CN102757510A - Low-molecular weight schisandra chinensis polysaccharides and preparation method and application thereof - Google Patents
Low-molecular weight schisandra chinensis polysaccharides and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses low-molecular weight schisandra chinensis polysaccharides and a preparation method and an application thereof, and belongs to the technical field of medicine. The molecular weight of the polysaccharide component is 3.4*10<3> Da, and the monosaccharide composition is as follows: mannose:glucose:galactose=1:11.38:3.55; and the polysaccharide component contains (1-3)-alpha-glucan and has a tri-helix structure. The study of in vivo activity against liver cancer of SCPP11 polysaccharide component shows that the polysaccharide component is significantly active against liver cancer (HepG-2), can significantly inhibit growth of liver cancer, and can significantly enhance the autoimmunity and in vivo antioxidant activity of mice as compared with the 5-Fu. The water-soluble low-molecular weight schisandra chinensis polysaccharides have the advantages of definite active components and controllable quality, and can be used as a drug against liver cancer or a health-care product for improving immunity.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of preparation method and the application aspect anti-liver cancer and enhance immunity power thereof of lower molecular weight shizandra berry polysaccharide.
Background technology
Malignant tumour is universally acknowledged to one of the most serious disease of human health risk, and its hazard rating is only second to cardiovascular disorder.According to the GLOBOCAN of international cancer research institution 2008 statistical reports (Jemal, A., Bray, F.; Center, M.M., Ferlay, J.; Ward, E., Forman; D..Global Cancer Statistics.CA:A Cancer Journal for Clinicians.2011,61 (2), 69-90; GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide:International Agency for Research on Cancer; Available at:
Http:// globocan.iarc.fr/.), 2008, there were 1,270 ten thousand New Development cases of cancers in the whole world, 7,600,000 people die from cancer, prediction the year two thousand thirty, the cancer mortality case estimate to rise 72%, death toll by 2008 7,600,000 rise to 1,300 ten thousand people.It is thus clear that malignant tumour has been that the world today influences one of significant problem of social development.Liver cancer is the common malignancy disease, and (GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide:International Agency for Research on Cancer according to statistics; Available at:
Http:// globocan.iarc.fr/.) whole world had 69.4 ten thousand people to die from liver cancer 2008 years, its mortality ratio ranked third the position in the world, and the sickness rate of liver cancer has rising tendency.The medicine of treatment tumour is a lot; But owing to lack the metabolism difference of essence between tumour cell and normal tissue cell; Most of antitumour drugs also can cause damage to some normal cell, tissue and the organ of body in inhibition or killing tumor cell; To the human body toxigenicity, restricted the use of medicine to a great extent.Therefore, the research and development low-poison efficient antineoplastic medicine is one of emphasis problem of current research.
Polysaccharide is a kind of natural high moleculer eompound; Have antitumor, immunomodulatory, various active such as anti-oxidant; And toxicity is low, safe; Have good antitumor drug application prospect (Pang Chunhong. the polysaccharide Advance on Pharmacological Activities. Zhejiang University of Traditional Chinese Medicine's journal, 2011,35 (4): 639-640.).Yu Rongmin etc. have applied for patent " a kind of Preparation method and use with yew amylose of antitumor action " (application number: 200910192993.6); The yew amylose solution of finding different concns is all inhibited to multiple cancer cells (HBT's cell MCF-7, people's fibroma cell HT1080, human cervical carcinoma cell Hela, the chronic marrow sexual cell of people K562, Human Prostate Cancer Cells PC3, human liver cancer cell SMMC7721, people's acute promyelocytic leukemia cell HL-60), and restraining effect and concentration are linear.The patent of applications such as Zhu Zhenyuan " a kind of water-soluble low-molecular-weight cordyceps polysaccharide " (application number: 200910070325.6) with anti-tumor activity; Find that this polysaccharide has the propagation of human hepatoma cell strain Bel-7204 and human stomach cancer cell line SGC-7901 and suppress effect significantly; Growth to implanting the intravital S180 tumour cell of mouse has the obvious suppression effect, but and enhance immunity function.
