CN108164615A - A kind of preparation method and application of Siberian solomonseal rhizome polysaccharide - Google Patents
A kind of preparation method and application of Siberian solomonseal rhizome polysaccharide Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 58
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 57
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 57
- 241000756042 Polygonatum Species 0.000 title claims abstract description 51
- 235000008737 Polygonatum biflorum Nutrition 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 10
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960004397 cyclophosphamide Drugs 0.000 claims abstract description 8
- 206010061598 Immunodeficiency Diseases 0.000 claims abstract description 5
- 235000008216 herbs Nutrition 0.000 claims abstract description 4
- 230000036039 immunity Effects 0.000 claims abstract description 3
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- 238000001035 drying Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
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- 206010028980 Neoplasm Diseases 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
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- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 8
- 102000004889 Interleukin-6 Human genes 0.000 description 8
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- 239000012980 RPMI-1640 medium Substances 0.000 description 8
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- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
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- 102000003777 Interleukin-1 beta Human genes 0.000 description 5
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- 210000004988 splenocyte Anatomy 0.000 description 3
- 206010011224 Cough Diseases 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
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- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- KVYRCBOUKXJXDK-UHFFFAOYSA-N 3,4-dimethylphenazine-1,2-diamine hydrochloride Chemical compound Cl.C1=CC=CC2=NC3=C(C)C(C)=C(N)C(N)=C3N=C21 KVYRCBOUKXJXDK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
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- 238000012449 Kunming mouse Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241001468611 Polygonatum cyrtonema Species 0.000 description 1
- 241000037826 Polygonatum kingianum Species 0.000 description 1
- 241000037831 Polygonatum sibiricum Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
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- 238000000227 grinding Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Sustainable Development (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to the preparation method and applications fields of effective component of chinese medicine, and in particular to a kind of preparation method of Siberian solomonseal rhizome polysaccharide and its application in medicament for immunity enhancement is prepared.By sealwort raw medicinal herbs by processing, Siberian solomonseal rhizome polysaccharide is obtained after extraction separation, extracting method using the present invention, Siberian solomonseal rhizome polysaccharide recovery rate is high, and total starches content is high, Siberian solomonseal rhizome polysaccharide increases to normal mouse Spleen cell proliferation vigor, also the spleen and thymus index of the mouse by cyclophosphamide induction immunocompromised can be improved, enhance the lower spleen of concanavalin A stimulation and Detection of thymocyte proliferation in vitro ability, enhance the phagocytic function of hypoimmunity mice macrophage and the ability of secretion IL 6 and TNF α, in terms of the treatment of the diseases such as immunologic deficiency disease and tumour, with actual application value.
Description
Technical field
The present invention relates to the preparation method and applications fields of effective component of chinese medicine, and in particular to a kind of system of Siberian solomonseal rhizome polysaccharide
Preparation Method and its application in medicament for immunity enhancement is prepared.
Background technology
Sealwort is liliaceous plant P. kingianum Polygonatum kingianum Coll.et Hemsl., sealwort
The dry rhizome of Polygonatum sibiricum Red. or David's-harp Polygonatum cyrtonema Hua.Nature and flavor
Gan Ping, returns spleen, lung, kidney channel have the effect of air making-up and spleen enlivening.Sealwort has the effects that edible, medicinal, ornamental, beauty, commonly uses
In deficiency of spleen-QI and stomach-QI, fatigue and asthenia, deficiency of stomach-Yin, dry deficiency of food, deficiency syndrome of the lung cough caused by dryness, overstrain cough hemoptysis, asthenia of essence and blood, soreness and weakness of waist and knees, palpus
Early white, the Heat Diabetes of hair.
Siberian solomonseal rhizome polysaccharide is one of principle active component of sealwort, has the work(such as antitumor, anti-aging, antiatherosclerosis
Energy.
