reduced in preparation after PNPS I and cyclophosphamide CTX mixing Spend the application in drug.

The present invention also provides the neutral notoginseng polysaccharides of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide to mention in preparation Application in the Therapeutic Results of Combination Chemotherapy drug of high tumour.

The present invention also provides a kind of neutral notoginseng polysaccharide applications of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide In pharmaceutical intermediate.

The present invention also provides a kind of neutral notoginseng polysaccharide applications of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide In health food.

The present invention also provides a kind of neutral notoginseng polysaccharide applications of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide In cosmetics.

The present invention, using water extraction and alcohol precipitation method, obtains neutrality using the Radix Notoginseng waste residue for extracting notoginsenoside in producing as raw material Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide content can reach 93.6%.It is handed over using DEAE Sepharose Fast Flow anion Chromatographic purifying Radix Notoginseng Thick many candies are changed, 5 components are obtained, through dialysis purification, neutral notoginseng polysaccharide PNPS I is obtained after freeze-drying And acid notoginseng polysaccharide PNPS II, PNPS III, 5 PNPS IV, PNPS V samples.Measure Radix Notoginseng Thick many candies, neutral Radix Notoginseng Polysaccharide PNPS I and acid notoginseng polysaccharide II-V (acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV's, PNPS V) contains Amount and molecular weight ranges.The present invention also provides a kind of neutral Radix Notoginseng of the method for extraction and purification preparation of above-mentioned neutral notoginseng polysaccharide is more Sugar is to bone marrow suppression and immunocompromised and combined chemotherapy to the application in terms of the treatment of tumour.The present invention also provides a kind of above-mentioned Neutral notoginseng polysaccharide have inhibiting effect to 7 kinds of Proliferation of Tumor Cells In Vitro in terms of application, 7 kinds of tumour cells are respectively that liver cancer is thin Born of the same parents H22, sarcoma cell S180, lymphoma cell YAC-1, humanized's breast cancer cell MCF-7, breast cancer cell 4T1, melanin Oncocyte B16, colon cancer cell CT26.WT 7.The present invention also provides a kind of above-mentioned neutral notoginseng polysaccharides to bone marrow suppression and to exempt from The low mouse of epidemic disease has the application for improving immunity and marrow DNA effect aspect.The present invention also provides a kind of above-mentioned neutral Radix Notoginseng Application in terms of polysaccharide combination chemotherapy tumour.

Detailed description of the invention

Fig. 1 is Radix Notoginseng Thick many candies DEAE Sepharose Fast Flow anion-exchange chromatography 0-2mol/LNaCl solution Gradient elution curve；

Fig. 2 is Radix Notoginseng Thick many candies HPGPC chromatogram；

Fig. 3 is neutral notoginseng polysaccharide PNPSI HPGPC chromatogram；

Fig. 4 is acid notoginseng polysaccharide PNPSII HPGPC chromatogram；

Fig. 5 is acid notoginseng polysaccharide PNPSIII HPGPC chromatogram；

Fig. 6 is acid notoginseng polysaccharide PNPSIV HPGPC chromatogram；

Fig. 7 is acid notoginseng polysaccharide PNPSV HPGPC chromatogram；

Fig. 8 neutrality notoginseng polysaccharide merges the influence experimental result picture of bone marrow suppression mouse platelets to immunocompromised；

Influence experimental result picture of Fig. 9 neutrality notoginseng polysaccharide to immunosuppressed mice macrophage phagocytic index；

Figure 10 neutrality notoginseng polysaccharide merges the influence experimental result picture of bone marrow suppression mouse bone marrow cells hyperplasia to immunocompromised: its Middle A is normal group marrow picture；B is model group marrow picture；C is Radix Notoginseng Thick many candies group marrow picture；D is that neutral Radix Notoginseng is more Sugared low dose group marrow picture；E is neutral notoginseng polysaccharide middle dose group marrow picture；F is neutral notoginseng polysaccharide high dose group bone Marrow picture.

Figure 11 is H22 tumor-bearing mice tumour pictorial diagram.

Specific embodiment

In order to further appreciate that technical characteristic of the invention, the present invention is explained in detail combined with specific embodiments below It states.Embodiment only has illustrative effect to the present invention, without the effect of any restrictions, those skilled in the art The modification for any unsubstantiality made on the basis of the present invention, all should belong to protection scope of the present invention.

Embodiment 1:

A kind of method for extraction and purification of neutrality notoginseng polysaccharide, specifically includes the following steps:

(1) extraction of Radix Notoginseng Thick many candies: weighing the waste residue 1kg for extracting arasaponin, 10 times of amount water be added for the first time, Boiling water bath boils 8h, collects filtrate, second of addition, 5 times of amount water, boiling water bath 8h.Merging filtrate twice, 4500rpm is centrifuged 5min, Take supernatant.80 DEG C of spin concentrations are stood for 24 hours, centrifugation abandons precipitating, is slowly added to three times dehydrated alcohol, side to the 1/8 of original volume Edged stirring, 4 DEG C of placement 12h filter to obtain precipitating.75% ethanol washing precipitates for several times, with appropriate water-soluble after ethyl alcohol volatilization is done Supernatant is collected in solution, centrifugation, and three times dehydrated alcohol is added, and 4 DEG C of placement 12h are filtered to obtain and be precipitated, 75% ethanol washing precipitating, and 50 DEG C Baking oven is dried to get Radix Notoginseng Thick many candies.

(2) DEAE Sepharose Fast Flow anion-exchange chromatography purifies Thick many candies

1. filling column: wet method dress post, pillar fill in cotton, and distilled water is added in column and keeps a bit of liquid level, glass bar is pressed Pressure cotton dispels bubble.It is poured slowly into column with glass bar guidance homogenate along column wall, bubble cannot be generated.It opens pillar and goes out liquid Mouthful, make gel free settling in column, washes with water chromatographic column edge.There cannot be tomography when filling column, then use distillation water balance, Column specification is 2.4 × 40cm.

2. balance: distilled water crosses 0.45 μm of filter membrane, pillar is rinsed with 5 times of cylinder ponding with certain flow rate, until efflux Conductivity and pH are constant, balance 48 hours.

3. loading: Radix Notoginseng Thick many candies are dissolved in a small amount of distilled water, are configured to 30mg/mL, and 4500rpm is centrifuged 10min, supernatant 0.45 μm of filter filtering of liquid, loading.When loading, chromatographic column liquid outlet is opened, it is to be distilled under water when moving to column bed surface, it closes Liquid outlet is added notoginseng polysaccharide solution along column wall with dropper, opens liquid outlet, sample liquid is allowed all to flow into column interior, and With a small amount of distilled water flushing column wall, then row elution.

4. elution: respectively with 0,0.1,0.2,0.3,0.4,0.5,2mol/L NaCl solution gradient elution, each elution about 5 Times column volume, flow velocity 0.8mL/min, every pipe meet 2mL, Anthrone-sulfuricacid method detection, until the detection of no polysaccharide, to elute pipe number For abscissa, absorbance value is that ordinate makees DEAE Sepharose Fast Flow anion exchange chromatography elution curve Figure, merges each eluting peak solution respectively, is concentrated under reduced pressure into 1/10 volume.Part is eluted with water as neutral notoginseng polysaccharide；Remaining It is acid notoginseng polysaccharide with various concentration NaCl solution elution fraction.Radix Notoginseng Thick many candies DEAE Sepharose Fast Flow Anion-exchange chromatography 0-2mol/LNaCl solution gradient elution curve is shown in Figure of description 1.