Shizandra berry is Magnoliaceae shizandra berry (Schisandra chinensis (Turcz.) Baill; Be commonly called as Schisandra chinensis) or schisandra chinensis (S.sphenanthera Rehd.et Wils; Be commonly called as Fructus Schisandrae Sphenantherae) dry mature fruit; Because of sweet, sour, hot, bitter, the salty five tastes of its fruit, thus the name shizandra berry, have another name called five plums, profound and, can and, Fructus Zanthoxyli Plansipini etc.Effect with astringent or styptic treatment for spontaneous sweating, the supplementing QI for promoting the production of body fluid of convergence and kidney calming; Be used for that chronic cough void is breathed heavily, emission, enuresis frequent micturition, incessant chronic diarrhea, spontaneous perspiration, night sweat, Tianjin wound is thirsty and disease such as palpitation and insomnia; For the traditional Chinese medical science strong kidney medicine of enriching yin commonly used, be widely used in sick treatment such as hepatitis, neurasthenia.The shizandra berry aboundresources contains lignanoid, polysaccharide, volatilization wet goods various active composition, is the Chinese medicinal materials of the wide dietotherapeutic of a kind of DEVELOPMENT PROSPECT.Multiple pharmacological effect (Yan Shu such as polysaccharide is as one of important activity composition in the shizandra berry, and modern pharmacology research shows, it has antitumor, immunological enhancement, leukocyte increasing, protects the liver, anti-ageing and hypoglycemic; Face upward pomegranate green grass or young crops, Zhao Ting, Zhao Jiangli; Zou Yanmin. the latest Progress of shizandra berry polysaccharide [J]. Jiangsu University's journal, (medicine), 2009; 19 (4): 366-368.), become the focus that people pay close attention to.The patent " preparation method of schisandra active component polysaccharides " of application such as Zhu Beiwei (application number: 200710159047.2), a kind of method of from shizandra berry, extracting active component polysaccharides is provided.Yu He etc. discover that the shizandra berry Crude polysaccharides has the anti-tumor in vivo activity, and can improve the immunity of organism of tumor-bearing mice.(in conspicuous, Li Ji, Qi Yan. the shizandra berry polysaccharide is to the restraining effect and the amynologic mechanism pre-test thereof of liver cancer mouse tumor growth. tcm-information, 2010,27 (2): 26-27; In conspicuous, Li Ji, Wang Yanjie. the shizandra berry polysaccharide is to H
22The intervention effect of tumor-bearing mice serum il-2, IL-10 and vegf expression grinds. tcm-journal 2010,38 (2): 39-41; In conspicuous, Li Ji, the refined lotus shizandra berry of Lee polysaccharide is to H
22Tumor-bearing mice serum Zn, Se level change in preact research. tcm-information, 2010,27 (3): 25-26.).A kind of pure shizandra berry polysaccharide of discoveries such as Xu Keyu (content 60.3mg/g) can suppress survivin effectively on cell levels expresses; Increase the apoptosis rate (Xu Keyu of cell; Xiao Jianying. the shadow of sub-polysaccharide of distinguishing the flavor of to thyroid carcinoma cell strain SW579 apoptosis and survivin expression. Jilin University's journal; 2011,37 (2): 279-284.).The inventor carries out separation and purification to the shizandra berry polysaccharide, confirms the constitutional features of polysaccharide fraction, anti-liver cancer (HepG-2) activity in the research shizandra berry polysaccharide purification component body.But the domestic and foreign literature report is not seen in the research of anti-human liver cancer cell HepG-2 in the at present relevant shizandra berry polysaccharide purification component body, does not see that corresponding patent is open yet.