Generally more than 10%, the extraction separation method currently used for extracting Siberian solomonseal rhizome polysaccharide includes polyoses content in sealwort
Hot water extraction, alcohol analysis separation, microwave radiation exaraction, ultrasonic wave extraction etc., but the purity of polysaccharide of extraction it is not high (<80%).Knot
Modern chemistry and pharmacological research achievement are closed, Siberian solomonseal rhizome polysaccharide constituents are developed and used, excavates new drug, it will be to traditional Chinese medicine Huang
The exploitation of essence has realistic meaning with application.
Cyclophosphamide as clinically common antitumor drug, can killing tumor cell, while have apparent immune
Inhibiting effect.It is immune that a large amount of pharmaceutical research shows that the compound of some polysaccharides can be adjusted, and activates the immune thin of body
Born of the same parents improve the immune function of body.The present invention is directed to probe into influence of the Siberian solomonseal rhizome polysaccharide to cyclophosphamide inducing mouse immunocompromised,
For Siberian solomonseal rhizome polysaccharide rational foundation is provided as the immunologic adjuvant of cyclophosphamide.
Invention content
There is provided a kind of preparation methods of Siberian solomonseal rhizome polysaccharide for first purpose of the present invention.
To achieve these goals, the technical solution adopted by the present invention is as follows：
A kind of preparation method of Siberian solomonseal rhizome polysaccharide, includes the following steps：
(1) sealwort raw medicinal herbs is cleaned, is deposited in 4-10h on clean table top after soaking (10-20 minutes), use is wet
Cloth covers, and during which sprays clear water, moistens to without the dry heart, then steaming in medicinal material to yellow black, soft for degree, closes fire and continue to boil in a covered pot over a slow fire to appearance
It is brownish black, glossy, take the dish out of the pot, dry in the air to it is outer it is dry in moisten, be cut into (3~5) mm sheets, dry；
(2) slice of step (1) is taken, is smashed, with ethanol water refluxing extraction 2-3 times of 80~95% (v/v),
Filtering, the drying of the gained dregs of a decoction；
(3) distilled water is added in into the dregs of a decoction after the drying of step (2) in 80 DEG C of refluxing extractions 2-4 time, is filtered, filtrate is dense
After being reduced to medicinal extract state, ethyl alcohol is added in, until ethanol content is 70%-90% (v/v), stirs evenly, is then placed at 4 DEG C
(8-12) h, filtering, precipitation drying is to get Thick many candies；
(4) Thick many candies obtained by step (3) are removed into deproteinized with Sevage methods；
(5) by step (4) except the polysaccharide water dissolution after deproteinized after, dialyse 48h in flowing water；Take dialyzate vacuum cold
It is lyophilized dry, obtains Siberian solomonseal rhizome polysaccharide.
Further, 4~8h is steamed after being soaked in the step (1) in food steamer, fire is closed and continues stewing 4~8h, take the dish out of the pot；
6h is preferably steamed in food steamer, fire is closed and continues stewing 6h, take the dish out of the pot.
Further, drying described in step (1) is is placed in 70 DEG C of drying of constant temperature drying box.
Further, drying temperature described in step (2) is less than 50 DEG C.
Further, it is with the process of the ethanol water refluxing extraction of 80~95% (v/v) described in step (2)：With
95% (v/v) ethyl alcohol is in 60 DEG C of reflux 3h, filtering；Then into obtained solid add in 80% (v/v) ethyl alcohol in 60 DEG C flow back 2h,
Filtering, dregs of a decoction drying；
Further, distilled water is added in described in step (3) is in 80 DEG C of refluxing extraction processes：After step (2) drying
The dregs of a decoction in add in distilled water refluxing extraction 3 times at 80 DEG C, each 3h, filtering, merging filtrate.
Further, the dregs of a decoction in the step (3) after drying and distilled water amount ratio during extraction every time are 1g：20-
30mL, preferably 1g：25mL.
Siberian solomonseal rhizome polysaccharide another object of the present invention is to provide a kind of preparation of above method is as antitumor drug
The application of adjuvant, the specially application in the drug of immunocompromised disease treated and induced by cyclophosphamide is prepared.