5. dialysis: bag filter being cut into every section of about 10cm, is put in distilled water and boils 30min, boiled again after washing three times 10min, distilled water wash clean.Bag filter can be used three times, clear after being boiled for the second time with 2%NaHCO3 and 1mmol/L EDTA It washes.Respectively by the eluent after concentration loaded in the bag filter of molecular weight 14000, about 1/2 volume clamps bag filter with dialysis clamp Both ends, are put in distilled water the 36h that dialyses, and every 4h changes a water.

6. freeze-drying: polysaccharide the meeting moisture absorption, notoginseng polysaccharide eluent lyophilized technique: by eluent in freezing dry process It is collected in 50mL centrifuge tube, fills about 16mL, -40 DEG C of pre-freeze 12h are put in freeze drier 36h, and about 1mL distillation is added after taking-up Water dissolution, shakes up.- 40 DEG C of pre-freeze 12h, are put in freeze drier for 24 hours.Sealing saves in drier after taking-up.

(3) molecular weight determination

1. standard solution prepare: Dextran T serial standards: dextran D0, D1, D2, D3, D4, D5, D6, D7, D8, D2000, standard molecular weight is respectively 180,2500,4600,7100,10000,21400,41100,84400,133800, 2000000Da.Each standard items 10mg is taken respectively, and 1ml mobile phase (0.1mol/L NaCl) is added to dissolve, through 0.22 μm of membrane filtration, It takes 20 μ L of filtrate to inject liquid chromatograph, records chromatogram, draw standard polysaccharide molecular weight-elution volume standard curve.

2. test solution is prepared: Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide PNPS I, acidity notoginseng polysaccharide II-V (10mg/ ML) through 0.22 μm of membrane filtration sample introduction, chromatogram is recorded, reference standard curve calculates molecular weight.

3. according to the liquid chromatogram of Series Molecules amount dextran standard items, the equation drawn are as follows:

log M_w=-0.7920V+12.1438, R²=0.9761.

Molecular weight according to the sample of standard curve calculating is as shown in table 1, Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I And acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V chromatogram are shown in Figure of description 2- attached drawing 7.

1 Radix Notoginseng Thick many candies of table, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II~V molecular weight determination knot Fruit

From the above results, neutral notoginseng polysaccharide PNPS I weight average molecular weight is 31590, molecular weight distribution spread factor D is 2.828, respectively less than other groups, shows neutral polysaccharide than other components sub- amount narrowly distributing.

(4) Anthrone-sulfuricacid method measures notoginseng polysaccharide content

Anthrone-sulfuricacid method measure Radix Notoginseng Thick many candies total sugar content, DNS method measure Radix Notoginseng Thick many candies in content of reducing sugar, three Seven Thick many candies contents=total sugar content-content of reducing sugar, the polyoses content of Radix Notoginseng Thick many candies are 63.86%；Anthrone-sulfuricacid method is surveyed Fixed neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide II-V content are shown in Table 2.

2 neutrality notoginseng polysaccharide PNPS I of table and acid notoginseng polysaccharide PNPS II~V polyoses content and yield

As can be seen from the above table, neutral notoginseng polysaccharide PNPS I content is in Radix Notoginseng raw slag material, content highest, up to 93.6%.

25 kinds of notoginseng polysaccharides of embodiment influence experiment to 7 kinds of Proliferation of Tumor Cells In Vitro

1 material

1.1 instruments and reagent

CO₂Incubator (German Heraeus company)；YJ-1450 type ultraviolet superclean bench SW-CJ-IF (the safe and sound sky in Suzhou Gas Technology Co., Ltd.)；680 type microplate reader of Bio-rad MODEL (Bio-rad company of the U.S.)；1.0R type high-speed low temperature from Scheming (German Heraeus company)；DK-98- II electric-heated thermostatic water bath (Tianjin Stettlen Instrument Ltd.)；Liquid relief Rifle (German Eppendorf company)；Inverted phase contrast microscope (Japanese Olympus company)；METTLER AE240 type electronics point Analyse balance (Shanghai plum Teller-support benefit Instrument Ltd. production)；HFC386STD-V12 type ultra low temperature freezer (Germany Hereaus company production).0.45 μm of miillpore filter (Tianjin Jin Teng experimental facilities Co., Ltd)；0.22 μm of filter (Tianjin Jin Teng experimental facilities Co., Ltd)；96 porocyte culture plates (Corning company of the U.S.)；Tissue Culture Dish (U.S. Corning Company)；The outer spiral cover of 2ml can found cryopreservation tube (Corning company of the U.S.)；(Shanghai section is into the limited public affairs of biotechnology for conical centrifuge tube Department).

Sterilize super blood serum of newborn calf without mycoplasma (Sijiqing Bioengineering Material Inst., Hangzhou City)；Fetal calf serum (Gibco company)；Calf serum (Gibco company)；DMEM culture solution (Gibco company)；(Gibco is public for PRMI-1640 culture solution Department)；Trypsase (Difco company)；Mycillin mixed liquor (Shanghai HyClone biochemistry corporation,Ltd.)；Dimethyl four Nitrogen azoles salt (MTT) (Sigma company)；Hydrochloric acid is analyzed pure (Chongqing Chuan Dong Chemical Co., Ltd.)；Isobutanol analyzes pure (western Gansu Province Work limited liability company)；Lauryl sodium sulfate (SDS) (Sigma company)；(pharmacy of Kunming South Sinkiang has physiological saline (N.S) Limit company production)；Without calcium and magnesium phosphate buffer (PBS) (Fuzhou Maixin biotechnology Development Co., Ltd)；Lentinan glue Capsule (Hubei China Chuan Li pharmaceutcal corporation, Ltd, national drug standard Z20080579)；PNPS (self-control)；Isolate 5 kind three of laboratory Seven polysaccharide: PNPS I, PNPS II, PNPS III, PNPS IV, PNPS I-A.

1.2 cell strain

Humanized's breast cancer cell MCF-7, murine hepatocarcinoma cell H22, mice sarcoma cell S180 (are stored in Kunming medical courses in general Natural drug pharmacology key lab of Yunnan Province of university)；Mouse mastopathy cell 4T1, B16 mouse melanoma cell line, mouse Colon cancer cell CT26.WT, mouse lymphoma cell YAC-1 (being purchased from Shanghai Inst. of Life Science, CAS).

1.3 animal

Kunming mouse, SPF grades, female, 18~22g is provided, experimental animal by Kunming Medical University's Experimental Animal Center Production licence number: SCXK (Yunnan) 2005-0008.