Summary of the invention
Based on the deficiency that exists in the above background technology, the object of the present invention is to provide a kind of preparation method and its medicinal application with herbal medicine efficient part-lower molecular weight shizandra berry polysaccharide fraction of oncotherapy effect.
One, first purpose of the present invention is to provide a kind of lower molecular weight shizandra berry polysaccharide fraction (SCPP11).
A kind of lower molecular weight shizandra berry polysaccharide (SCPP11), this polysaccharide molecular weight are 3.4 * 10
3Da; Monose consists of: seminose (man): glucose (glu): semi-lactosi (gal)=1: 11.38: 3.55; This polysaccharide has α-glycosidic link and β-glycosidic link; This polysaccharide has (1 → 3)-α-glucan (VISOSE); This polysaccharide has the triple helix structure.
Two, second purpose of the present invention is to provide the preparation method of above-mentioned lower molecular weight shizandra berry polysaccharide fraction (SCPP11), carries out according to following step:
(1) process for extracting of shizandra berry Crude polysaccharides:
With the raw material washing, 60 ℃ of oven dry are pulverized, and are put in the apparatus,Soxhlet's, and sherwood oil (boiling range 60-90 ℃) degreasing 15h filters, and 60 ℃ of oven dry of filter residue are subsequent use; The degreasing Chinese Magnolivine Fruit is with solid-liquid ratio 1: 8-1: 12; In temperature 80-100 ℃ water, extract 3-5h, extract 1-3 time, merge supernatant; Supernatant is after concentrating; Add the ethanol alcohol precipitation, make that alcohol concn is 80% in the liquid concentrator of polysaccharide, the gained deposition is centrifugal, behind lyophilize 24-48h the shizandra berry Crude polysaccharides; It is an amount of to get the shizandra berry Crude polysaccharides, and the back use trichloroacetic acid method that redissolves removes albumen, except that the dialysis of the liquid glucose behind albumen freeze-drying, obtains deproteinated shizandra berry polysaccharide.
(2) preparation of lower molecular weight shizandra berry polysaccharide
Get deproteinated shizandra berry polysaccharide, with the suitable quantity of water dissolving, the centrifugal insolubles of removing, supernatant separates through the DEAE-52 ion exchange column; Progressively with the NaCl aqueous solution wash-out of 0-0.2mol/L; Draw elution curve with sulfuric acid-phynol method, collect the merging elutriant respectively according to elution curve, wherein the elutriant of the 0mol/L NaCl aqueous solution (being water) is after concentrating, dialysing; Last sephadex column; With 0.1mol/L NaCl aqueous solution wash-out, collect the merging elutriant according to elution curve, through concentrate, dialysis, freeze-drying make low score son amount shizandra berry polysaccharide.
Three, the 3rd purpose of the present invention is to provide lower molecular weight shizandra berry polysaccharide fraction (SCPP11) as the application in medicines resistant to liver cancer and the enhance immunity functional health care product.
Four, according to the present invention, this polysaccharide mainly uses with oral form of medication, and the oral administration form mainly contains tablet, capsule and granule etc.
The advantage of this invention is: the present invention is to be raw material with five tastes of medicinal herb, through petroleum ether degreasing, hot water lixiviate, filtration, concentrate, prepared such as alcohol precipitation, ion exchange chromatography form, and are simple to operate, with low cost; Anti-liver cancer (HepG-2) activity research shows in the body, and this water-soluble low molecular weight shizandra berry polysaccharide fraction can significantly suppress tumor propagation, and toxicity is low, can significantly improve anti-oxidant activity in mouse autoimmunization and the body; This polysaccharide fraction can be used as medicines resistant to liver cancer and enhance immunity functional health care product.
Description of drawings
Fig. 1 is the IR spectrogram of SCPP11;
Fig. 2 is SCPP11
13The C-NMR spectrogram;
Fig. 3 is the maximum absorption wavelength changing trend diagram of SCPP11-Congo red mixture;
Fig. 4 is the influence of SCPP11 to tumor-bearing mice TNF secretion-α and IL-2, and wherein (a) is to the influence of TNF-α; (b) to the influence of IL-2.