There is provided Siberian solomonseal rhizome polysaccharides prepared by a kind of above method to prepare immunopotentiating drug for third object of the present invention
Application in object.
Experiment shows that Siberian solomonseal rhizome polysaccharide increases to normal mouse Spleen cell proliferation vigor, can also improve and be lured by cyclophosphamide
Lead the spleen and thymus index of the mouse of immunocompromised, the lower spleen of enhancing concanavalin A stimulation and Detection of thymocyte proliferation in vitro energy
Power enhances the phagocytic function of hypoimmunity mice macrophage and the ability of secretion IL-6 and TNF-α, in immunologic deficiency disease and swells
There is actual application value in terms of the treatment of the diseases such as knurl.
Description of the drawings
Fig. 1 is influence figure of the Siberian solomonseal rhizome polysaccharide to hypoimmunity mice macrophages phagocytic capacity, compared with normal group#P<
0.05,##P<0.01；The * P compared with model group<0.05, * * P<0.01.
Fig. 2 is influence figure of the Siberian solomonseal rhizome polysaccharide to normal mouse splenocyte in-vitro multiplication vigor, compared with blank group, * P<
0.05, * * P<0.01, * * * P<0.001.
Specific embodiment
Applicant will in conjunction with specific embodiments be described in further detail technical scheme of the present invention below.
Sealwort medicinal material in embodiment comes from enshi.
Embodiment 1
A kind of preparation method of Siberian solomonseal rhizome polysaccharide, includes the following steps：
(1) sealwort raw medicinal herbs is cleaned, is deposited in 6h on clean table top after soaking 15 minutes, is covered with wet cloth,
Period sprays proper amount of clear water, keeps moisture state, and the moisture outside medicinal material is made to penetrate into inside drug entities slowly, moistens medicinal material
To without the dry heart；It is subsequently placed in food steamer and steams, water continues to steam 6h after opening, until medicinal material closes fire and continue stewing 6h extremely in yellow black, soft shape
It is appearance brownish black, glossy, it takes the dish out of the pot, dries in the air to moistening in dry outside sealwort, be then cut into 3~5mm sheets, be placed in 70 DEG C of constant temperature drying boxes
Drying.
(2) slice is taken to clay into power, first ((V/V, the percent concentration of ethyl alcohol described in this specification are equal with 95% ethyl alcohol
Refer to concentration of volume percent, do not repeat hereinafter) flow back 3h at 60 DEG C, degreasing, then is taken off single in 60 DEG C of reflux 2h with 80% ethyl alcohol
Sugar and oligosaccharide, then filter, the dregs of a decoction are dried (45 DEG C), add in distilled water (dregs of a decoction and the distilled water amount ratio added in every time
For 1g:25mL), refluxing extraction 3 times at 80 DEG C, each 3h.Filtering, merging filtrate are concentrated into medicinal extract state, add in ethyl alcohol, make
Alcohol content is 80%, is stirred evenly, and is stood overnight at 4 DEG C, filtering, by precipitation drying to get Thick many candies.Gained Thick many candies are used
Sevage methods remove deproteinized, remove the polysaccharide after deproteinized, add (60 DEG C) dissolvings of appropriate hot water, dialyse 48h in flowing water；It takes
Liquid vacuum freeze drying is analysed to get smart polysaccharide (Siberian solomonseal rhizome polysaccharide).Gained Siberian solomonseal rhizome polysaccharide according to H2SO4-anthrone method develop the color after
Absorbance is surveyed at 627nm, polysaccharide standard curve is done with dextrose standard sample, it is 96.9% to measure sealwort total starches content.
2 Siberian solomonseal rhizome polysaccharide of embodiment is to the adjustment effect of cyclophosphamide induction hypoimmunity mice
1. experimental animal and reagent
8 SPF grades of week old male mouse of kunming (22 ± 2) g, purchased from Disease Prevention Control Center, Hubei Prov, mouse adaptability
Start to test after feeding 3 days.