2 prepare solution

2.1 compounding pharmaceutical solution

Lentinan drug solution: the lentinan 56mg after weighing constant weight with assay balance adds physiological saline 2.5mL, It obtains the polysaccharide medicine solution that concentration is 22.4 mg/mL and is filtered with 0.22 μm of sterile filter spare.PNPS drug solution: it uses and divides Analysis balance weighs the PNPS 98.76mg after constant weight, and other methods are the same as lentinan drug solution configuration method.PNPS I drug Solution: the PNPS I 66.74mg after weighing constant weight with assay balance, other methods are the same as lentinan drug solution configuration method. PNPS II drug solution: the PNPS II 71.33mg after weighing constant weight with assay balance, other methods are the same as lentinan drug Solution allocation method.PNPS III drug solution: the PNPS III 113.36mg after weighing constant weight with assay balance, other party Method is the same as lentinan drug solution configuration method.PNPS IV drug solution: the PNPS after weighing constant weight with assay balance IV131.76mg, other methods are the same as lentinan drug solution configuration method.PNPS I-A drug solution: it is weighed with assay balance PNPS I-A 66.03mg after constant weight, other methods are the same as lentinan drug solution configuration method.

2.2 prepare MTT solution

5mg MTT powder is weighed with assay balance, the solution that 1mL physiological saline is made into 5mg/mL concentration is added.4 DEG C are put to keep away Light saves.Pay attention to being protected from light during preparation and preservation, container is encased with aluminium-foil paper.

2.3 prepare three liquid

36%~37% concentrated hydrochloric acid of 1.2mL, 100g SDS, 50mL isobutanol are obtained with 1L tri-distilled water stirring and dissolving 0.012mol/L HCl, three liquid of 10% lauryl sodium sulfate (SDS), 5% isobutanol, saves backup.

2.4 prepare cell culture fluid

DMEM culture solution: 90%DMEM culture medium+10%NBS

PRMI-1640 culture solution: 90%PRMI-1640 culture medium+10%NBS

2.5 prepare cells frozen storing liquid

DMEM frozen stock solution: 50%DMEM culture medium+40%FBS+10%DMSO

PRMI-1640 frozen stock solution: 50%PRMI-1640 culture medium+40%FBS+10%DMSO

The basic procedure of 3 cell culture

3.1 cell recoveries and passage

3.1.1 the recovery and passage of H22 cell strain

H22 cell cryopreservation tube is taken out from Liquid nitrogen storage tank, is quickly put into 37 DEG C of water-baths, slowly rocking makes cell solution Freeze, in course of defrosting, the sealed membrane of cryopreservation tube cannot rupture, and prevent cell contaminated.After defrosting, cryopreservation tube table is dried with paper The water droplet in face, alcohol watering can spray appropriate 75% alcohol disinfecting cryopreservation tube sealing part.Cryopreservation tube puts super-clean bench, will with aseptic straw Cell is all sucked out to 15mL sterile EP tube, adds 4mL DMEM culture solution, and centrifugation (1000r/min, 5min) makes cell precipitation, Supernatant is abandoned, with a small amount of physiological saline suspension sedimentation cell, is injected in right amount to mouse peritoneal with the absorption of 1mL needle tubing, to 8-10 days Afterwards, the abdominal cavity enlargement of Mice Inoculated, is further continued for propagating.

3.1.2 the recovery and passage of S180 cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.1.3 the recovery and passage of YAC-1 cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.1.4 the recovery and passage of MCF-7 cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.1.5 the recovery and passage of 4T1 cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.1.6 the recovery and passage of B16 cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.1.7 the recovery and passage of CT26.WT cell strain

And the recovery of H22 cell strain is identical as propagating method.

3.2 cell cryopreservation

When cell density about 70%, cell is in logarithmic growth phase at this time, and vigor is good, is most suitable for for freezing.

3.2.1 H22 cell strain freezes

Mouse is cultivated in tumour cell abdominal cavity, and skin of abdomen aspirates the thick of white after 75% alcohol disinfecting, with syringe Bloody ascites should be avoided during extracting ascites in ascites.Centrifugation (1000r/min, 5min) makes cell precipitation, abandons supernatant, uses PBS buffer solution cleaning, continues to be centrifuged, and abandons supernatant, is diluted and mixed with the DMEM frozen stock solution of Fresh, adjusts cell concentration, Make cell ultimate density 5 × 10⁶A/mL~1 × 10⁷Cell suspension is transferred in cryopreservation tube (1mL/ with suction pipe by a/mL Pipe), it indicates the title of cell, operator, freeze the time.Using the method for gradient cooling, cryopreservation tube is put into 4 DEG C of refrigerators 30min, then cryopreservation tube is finally put into liquid nitrogen container by -20 DEG C of refrigerator 90min, -80 DEG C of refrigerator overnights.

3.2.2 the freezing of S180 cell strain, the freezing of YAC-1 cell strain, the freezing of YAC-1 cell strain, 4T1 cell strain Freeze, the freezing of B16 cell strain, CT26.WT cell strain freeze

It is all the same with the cryopreservation methods of H22 cell strain.

4 methods

Influence of 4.1 5 components to H22 cell-proliferation activity

It takes third generation H22 tumour cell abdominal cavity to cultivate mouse, dips 75% ethanol mouse web portion with cotton balls, will inject Sodium chloride injection in device is pushed into mouse peritoneal, extracts cell in mouse peritoneal immediately.It is repeated several times operation, obtains cell suspension Appropriate liquid.The cell suspension in syringe is transferred in centrifuge tube, 1000r/min is centrifuged 5min, removes supernatant, then use Brine tumour cell twice, is diluted with DMEM culture solution.Cell concentration is adjusted to 3.33 × 10 by cell count⁴A/ mL.It is inoculated in 96 orifice plates, 90 holes μ l/, 54 holes/plate.Kind of 8 blocks of plates altogether.96 orifice plates are put to CO₂Incubator culture.

After 12h, 96 orifice plates are taken out, 8 blocks of plates add different drug solutions respectively.Every hole adds 10 μ L drug solutions, is administered dense Degree is respectively 2.24mg/mL, 1.12mg/mL, 560 μ g/mL, 280 μ g/mL, 140 μ g/mL, 70 μ g/mL, 35 μ g/mL, 17.5 μ G/mL, each concentration set 6 parallel holes, and remaining 6 holes add 10 μ L physiological saline as negative control group.After carrying out respective markers, 96 orifice plates are put into CO₂Incubator continues after cultivating 48h, and 20 hole μ l/ of MTT solution is added, puts CO₂After incubator 4h, 100 μ L are added Three liquid/hole, put CO₂After incubator 12h, absorbance (OD) is surveyed under conditions of wavelength 570nm.4.2 5 components are to S180 The influence of cell-proliferation activity

Third generation S180 tumour cell abdominal cavity is taken to cultivate mouse.Specific method is the same as 4.1.

Influence of 4.3 5 components to YAC-1 cell-proliferation activity

The YAC-1 cell of logarithmic growth phase after centrifuge washing cell, is diluted with PRMI-1640 culture solution, cell note Number, is adjusted to 3.33 × 10 for cell concentration⁴A/mL.It is inoculated in 96 orifice plates, 90 holes μ l/, 54 holes/plate.Kind of 8 blocks of plates altogether.By 96 holes Plate is put to CO₂Incubator culture.