Embodiment
With the raw material washing, 60 ℃ of oven dry are pulverized, and are put in the apparatus,Soxhlet's, and sherwood oil (boiling range 60-90 ℃) degreasing 15h filters, and 60 ℃ of oven dry of filter residue are subsequent use.Take by weighing 100g degreasing Chinese Magnolivine Fruit, place round-bottomed flask, 80 ℃ of press solid-liquid ratio 1: 8, extraction time 3h, extraction temperature, the backflow lixiviate is 1 time in hot water bath, and the cooling back is centrifugal, merges supernatant; Above-mentioned clear liquid adds 4 times of volume 95% ethanol and precipitates through concentrating under reduced pressure, makes that alcohol concn is 80% in the liquid concentrator of polysaccharide, the gained deposition behind centrifugal, lyophilize 24h shizandra berry Crude polysaccharides sample powder; It is an amount of to get the shizandra berry Crude polysaccharides, redissolves the back to use trichloroacetic acid method to remove albumen, falls the liquid glucose dialysis freeze-drying behind the albumen, obtains deproteinated shizandra berry polysaccharide.
With the raw material washing, 60 ℃ of oven dry are pulverized, and are put in the apparatus,Soxhlet's, and sherwood oil (boiling range 60-90 ℃) degreasing 15h filters, and 60 ℃ of oven dry of filter residue are subsequent use.Take by weighing 100g degreasing Chinese Magnolivine Fruit, place round-bottomed flask, 90 ℃ of press solid-liquid ratio 1: 10, extraction time 4h, extraction temperature, the backflow lixiviate is 2 times in hot water bath, and the cooling back is centrifugal, merges supernatant; Above-mentioned clear liquid adds 4 times of volume 95% ethanol and precipitates through concentrating under reduced pressure, makes that alcohol concn is 80% in the liquid concentrator of polysaccharide, the gained deposition behind centrifugal, lyophilize 36h shizandra berry Crude polysaccharides sample powder; It is an amount of to get the shizandra berry Crude polysaccharides, redissolves the back to use trichloroacetic acid method to remove albumen, falls the liquid glucose dialysis freeze-drying behind the albumen, obtains deproteinated shizandra berry polysaccharide.
Embodiment 3
With the raw material washing, 60 ℃ of oven dry are pulverized, and are put in the apparatus,Soxhlet's, and sherwood oil (boiling range 60-90 ℃) degreasing 15h filters, and 60 ℃ of oven dry of filter residue are subsequent use.Take by weighing 100g degreasing Chinese Magnolivine Fruit, place round-bottomed flask, 100 ℃ of press solid-liquid ratio 1: 12, extraction time 5h, extraction temperature, the backflow lixiviate is 3 times in hot water bath, and the cooling back is centrifugal, merges supernatant; Above-mentioned clear liquid adds 4 times of volume 95% ethanol and precipitates through concentrating under reduced pressure, makes that alcohol concn is 80% in the liquid concentrator of polysaccharide, the gained deposition behind centrifugal, lyophilize 48h shizandra berry Crude polysaccharides sample powder; It is an amount of to get the shizandra berry Crude polysaccharides, redissolves the back to use trichloroacetic acid method to remove albumen, falls the liquid glucose dialysis freeze-drying behind the albumen, obtains deproteinated shizandra berry polysaccharide.
The preparation of embodiment 4 shizandra berry polysaccharide:
The purifying of step 1, deproteinated shizandra berry polysaccharide
1, experiment material
1.1 medicine and reagent
Deproteinated shizandra berry polysaccharide;
DEAE-52 Mierocrystalline cellulose exchange resin, Britain Whatman company;
Sephadex G-100, Britain Whatman company.
Dextran T-10, T-40, T-70, T-500, T-2000 and Blue Dextran T-2000, Pharmacia company.