Siberian solomonseal rhizome polysaccharide：Prepared by embodiment 1, concanavalin A (ConA) and lipopolysaccharides (LPS) are purchased from Sigma-Adrich
(St.Louis, Missouri, USA)；IL-1 β, IL-6 and TNF-α assay ELISA kit are purchased from Nanjing and build up biology
Research institute.
PBS buffer solution：0.01M, pH=7.4.
MTT solution：250mg MTT are added in 50mL PBS buffer solution and are made it completely dissolved, after filtering with microporous membrane, are put
Enter -20 DEG C of refrigerators to be kept in dark place.
2. method
2.1 animal packets and model preparation method
Kunming mice 50 is taken, is randomly divided into the basic, normal, high dosage group (dosage of Normal group, model group, Siberian solomonseal rhizome polysaccharide
Respectively 50,100,150mg/kg), every group 10.
The normal group daily intraperitoneal injection of saline of mouse (0.01mL/g), every mouse peritoneal injection ring phosphorus of other each groups
Amide (2mg/d), continuous 3d establish immunosuppression model.Start within 4th day, normal group and the daily gavage physiology of model group mouse
Brine (0.01mL/g), other each groups distinguish daily gavage corresponding dosage Siberian solomonseal rhizome polysaccharide solution (0.01mL/g), and (prepared by embodiment 1
Siberian solomonseal rhizome polysaccharide be formulated with distilled water), continuous 1 week.
The preparation of 2.2 macrophage suspensions
(1) the RPMI-1640 culture solutions of 5mL are injected into mouse peritoneal, gently rub mouse web portion, abdominal cavity is collected after 15min
Liquid rinses cell suspension 2 times with RPMI-1640 culture solutions；
(2) and then by cell suspension it is resuspended in RPMI-1640 culture solutions, Trypan Blue determines that cell survival rate is more than
95%.Finally with RPMI-1640 complete culture solutions (containing 10% fetal calf serum) dilution macrophage suspension, it is 2 to make cell density
×106A/mL, it is spare.
2.3 spleens/thymus index measures and prepared by cell suspension
Cervical dislocation puts to death mouse, takes out spleen respectively and weighs with thymus gland, and calculate thymus index and index and spleen index：
Spleen (thymus gland) index=spleen (thymus gland) quality (mg)/mouse weight (g).
Appropriate spleen and thymus gland are taken, is positioned in the culture dish containing ice-cold RPMI-1640 culture solutions, after shredding grinding, mistake
Filter obtains the single cell suspension of spleen or thymus gland.Spleen and thymus cell suspension are obtained by step (2) method under " 2.2 " item again, it is standby
With.
2.4 phagocytosis function detection of macrophages
The macrophage suspension that 2.2 are obtained respectively is inoculated in 96 orifice plates, and it is 2 × 10 to make cell density5A/hole, in
37 DEG C, 5%CO2Under the conditions of cultivate 4h.Original fluid is discarded, non-attached cell is washed away with PBS buffer solution, 100 μ are added in every hole
The dimethyl diaminophenazine chloride physiological saline of L 0.04%, continues to cultivate.Supernatant is abandoned after 4h, is cleaned 3 times, added with 100 μ L physiological saline per hole
Enter 200 μ L Ethanol-Acetic Acids (volume ratio 1: 1) mixed liquors, stand 12h.After cell is completely dissolved, with microplate reader at 570nm
The OD values in each hole are measured, the phagocytic function of macrophage is compared with the size of OD values.
2.5ConA stimulates spleen to be detected with thymocyte proliferation
The spleen or thymus cell suspension that 2.3 are prepared after removing red blood cell, are inoculated in 96 orifice plates, make cell close
Spend is 5 × 105A/hole, culture is for 24 hours.Then the RPMI-1640 cultures that 100 μ L contain ConA (2 μ g/mL) are added in into each hole
Liquid, while negative control hole (per 100 μ L RPMI-1640 culture solutions of hole) is set.In 37 DEG C, 5%CO2After lower culture 72h, often
Hole adds in 10 μ L, 5.0mg/mL MTT solution, continues to cultivate 4h, discards culture solution, and 150 μ L DMSO are added in, and fully to every hole
Mixing.Finally, the OD values in each hole are measured at 570nm.