After 12h, 96 orifice plates are taken out, 8 blocks of plates add different drug solutions respectively.Specific method is the same as 4.1.

Influence of 4.4 5 components to MCF-7 cell-proliferation activity

The MCF-7 cell of logarithmic growth phase discards culture solution, and 1mL PBS buffer solution rinse cell is added, discards buffering Liquid is repeated 3 times.Make cell detachment with 1mL trypsin digestion and cell, 2mL PRMI-1640 culture solution is added and terminates digestion, it will Cell suspension is transferred to 15mL sterile EP tube, and centrifugation (1000r/min, 5min) makes cell precipitation, abandons supernatant, is added appropriate PRMI-1640 culture solution, mixes gently, cell count, and cell concentration is adjusted to 3.33 × 10⁴A/mL.96 orifice plates are inoculated in, 90 holes μ l/, 54 holes/plate.Kind of 8 blocks of plates altogether.96 orifice plates are put to CO₂Incubator culture.

After 12h, 96 orifice plates are taken out, 8 blocks of plates add different drug solutions respectively.Specific method is the same as 4.1.

4.5 5 components to 4T1 cell-proliferation activity, B16 cell-proliferation activity, CT26.WT cell-proliferation activity shadow It rings

The 4T1 cell of logarithmic growth phase, specific method is the same as 4.4.

4.6 test datas statistics

All data are all made of mean ± standard deviationIt is provided using data of the SPSS17.0 statistics software to record Material carries out variance analysis statistical disposition.

5 results

Influence of 5.1 5 components to H22 cell-proliferation activity

3 five components of table to H22 cell-proliferation activity influence result (N=6)

Table2.1 Effects of five components on the proliferation of H22cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

It as shown in table 3, is in inhibiting effect to the growth of H22 cell when the administration concentration of PNPS is greater than 0.07mg/mL.PNPS It is in inhibiting effect to the growth of H22 cell when the administration concentration of I is greater than 0.56mg/mL.The administration concentration of PNPS IV 0.07~ When 0.28mg/mL, there is slight proliferation, concentration is in inhibiting effect in 1.12~2.24mg/mL.

Influence of 5.2 5 components to S180 cell-proliferation activity

4 five components of table to S180 cell-proliferation activity influence result (N=6)

Table2.2 Effects of five components on the proliferation of S180cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 4, PNPS is in inhibiting effect to the growth of S180 cell；The administration concentration of PNPS I 0.035~ The administration concentration of 2.24mg/mL, PNPS II and PNPS I-A are in 0.28~2.24mg/mL, to the growth of S180 cell in suppression Production is used.

Influence of 5.3 5 components to YAC-1 cell-proliferation activity

5 five components of table to YAC-1 cell-proliferation activity influence result (N=6)

Table2.3 Effects of five components on the proliferation of YAC- 1cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 5, PNPS administration concentration has the growth of YAC-1 cell and promotees proliferation in 0.0175~0.14mg/mL Effect, it is inhibited in 0.28~2.24 mg/mL；The administration concentration of PNPS I is in 0.56~2.24mg/mL, PNPS For the administration concentration of II in 1.12~2.24mg/mL, the administration concentration of PNPS III is right in 0.28~2.24mg/mL The growth of YAC-1 cell is in inhibiting effect；The administration concentration of PNPS IV and PNPS I-A is right in 0.0175~0.28mg/mL The growth of YAC-1 cell has slight proliferation；The administration concentration of PNPS IV is in 0.56~2.24mg/mL, to YAC-1 Cell growth is in inhibiting effect.

Influence of 5.4 5 components to MCF-7 cell-proliferation activity

6 five components of table to MCF-7 cell-proliferation activity influence result (N=6)

Table2.4 Effects of five components on the proliferation of MCF- 7cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 6, PNPS administration concentration has the growth of MCF-7 cell and promotees proliferation in 0.0175~1.12mg/mL Effect, it is inhibited in 2.24 mg/mL；The administration concentration of PNPS I is in 0.035~2.24mg/mL, to MCF-7 Cell growth is inhibited；In 0.28~2.24mg/mL, PNPS III administration concentration exists PNPS II administration concentration When 0.56~2.24mg/mL, PNPS IV administration concentration has the growth of MCF-7 cell and promotees in 0.070~2.24mg/mL Proliferation function；The administration concentration of PNPS I-A is in inhibiting effect to the growth of MCF-7 cell in 0.035~2.24mg/mL.

Influence of 5.5 5 components to 4T1 cell-proliferation activity

7 five components of table to 4T1 cell-proliferation activity influence result (N=6)

Table2.5 Effects of five components on the proliferation of 4T1cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 7, the administration concentration of PNPS and PNPS II grows 4T1 cell in 0.0175~0.28mg/mL It is inhibited in 0.56~2.24mg/mL with proliferation；PNPS I and PNPS III grows 4T1 cell It is inhibited；PNPS IV administration concentration has the growth of 4T1 cell and promotees proliferation work in 0.0175~0.14mg/mL With inhibited in 0.56~2.24mg/mL；The administration concentration of PNPS I-A in 0.0175~0.56mg/mL, There is proliferation to the growth of 4T1 cell.

Influence of 5.6 5 components to B16 cell-proliferation activity

8 five components of table to B16 cell-proliferation activity influence result (N=6)

Table2.6 Effects of five components on the proliferation of B16cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 8, PNPS administration concentration is in 0.070~2.24mg/mL, the administration concentration of PNPS I 0.14~ When 2.24mg/mL, PNPS II administration concentration in 0.56~2.24mg/mL, PNPS III administration concentration 0.070~ When 2.24mg/mL, PNPS IV and PNPS I-A administration concentration are in 0.14~2.24mg/mL, to the growth of B16 cell in inhibition Effect.

Influence of 5.7 5 components to CT26.WT cell-proliferation activity

9 five components of table to CT26.WT cell-proliferation activity influence result (N=6)

Table2.7 Effects of five components on the proliferation of CT26.WT cells

Note: compared with the control group, * table promotes, P < 0.05 *, P < 0.01 * *；# table inhibits, #P < 0.05, ##P < 0.01.

As shown in table 9, PNPS administration concentration has the growth of CT26.WT cell and promotees to increase in 0.0175~0.28mg/mL The effect of growing, it is inhibited in 0.56~2.24 mg/mL；The administration concentration of PNPS I in 1.12~2.24mg/mL, CT26.WT cell is grown inhibited；PNPS II administration concentration is thin to CT26.WT in 0.070~2.24mg/mL Intracellular growth has proliferation；PNPS III administration concentration grows CT26.WT cell in 0.0175~0.56mg/mL It is inhibited when 2.24mg/mL with proliferation；PNPS IV administration concentration in 0.035~0.56mg/mL, There is proliferation to the growth of CT26.WT cell, it is inhibited when 1.12~2.24mg/mL；The administration of PNPS I-A Concentration has proliferation in 0.0175~2.24mg/mL, to the growth of MCF-7 cell.

Embodiment 3:

Neutral notoginseng polysaccharide causes the Protection of bone marrow suppression and hypoimmunity mice to chemical drug

1. experiment content

1.1 materials and reagent

1.1.1 instrument

(Japanese Olympus is public for 680 microplate reader of Bio-rad MODEL (Bio-rad company of the U.S.), inverted phase contrast microscope Department), 1.0R type low-temperature and high-speed centrifuge (German Heraeus company), the ultraviolet superclean bench SW-CJ-IF of YJ-1450 type (Soviet Union The safe and sound air technique Co., Ltd in state), CO₂(German Hereaus is public for incubator (German Heraeus company), ultra low temperature freezer Department, HFC386STD-V12 type), Automatic Blood Cell Analyzer (Sysmex, XT-2000i).

1.1.2 material

Radix Notoginseng Thick many candies (self-control), neutral notoginseng polysaccharide (NPNPS, self-control), (enlightening is contained in CTX, Jiangsu to syklofosfamid ampoule Pharmaceuticals Ltd), RPMI-1640 culture medium (Gibco, sheep red blood cell (SRBC) SRBC, erythrocyte cracked liquid, calf serum, ConA, MTT, Hank ' s liquid (pH 7.2~7.4), PBS buffer solution (pH7.2~7.4), complement (guinea pig serum), india ink (Solarbio), 0.1% sodium carbonate, SA (sodium acetate) buffer, 1%NP40, lactic dehydrogenase (LDH) kit (Nanjing Build up Bioengineering Research Institute), anticoagulant tube, Wright's staining liquid.

1.1.3 animal and cell

SPF grades of healthy Kunming mouses (are provided, credit number: SCXK (Yunnan) by Kunming Medical University's Experimental Animal Center K2015-0002 is raised in SPF grades of Animal Lab.s.Zoopery obtains Kunming Medical University medical animal experiment ethics committee member Can ratify), female, 18-22g, YAC-1 cell strain (Shanghai Inst. of Life Science, CAS).

1.2 experimental methods and result

1.2.1 CTX causes the foundation and grouping of immunocompromised merging bone marrow suppression mouse model

SPF Kunming mice divides 6 groups at random, and every group 10, neutral notoginseng polysaccharide low, middle and high dose groups (93mg/Kg, 188mg/Kg, 375mg/Kg), Radix Notoginseng Thick many candies group (545mg/Kg), model group (physiological saline), normal group (physiological saline). Each group uses neutral notoginseng polysaccharide, Radix Notoginseng Thick many candies and continuous normal saline stomach-filling 10d respectively, and stomach-filling the 6th day, abdomen except for the normal group Chamber injects cyclophosphamide (CTX)

(80mg/Kg/d), continuous 5d, the 11st day measurement indices.

1.2.2 organ index measures

Mouse is put to death after weighing, takes out thymus gland and spleen, and weighing calculates dirty/body ratio.Index and spleen index=spleen weight (mg)/weight (g) thymus index=thymic factor D injection (mg)/weight (g).Neutral notoginseng polysaccharide merges marrow to immunocompromised The influence of mouse thymus, Spleen coefficient is inhibited to be shown in Table 10:

The neutral notoginseng polysaccharide of table 10 to immunocompromised merge bone marrow suppression mouse thymus, Spleen coefficient influence (n =10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01。

Compared with normal group: model group mouse spleen and thymus index significantly reduce (P < 0.01), show immunocompromised Model modeling success；Compared with model group: neutral notoginseng polysaccharide high dose group thymus index obviously increases (P < 0.05)；Radix Notoginseng is thick Polysaccharide group spleen index increases (P < 0.05).

1.2.3 hematological indices measure

Before animal is put to death, eye socket angular vein takes blood, EDTA-K2 anticoagulation, and Automatic Blood Cell Analyzer measures white thin Born of the same parents count (WBC), red blood cell (RBC), lymphocyte (LYM), blood platelet (PLT) routine blood indexes.

The neutral notoginseng polysaccharide of table 11 immunocompromised is merged bone marrow suppression mouse blood index influence (N= 10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01。

As shown in table 11, WBC, RBC, PLT and lymphocyte are substantially less than normal group (P < 0.01) in model group, show bone Marrow inhibits model modeling success；Compared with model group: the quantity of the neutral middle and high dosage group WBC of notoginseng polysaccharide increases (P < 0.05 Or P < 0.01), high dose group RBC is apparently higher than model group (P < 0.05)；Middle and high dosage group lymphocyte is all remarkably higher than model Group (P < 0.01)；Neutral notoginseng polysaccharide low, middle and high dose groups PLT and normal group indifference, wherein high dose group PLT is significantly high In model group (P < 0.01)；Radix Notoginseng Thick many candies group WBC and RBC quantity increases (P < 0.05), PLT quantity and normal group indifference And it is significantly higher than model group (P < 0.01).(see Fig. 8, it is small that neutral notoginseng polysaccharide merges bone marrow suppression mouse blood to immunocompromised The influence of plate) show that notoginseng polysaccharide and Radix Notoginseng Thick many candies can improve the quantity of immunocyte in immunosuppressed mice immune function, Also illustrate that neutral notoginseng polysaccharide and Radix Notoginseng Thick many candies promote bone marrow suppression mouse hemopoietic function.

1.2.4 mouse carbonic clearance is tested

Each group mouse dilutes 4 times of india ink through tail vein injection saline, by 0.1mL/ in after the last administration Timing immediately after 10g injection, the 2nd, 10min after injecting prepared Chinese ink take 20 μ L of blood to be added to 2mL 0.1%Na₂CO₃It is shaken up in solution. With Na₂CO₃Solution makees blank control, in 600nm wavelength densitometric value (OD), respectively sets 3 parallel holes.Mouse is put to death, is taken Liver, spleen and thymus gland, weighing.Phagocytic index α is calculated as follows.α=K^1/3K=in × weight/(liver weight+spleen weight) formula (lgOD₁- lgOD₂)/(T₂-T₁).Neutral notoginseng polysaccharide merges bone marrow suppression mouse macrophage phagocytic activity to immunocompromised Influence be shown in Table 12 and Fig. 9:

The neutral notoginseng polysaccharide of table 12 immunocompromised is merged bone marrow suppression mouse macrophage phagocytic activity influence (N=10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01.Experiment hair Existing, model group phagocytic index α and Grazing rate K are significantly lower than normal group (P < 0.05)；Neutral low dose of notoginseng polysaccharide after administration Amount group Grazing rate is apparently higher than model group (P < 0.05)；Neutral notoginseng polysaccharide low, middle and high dose groups are equal compared with normal group Indifference, wherein neutral notoginseng polysaccharide is low, high dose group Grazing rate is significantly higher than model group (P < 0.05 or P < 0.01)；It is neutral Notoginseng polysaccharide high dose group phagocytic index is significantly higher than model group (P < 0.05).Radix Notoginseng Thick many candies group equal indifference compared with normal group It is different.(Fig. 9, influence of the neutral notoginseng polysaccharide to immunosuppressed mice macrophage phagocytic index)

1.2.5 serum hemolysin measures

2% sheep red blood cell (SRBC) (SRBC) intraperitoneal injection 0.2mL/ only carries out sensitization, after 4d is immunized, plucks eyeball and takes blood in centrifugation In pipe, about 1h is placed, 4000r/min is centrifuged 10min, separates and collects serum.The 1mL through 100 times of dilution is sequentially added in test tube Serum, 10%SRBC 0.5mL, 1mL10% complement.Make blank control with the physiological saline of same volume, shakes up 37 DEG C of water of postposition 30min is kept the temperature in bath, moves to and terminates reaction in ice bath, after 2000r/min centrifugation 10min, supernatant is taken respectively to set 3 parallel holes, OD value is measured with microplate reader (540nm).