Stimulus index is experimental port and the ratio of negative control hole optical density OD, and ConA stimulations are represented with ConA stimulus indexes
The proliferative capacity of lower immunocyte.
2.6 macrophage IL-1 β, IL-6 and the detection of TNF-α secretion ability
The macrophage that 2.2 are obtained is inoculated in 24 orifice plates, and it is 1 × 10 to make cell density6A/hole, to each Kong Zhongjia
Enter to contain the RPMI1640 culture solutions 0.5mL of LPS (20 μ g/mL).In 37 DEG C, 5%CO2Supernatant is collected in lower culture afterwards for 24 hours, is pressed
ELISA kit specification operates, and detects the content of IL-1 β, IL-6 and TNF-α in each group mouse macrophage.
2.7 Siberian solomonseal rhizome polysaccharides are to the facilitation of Cultured Mouse spleen lymphocyte proliferation
The spleen of normal mouse is taken, obtains splenocyte suspension, after removing red blood cell, is inoculated in 96 orifice plates, makes cell close
Spend is 2 × 105A/hole is divided into 6 groups, and every group sets 6 multiple holes.Each group is separately added into 0,25,50,100,200,400 μ g/mL's
Siberian solomonseal rhizome polysaccharide solution represents to add in per 100 μ L of hole, when Siberian solomonseal rhizome polysaccharide solution is 0 100 μ L distilled waters as blank group.37 DEG C,
5%CO2After lower culture 48h, 10 μ L, 5mg/mL MTT solution are added in into every hole, continues to cultivate 4h, culture solution is discarded, to every
150 μ L DMSO are added in hole, and are sufficiently mixed.Finally, the OD values in each hole are measured at 570nm.
2.8 statistical procedures
Experimental data is represented with mean ± standard deviation (Mean ± SE), for statistical analysis to data.It is used between two groups
Double tail t are examined, and are analyzed.First using one-way analysis of variance between multigroup, then compared two-by-two with SNK.P ＜ 0.05 are represented
Data have statistical significance；P<0.01, represent that data have significant difference.
3. result
Influence of 3.1 Siberian solomonseal rhizome polysaccharides to hypoimmunity mice index and spleen index and ConA stimulation Spleen cell proliferations
As shown in table 1, compared with Normal group, model group mouse spleen index, ConA stimulus indexes reduce (P<
0.05).Relative to model group, Siberian solomonseal rhizome polysaccharide can increase index and spleen index, ConA stimulus indexes, and in dosage correlation；Wherein,
Siberian solomonseal rhizome polysaccharide high dose group (150mgkg-1) index and spleen index is made to dramatically increase (P<0.01) ConA stimulus indexes, is made to increase (P<
0.05)。
Table 1：Siberian solomonseal rhizome polysaccharide to hypoimmunity mice index and spleen index and ConA stimulation Spleen cell proliferation influence (Mean ±
SE, n=10)
Compared with normal group#P<0.05,##P<0.01；The * P compared with model group<0.05,**P<0.01.
Influence of 3.2 Siberian solomonseal rhizome polysaccharides to hypoimmunity mice thymus index and ConA stimulation thymocyte proliferations
As shown in table 2, model group mouse thymus index, ConA stimulus indexes are declined (P compared with Normal group<
0.05).And Siberian solomonseal rhizome polysaccharide high dose group (150mgkg-1) thymus index apparent increase (P<0.01), ConA stimulus indexes increase
(P<0.05)；And Siberian solomonseal rhizome polysaccharide group, with the increase of dosage, thymus index, ConA stimulus indexes accordingly increase.