The neutral notoginseng polysaccharide of table 13 immunocompromised is merged bone marrow suppression mice serum haemolysis cellulose content influence (n =10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01。

Compared with Normal group, hemolytic antibody content significantly reduces (P < 0.01) in model group serum, and table 13 shows Immunodeficiency models modeling success；Compared with model group, the content of the neutral high, medium and low dosage group hemolysin of notoginseng polysaccharide is aobvious It writes and increases (P < 0.01), and low, middle and high dose groups haemolysis cellulose content and normal group indifference.

1.2.6 NK cytoactive detection

Target cell: taking the Yac-1 cell of culture for 24 hours, adjusts cell concentration to 4 × 10 with complete culture solution⁵A/mL is standby With.Effector cell: separating mouse spleen under aseptic condition is placed in the plate for filling appropriate sterile Hank ' s liquid, passes through after grinding 200 mesh net filtrations, collect filtrate, and 1000r/min is centrifuged 10min, 3 times of volume erythrocyte cracked liquids are added, sets and cracks on ice 10min abandons supernatant after 4 DEG C of centrifugations, Hank ' s is washed centrifuge cell 2 times, and it is 2 × 10 that viable count is adjusted after resuspension⁷A/ mL.By each 100 μ L of effector cell and target cell, ((E/T=50:1) is added in 96 orifice plates, while setting target cell Spontaneous release hole (+100 μ L complete culture solution of 100 μ l target cell) and maximum relief hole (+100 μ L1%NP-40 liquid of 100 μ L target cell), respectively sets 3 After a parallel hole is incubated for 48h.Each hole supernatant is drawn, with lactic dehydrogenase (LDH) kit measurement LDH activity, is surveyed in 450nm Determine OD value.NK cell activity (%)=(measurement hole OD- Spontaneous release hole OD)/(maximum relief hole OD- Spontaneous release hole OD) × 100%.

The neutral notoginseng polysaccharide of table 14 on immunocompromised merge bone marrow suppression NK cells in mice it is active influence (N= 10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01。

Model group NK cells in mice activity is substantially less than normal group (P < 0.01), compared with model group, Radix Notoginseng Thick many candies group NK cell activity is significantly raised (P < 0.05), remaining group no significant difference.(table 14)

1.2.7 lymphopoiesis measures

The preparation of mouse spleen lymphocyte: separating mouse spleen under aseptic condition is placed in and fills appropriate sterile Hank ' s liquid Plate in, through 200 mesh net filtrations after grinding, collect filtrate, 1000r/min is centrifuged 10min, 3 times of volume red blood cells are added Lysate is set and cracks 10min on ice, and supernatant is abandoned after 4 DEG C of centrifugations, and Hank ' s is washed centrifuge cell 2 times, adjusts after resuspension living thin Born of the same parents' number is 2 × 10⁶A/mL.Mouse boosting cell suspension presses 200 holes μ L/, if stimulation hole and each 3 multiple holes of control wells, stimulation hole adds Enter the 15 μ L of ConA culture solution containing 0.1mg/mL.20 μ L of MTT solution is added after culture 44h to continue to cultivate 4h.It inhales and abandons supernatant 150 μ L, tri- liquid is added in 100 μ L, each hole, and microplate reader detects the OD value at 570nm wavelength.Calculate stimulus index (SI): SI=thorn Swash hole OD value/control wells OD value.

The neutral notoginseng polysaccharide of table 15 immunocompromised is merged bone marrow suppression mice spleen lymphocytes proliferation power influence (N=10)

Note: compared with normal group, P < 0.01 * P < 0.05, * *；Compared with model group,^ΔP < 0.05,^ΔΔP<0.01。

Experiment shows that model group stimulus index is substantially less than normal group (P < 0.01), and neutral notoginseng polysaccharide is low, middle dose group It is higher than model group (P < 0.05) with Radix Notoginseng Thick many candies group stimulus index.(table 15)

1.2.8 protective effect of the neutral notoginseng polysaccharide to bone marrow suppression mouse bone marrow cells

Last dose takes the femur bone marrow smear of mouse after 24 hours, Wright's staining counts mature red thin under microscope The ratio of born of the same parents and karyocyte, calculating myelosis degree, (16) five-category method, is shown in Table.

16 myelosis rank of table

Tab.7 Levels of myeloproliferation

The neutral notoginseng polysaccharide of table 17 immunocompromised is merged bone marrow suppression mouse bone marrow cells hyperplasia influence (N=6)

Find out from table 17 and Figure 10, normal to organize mouse bone marrow cells polar hyperplasia, granulocyte, erythrocyte size, form are just Often；Model group mouse bone marrow cells hyperplasia lowers, and degenerated cell endochylema is imperfect, the dissolution of endochylema karyon, has vacuole in smear；It is neutral Notoginseng polysaccharide low, middle and high dose groups have 3,1,0 mouse bone marrow cells hypoplasias respectively, there is 3 active proliferations, 5 hyperplasia respectively Obvious active, 3 obvious proliferations and active proliferation, and cell cytosol, karyon are imperfect gradually reduces are granulocyte, red thin Born of the same parents gradually increase, form normal in size；5 active proliferations of mouse bone marrow cells of Radix Notoginseng Thick many candies group, granulocyte and erythroneocytosis by Step increases.

1.2.9 experimental data counts

One-way analysis of variance is carried out using SPSS17.0 statistical software.

Embodiment 4:

Experiment on therapy of the neutral notoginseng polysaccharide combined chemotherapy to tumor-bearing mice

1 material

1.1 instruments and reagent

BH-2 type microplate reader (Bio-rad company of the U.S.)；TGL18C low-temperature and high-speed centrifuge (Hunan Xiang Li Co., Ltd)； XT-2000i type Automatic Blood Cell Analyzer (Japanese Sysmex company)；96 orifice plates (Corning company of the U.S.)；BSA224S Assay balance (Sai Duolisi scientific instrument (Beijing) Co., Ltd)；EDTA-K₂Anticoagulant tube (the medical development in science and technology of three power of Liuyang City Co., Ltd)；Mouse fixing device；Dispensable 1 ml syringe (upper Haikang longevity Medical Devices Co., Ltd.)；Liquid-transfering gun (Germany Eppendorf company)；Operating scissors；Tweezers；No. 12 intragastric administration on mice needles；EP pipe.

India ink (Beijing Suo Laibao Biotechnology Co., Ltd)；Natrium carbonicum calcinatum analyzes pure (Chengdu section dragon chemical industry Chemical reagent work)；Sheep red blood cell (SRBC) (Beijing Solarbio company, concentration: 15%, 100mL/ bottles)；Liquid guinea pig serum (complement) (north Capital Solarbio company, 1mL/ bottles)；0.9% sodium chloride injection (Kuming Nanjiang Pharmacy Co., Ltd)；Injection ring phosphinylidyne Amine (Jiangsu Sheng Di Pharmaceuticals Ltd, national drug standard HB2020857,0.2g/ bottle)；Radix Notoginseng Thick many candies (self-control)；PNPS I (self-control).

1.2 animal

Kunming mouse, SPF grades, female, 18~22g is provided, experimental animal by Kunming Medical University's Experimental Animal Center Production licence number: SCXK (Yunnan) 2005-0008.

1.3 cell strain

Murine hepatocarcinoma cell H22 (is stored in natural drug pharmacology key lab of Yunnan Province of Kunming Medical University).

2 prepare solution

2.1 compounding pharmaceutical solution

PNPS I low-dose drugs solution: precision weighs the PNPS I of 0.1192g, is dissolved in 10mL physiological saline.80 DEG C of water It bathes half an hour, centrifugation removal precipitating obtains PNPS I low-dose drugs solution.PNPS I middle dosage drug solution: precision weighs The PNPS I of 0.2383g, specifically with PNPS I low-dose drugs solution allocation method.PNPS I high dose medicament solution: accurate The PNPS I of 0.3575g is weighed, specifically with PNPS I low-dose drugs solution allocation method.PNPS drug solution: precision weighs The PNPS of 0.3539g, specifically with PNPS I low-dose drugs solution allocation method.Cyclophosphamide (CTX) drug solution: precision claims The cyclophosphamide for taking 0.0200g is settled to 10mL with physiological saline in 10mL volumetric flask, mixes, obtains CTX drug solution.

2.2 prepare common agents

India ink: taking india ink 12.5mL, and physiological saline is settled to the print in 50mL volumetric flask to get 4 times of dilution Spend prepared Chinese ink.0.1% Na₂CO₃Solution: 0.1g Na is taken₂CO₃To 100mL volumetric flask, with distilled water constant volume.2% sheep red blood cell (SRBC): With pipettor draw 1mL15% sheep red blood cell (SRBC), be transferred in clean test tube, then plus 6.5mL physiological saline, mix, obtain 2% Sheep red blood cell (SRBC) faces with newly matching.10% sheep red blood cell (SRBC): 1mL15% sheep red blood cell (SRBC) is drawn with pipettor, is transferred to clean In test tube, then plus 0..5mL physiological saline, mix, obtain 10% sheep red blood cell (SRBC), face with newly matching.10% complement: 1mL liquid is taken Guinea pig serum obtains 10% complement by the dilution proportion of 1:10, faces with newly matching.

3 methods

3.1 groupings and administration

By 35 Kunming kind SPF mouse, 7 groups are randomly divided into, every group 5, is followed successively by (1) normal group, (2) physiological saline Group, (3) CTX group (20mg/kg), (4) PNPS (100mg/kg)+CTX (20mg/kg) group, (5) PNPS I low dosage (100mg/ Kg)+CTX (20 mg/kg) group, (6) PNPS I middle dosage (200mg/kg)+CTX (20mg/kg) group, (7) PNPS I high dose (300mg/kg)+CTX (20mg/kg) group.During test, (1), (2), (3) group intragastric administration on mice give physiological saline.Other groups Gastric infusion.

3.2 establish animal model for tumour and administration

The 6th day morning took H22 liver cancer cells abdominal cavity to cultivate mouse, and dipped 75% ethanol mouse web portion with cotton balls, will Sodium chloride injection in syringe is pushed into mouse peritoneal, extracts the H22 liver cancer cells in mouse peritoneal immediately.It is repeated several times behaviour Make, it is appropriate to obtain H22 liver cancer cells suspension.

It takes 10 μ L cell suspensions in clean EP pipe, 1990 μ L physiological saline is added, mix, numeration.

Cell suspension calculation formula: 4 middle lattice inner cell number/4 × 10⁴× extension rate=H22 liver cancer cells number/mL

Appropriate H22 cell suspension is taken, its concentration is adjusted to 1 × 10 with physiological saline⁷A/mL.

(2) group to (7) group mouse right axillary inoculates tumor cell suspension 0.2mL,

After inoculation, (3) group to (7) group mouse peritoneal injects cyclophosphamide drug solution, continuous injection 10 days.

The measurement of 3.3 H22 tumor-bearing mice Immune Organs Indexes

After the last administration, it is deprived of food but not water 12h, cervical dislocation puts to death mouse, dissects mouse, takes out spleen and thymus gland, uses Filter paper is weighed after blotting blood, the ratio between Computation immunity organ and weight.

Index and spleen index=spleen weight (mg)/weight (g) thymus index=thymic factor D injection (mg)/weight (g).

The measurement of 3.4 H22 tumor-bearing mice tumour inhibiting rates

After the last administration, it is deprived of food but not water 12h, cervical dislocation puts to death mouse, removes tumor tissues, weighing.Tumour inhibiting rate IR (%)=(1- treatment group average knurl weight/physiological saline group average knurl weight) × 100%.

The test of 3.5 H22 tumor-bearing mice carbonic clearances

After the last administration, it is deprived of food but not water 12h, through tail vein to 4 times of mouse injection normal saline dilution of indian ink Juice, 0.1mL/10g, timing immediately after prepared Chinese ink injection, the 2nd, 10min after injecting prepared Chinese ink take 20 μ of blood from angular vein clump respectively L is added to 2mL 0.1%Na₂CO₃In solution, shake up.With Na₂CO₃Solution makees blank control, with 600nm wavelength densitometric It is worth (OD).

Cervical dislocation puts to death mouse, takes liver, spleen, weighs.Phagocytic index a is calculated as follows.

A=K^1/3K=(lgOD in × weight/(liver weight+spleen weight) formula₁- lgOD₂)/(T₂-T₁) (in formula, OD₁It is adopted for the 1st time The absorbance of blood, OD₂The absorbance taken a blood sample for the 2nd time；T₁The time taken a blood sample for the 1st time, T₂The time taken a blood sample for the 2nd time).

The measurement of 3.6 H22 tumor-bearing mice serum hemolysins

Establishing the 7th day of animal model for tumour, every mouse peritoneal inject 2% sheep red blood cell (SRBC) (SRBC) 0.2mL into Row sensitization, and continue to be administered.Continuous immunity 4d is deprived of food but not water 12h after the last administration, extracts the eyeball of mouse, take blood in from In heart pipe, about 1h is placed, is centrifuged (4000r/min 10min), serum is collected in separation.In sequentially adding in test tube through diluting 100 times of 1mL serum, 10%SRBC 0.5mL, 1 mL10% complement.Blood serum sample is replaced with the physiological saline of same volume, Prepare blank control.It after shaking up the solution in test tube, sets in 37 DEG C of water-baths and keeps the temperature 30min, then move to and terminate reaction in ice bath, It is centrifuged (2000r/min 10min), takes supernatant to draw 200 μ L in 96 hole transparent panels, measure OD value with microplate reader (540nm).

The measurement of 3.7 H22 tumor-bearing mice physiochemical indices

After the last administration, it is deprived of food but not water 12h, eye socket angular vein takes blood 0.5mL, is stored in EDTA-K2 anticoagulant tube, uses XT-2000i type Automatic Blood Cell Analyzer surveys blood routine.