Table 2：Siberian solomonseal rhizome polysaccharide to hypoimmunity mice thymus index and ConA stimulation thymocyte proliferation influence (Mean ±
SE, n=10)
Compared with normal group#P<0.05,##P<0.01；The * P compared with model group<0.05, * * P<0.01.
Influence of 3.3 Siberian solomonseal rhizome polysaccharides to hypoimmunity mice macrophages secrete IL-1 β, IL-6 and TNF-α
As shown in table 3, compared with Normal group, the content drop of model group mouse macrophage secretion IL-6 and TNF-α
Low (P ＜ 0.05).Compared with model group, Siberian solomonseal rhizome polysaccharide high dose group (150mgkg-1) mouse macrophage secretion IL-6 and
The content of TNF-α significantly increases (P ＜ 0.01).
Table 3：Influence of the Siberian solomonseal rhizome polysaccharide to hypoimmunity mice macrophages secrete IL-1 β, IL-6 and TNF-α
Compared with normal group#P<0.05,##P<0.01；The * P compared with model group<0.05, * * P<0.01.
Influence of 3.4 Siberian solomonseal rhizome polysaccharides to hypoimmunity mice macrophages phagocytic capacity
As shown in Figure 1, compared with Normal group, model group mouse phagocytic activity significantly reduce (##P ＜ 0.01).With mould
Type group compares, and the basic, normal, high dosage group mouse of Siberian solomonseal rhizome polysaccharide is increased (* P ＜ 0.05), and good dose-effect relationship is presented.
3.5 Siberian solomonseal rhizome polysaccharides are to the facilitation of normal mouse splenocyte in-vitro multiplication activity
As shown in Fig. 2, compared with blank group, Siberian solomonseal rhizome polysaccharide can promote normal mouse Spleen cell proliferation, with dosage in dependence
Relationship；And dosage be 200,400 μ g/mL when, Spleen cell proliferation (* * * P ＜ 0.001) can be obviously promoted.
Claims (10)
1. a kind of preparation method of Siberian solomonseal rhizome polysaccharide, which is characterized in that include the following steps：
（1）Sealwort raw medicinal herbs is cleaned, is deposited on clean table top after adding bubble, is covered with wet cloth, during which sprays clear water, profit
It without the dry heart in medicinal material, then steams to yellow black, soft for degree, closes fire and continue to boil in a covered pot over a slow fire to appearance brownish black, it is glossy, it takes the dish out of the pot, dries in the air
To outer dry interior profit, 3~5 mm sheets are cut, are dried；
（2）Take step（1）Slice, smash, with ethanol water refluxing extraction 2-3 times of 80 ~ 95 v/v%, filtering, institute
Obtain dregs of a decoction drying；
（3）To step（2）Drying after the dregs of a decoction in add in distilled water, in 80 DEG C of refluxing extractions 2-4 times, filtering, filtrate concentration
To medicinal extract state, ethyl alcohol is added in, until ethanol content is 70-90 v/v%, is stirred evenly, 8-12 h are then placed at 4 DEG C,
Filtering, precipitation drying is to get Thick many candies；
（4）By step（3）Gained Thick many candies remove deproteinized with Sevage methods；
（5）By step（4）After the polysaccharide water dissolution after deproteinized, dialyse 48 h in flowing water；Take dialyzate vacuum refrigeration
It is dry, obtain Siberian solomonseal rhizome polysaccharide.
2. preparation method according to claim 1, which is characterized in that the step（1）In in food steamer steam 4~8 h, close
Fire continues stewing 4~8 h, takes the dish out of the pot.
3. preparation method according to claim 2, which is characterized in that step（1）Described in drying to be placed in constant temperature drying
70 DEG C of drying of case.
4. preparation method according to claim 3, which is characterized in that step（2）Described in drying temperature be less than 50 DEG C.
5. preparation method according to claim 4, which is characterized in that step（2）Described in 80 ~ 95 v/v% ethanol waters
The mistake of solution refluxing extraction is known as：First with 95 v/v% ethyl alcohol in 60 DEG C of 3 h of reflux, then filtering is added in into obtained solid
80 v/v% ethyl alcohol are in 60 DEG C of reflux 2h, filtering, dregs of a decoction drying.
6. preparation method according to claim 5, which is characterized in that step（3）Described in add in distilled water in 80 DEG C return
Flowing extraction process is：To step（2）Distilled water refluxing extraction 3 times at 80 DEG C are added in the dregs of a decoction after drying, every time 3 h, mistake
Filter, merging filtrate.
7. preparation method according to claim 6, which is characterized in that the step（3）The dregs of a decoction after middle drying with every time
Distilled water amount ratio is 1 g during extraction：20-30 mL.
8. Siberian solomonseal rhizome polysaccharide is in the adjuvant for preparing antitumor drug made from the preparation method according to one in claim 1-7
In application.
9. the answering in medicament for immunity enhancement is prepared of Siberian solomonseal rhizome polysaccharide made from the preparation method according to one in claim 1-7
With.
10. Siberian solomonseal rhizome polysaccharide made from the preparation method according to one in claim 1-7 is lured in preparation treatment by cyclophosphamide
Application in the immunocompromised drug led.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110655591A (en) * | 2019-10-29 | 2020-01-07 | 广西壮族自治区中国科学院广西植物研究所 | Extraction method of polygonatum polysaccharide |
| CN113966834A (en) * | 2021-07-02 | 2022-01-25 | 厦门医学院 | Polygonatum cyrtonema processing or preprocessing method and application thereof |
| CN115043955A (en) * | 2022-06-29 | 2022-09-13 | 西南林业大学 | Polygonatum polysaccharide extract and preparation method thereof |
| CN115844017A (en) * | 2022-11-07 | 2023-03-28 | 安徽农业大学 | Yellow big tea polysaccharide composition for improving immunity and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1796418A (en) * | 2004-12-30 | 2006-07-05 | 中国科学院上海药物研究所 | Immune polysaccharide of rhizome of manyflower solomonseal, composition and application |
| CN104106763A (en) * | 2014-06-19 | 2014-10-22 | 无限极（中国）有限公司 | Application of polygonatum polysaccharide in preparation of drugs for assisting inhibition of colon cancer |
-
2018
- 2018-02-12 CN CN201810151879.8A patent/CN108164615A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1796418A (en) * | 2004-12-30 | 2006-07-05 | 中国科学院上海药物研究所 | Immune polysaccharide of rhizome of manyflower solomonseal, composition and application |
| CN104106763A (en) * | 2014-06-19 | 2014-10-22 | 无限极（中国）有限公司 | Application of polygonatum polysaccharide in preparation of drugs for assisting inhibition of colon cancer |
Non-Patent Citations (3)
| Title |
|---|
| GUANGPU LIU等: "Hepatoprotective effects of polysaccharides extracted from Zizyphus jujube cv. Huanghetanzao", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| NA LIU等: "Characterization and protective effect of Polygonatum sibiricum polysaccharide against cyclophosphamide-induced immunosuppression in Balb/c mice", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 叶定江主编: "《中药炮制学（第1版）》", 31 May 1996, 上海科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110655591A (en) * | 2019-10-29 | 2020-01-07 | 广西壮族自治区中国科学院广西植物研究所 | Extraction method of polygonatum polysaccharide |
| CN113966834A (en) * | 2021-07-02 | 2022-01-25 | 厦门医学院 | Polygonatum cyrtonema processing or preprocessing method and application thereof |
| CN113966834B (en) * | 2021-07-02 | 2023-10-20 | 厦门医学院 | Polygonatum cyrtonema processing or preprocessing method and application thereof |
| CN115043955A (en) * | 2022-06-29 | 2022-09-13 | 西南林业大学 | Polygonatum polysaccharide extract and preparation method thereof |
| CN115844017A (en) * | 2022-11-07 | 2023-03-28 | 安徽农业大学 | Yellow big tea polysaccharide composition for improving immunity and application thereof |
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