3.8 test datas statistics

All data are all made of mean ± standard deviationIt is provided using data of the SPSS17.0 statistics software to record Material carries out variance analysis statistical disposition.

4 results

The measurement of 4.1 H22 tumor-bearing mice Immune Organs Indexes

18 PNPS I of table to H22 tumor-bearing mice Immune Organs Index influence (N=5)

Note: compared with physiological saline group, #P < 0.05, ##P < 0.01；Compared with CTX group, P < 0.01 * P < 0.05, * *.

As shown in table 18, compared with physiological saline group, spleen index, the thymus index of CTX group are reduced, and are had extremely significant Difference (P < 0.01)；Compared with CTX group, the index and spleen index value of PNPS I low dosage+CTX group is increased, and has extremely significant sex differernce (P < 0.01), thymus index value increase, and have significant difference (P < 0.05)；The thymus index value of PNPS I middle dosage+CTX group It increases, has significant difference (P < 0.05).

The measurement of 4.2 H22 tumor-bearing mice tumour inhibiting rates

19 PNPS I of table to H22 tumor-bearing mice tumour influence (N=5)

Note: compared with physiological saline group, ##P < 0.01；Compared with cyclophosphamide group, P < 0.01 * *.

Each group tumor mass weight and tumour inhibiting rate are shown in Table 19, each group tumour material object such as Figure 11, negative control group (physiology salt in test Water group) mouse knurl weight is all larger than 400mg, transplantable tumor success.Compared with physiological saline group, the knurl weight of each experimental group is reduced, and With extremely significant sex differernce (P < 0.01)；Compared with cyclophosphamide group, PNPS I low dosage+CTX group with PNPS I high dose+ The knurl weight of CTX group reduces, and has extremely significant sex differernce (P < 0.01).

4.3 H22 tumor-bearing mice monocytes/macrophages carbonic clearance ability tests

20 PNPS I of table to H22 tumor-bearing mice monocytes/macrophages carbonic clearance ability influence (N=5)

Note: compared with physiological saline group, ##P < 0.01；Compared with CTX group, P < 0.05 *.

As shown in table 20, compared with physiological saline group, the phagocytic index of CTX group is reduced, and have extremely significant difference (P < 0.01)；PNPS+CTX group, PNPS I low dosage+CTX group, the phagocytic index of PNPS I middle dosage+CTX group and physiological saline group There was no significant difference, and PNPS I high dose+CTX group phagocytic index value reduce, and have extremely significant sex differernce (P < 0.01).Compared with CTX group, the phagocytic index value of PNPS I low dosage+CTX group is increased, and is had significant difference (P < 0.05).

The measurement of 4.4 H22 tumor-bearing mice serum hemolysins

Influence that 21 PNPS I of table generates H22 tumor-bearing mice serum hemolysin (N=5)

Note: compared with physiological saline group, ##P < 0.01；Compared with CTX group, P < 0.01 * P < 0.05, * *.

As shown in table 21, compared with physiological saline group, PNPS I middle dosage+CTX group, PNPS I high dose+CTX group are in lotus It is above control group in the influence of tumor mice serum Hemolysin formation, and there is significant difference (P < 0.01)；With CTX group ratio Compared with PNPS I low dosage+CTX group, PNPS I middle dosage+CTX group, PNPS I high dose+CTX group are molten in tumor-bearing mice serum CTX group is above in the influence that sanguinin generates, difference has statistical significance.

The measurement of 4.5 H22 tumor-bearing mice physiochemical indices

22 PNPS I of table to H22 tumor-bearing mice physiochemical indice influence (N=5)

Note: compared with physiological saline group, ##P < 0.01；Compared with CTX group, P < 0.01 * P < 0.05, * *,^ΔP<0.05。

As shown in table 22, compared with physiological saline group, CTX group, PNPS I low dosage+CTX group, PNPS I middle dosage+CTX Group, the leucocyte of PNPS I high dose+CTX group, red blood cell, lymphocyte concentration reduce.Compared with CTX group, PNPS+CTX Group is increased with the white blood cell concentration that PNPS I low dosage+CTX is organized, and has significant difference (P < 0.05)；PNPS+CTX group, PNPS I middle dosage+CTX group, PNPS I high dose+CTX group red blood cell concentration increase, and have significant difference (P < 0.05), the red blood cell concentration of PNPS I low dosage+CTX group increases, and has extremely significant sex differernce (P < 0.01)；PNPS+CTX Group, PNPS I low dosage+CTX group, the hemoglobin concentration of PNPS I high dose+CTX group reduce, and have significant difference (P <0.05)；PNPS+CTX group, PNPS I low dosage+CTX group lymphocyte concentration increase, and have extremely significant sex differernce (P < 0.01)。

Claims (10)

1. a kind of method for extraction and purification of neutrality notoginseng polysaccharide, uses the Radix Notoginseng waste residue for extracting notoginsenoside for raw material, special Sign is: the following steps are included:

(1) it takes Radix Notoginseng to be ground into coarse powder, is extracted, filtered with 70% ethyl alcohol, extracted, filtered 4 times, obtain Radix Notoginseng waste residue, it is dry.

2.(2) water extraction and alcohol precipitation method extracts Radix Notoginseng Thick many candies；

(3) DEAE Sepharose Fast Flow anion-exchange chromatography and dialysis purify Thick many candies, and freeze-drying obtains To neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acid notoginseng polysaccharide PNPS IV, acid notoginseng polysaccharide PNPS V freeze-dried powder.

3. the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is controlled in preparation Treat, inhibit or promote the application in Proliferation of Tumor Cells In Vitro drug.

4. neutrality notoginseng polysaccharide according to claim 2 is in preparation treatment, inhibition Proliferation of Tumor Cells In Vitro drug Using the tumour cell includes at least liver cancer cells, sarcoma cell, lymphoma cell, humanized's breast cancer cell, breast cancer Cell, melanoma cells, colon cancer cell.

5. neutrality notoginseng polysaccharide according to claim 3 is in preparation treatment, inhibition Proliferation of Tumor Cells In Vitro drug Using the tumour cell includes at least liver cancer cells H22, sarcoma cell S180, lymphoma cell YAC-1, humanized's mammary gland Cancer cell MCF-7, breast cancer cell 4T1, melanoma cells B16, colon cancer cell CT26.WT 7

The neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is treated in preparation Application in bone marrow suppression drug.

6. the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is mentioned in preparation Application in high immunity and marrow DNA drug.

7. the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is mentioned in preparation Application in high tumour inhibiting rate, raising immunity and routine blood indexes drug.

8. the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is mentioned in preparation High immunocompromised merges bone marrow suppression thymus gland, Spleen coefficient, promotes application in bone marrow suppression hematopoiesis function drug.

9. the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 is mentioned in preparation Application in the Therapeutic Results of Combination Chemotherapy drug of high tumour.

10. prepared by the neutral notoginseng polysaccharide of the method for extraction and purification preparation of neutrality notoginseng polysaccharide according to claim 1 Improve the application in tumor blood leucocyte, red blood cell, lymphocyte concentration or reduction hemoglobin concentration drug.