1.2 instrument
The UV-7502PC visible spectrophotometer, the luxuriant glad Instr Ltd. in Shanghai
Chromatography column: 1.6cm * 50cm, Shanghai Hu Xi analytical instrument Co., Ltd., Factory
DHL-A computer constant flow pump, Shanghai Hu Xi analytical instrument Co., Ltd., Factory
DBS-100 computer automatic fraction collector, Shanghai Hu Xi analytical instrument Co., Ltd., Factory
2, experimental technique
Take by weighing the deproteinated shizandra berry polysaccharide 200mg for preparing among the foregoing description 1-3; Be dissolved in the 10mL zero(ppm) water, fully the dissolving back is centrifugal, and supernatant is added drop-wise in the DEAE-52 chromatography column; 0-0.2mol/L the NaCl aqueous solution carry out wash-out; Use the DHL-A constant flow pump, control flow velocity 1mL/min collects with automatic fraction collector.6min collects a pipe, and every pipe is collected 6mL, and collect the every effective phenolsulfuric acid method tracking of liquid and detect, be blank with zero(ppm) water, measure absorbancy down in the wavelength of 490nm, detect up to no longer including sugar.According to elution curve, merge the elutriant of each elution peak, water elution liquid part concentrating under reduced pressure wherein, freeze-drying; Sephadex column on the above-mentioned steps gained polysaccharide fraction with 0.1mol/LNaCl aqueous solution wash-out, uses the DHL-A constant flow pump, control flow velocity 0.3mL/min; Collect with branch's automatic collector, every pipe is collected 3mL, collects the every effective phenolsulfuric acid method of liquid and follows the tracks of detection; With zero(ppm) water is blank, measures absorbancy down in the wavelength of 490nm, detects up to no longer including sugar.Draw elution curve, merge the elutriant of elution peak, use distill water dialysis 72h, concentrating under reduced pressure, freeze-drying obtains shizandra berry polysaccharide fraction (SCPP11).
3, experimental result
Obtain water-soluble low molecular weight shizandra berry polysaccharide fraction (SCPP11).
The physico-chemical property and the constitutional features of step 2, water-soluble low molecular weight shizandra berry polysaccharide
1, experiment material
1.1 medicine and reagent
The shizandra berry polysaccharide; Sodium-chlor, sulfuric acid, trichoroacetic acid(TCA), phenol, sodium hydroxide, hydrochloric acid, sodium-acetate, oxammonium hydrochloride, inositol, pyridine, acetic anhydride, glucose, rhamnosyl, wood sugar, pectinose, seminose, sodium periodate, the analytical pure that is such as Congo red.
1.2 instrument
The LC-10AVP highly effective liquid phase chromatographic system, day island proper Tianjin
TSK-GEL G4000PW (7.5 * 300mm), TOSOH company
Pre-column: TSK-GUARD COLUMN PWH (7.5 * 75mm), TOSOH company
The ALPHA2-4 freeze drying equipment, German CHRIST company
PHS-2C type acidometer, Shanghai analytical instrument factory
R-200 type rotatory evaporator, Switzerland B ü chi company
GC 2010 gas chromatography systems, day island proper Tianjin
RTS-5 gas phase capillary column, (30m * 0.32mm)
NEXUS 670 intelligent Fourier infrared spectrographs, U.S. Ni Gaoli company
NMR, Braker
2, experimental technique
2.1 the mensuration of molecular weight
According to the ordinary method of polysaccharide, with the known T-10 of molecular weight, T-40; T-70, T-500 and T-2000 are standard, Tianjin, LC-10AVP island highly effective liquid phase chromatographic system; Chromatographic column is that (7.5 * 300mm), moving phase is the sodium acetate soln of 0.003mol/L to TSK-GEL G4000PW, flow velocity 0.8mL/min; Detector SHIMADZU RID-10A, 25 ℃ of temperature.Polysaccharide soln is crossed the filter membrane of 0.45 μ m, sample introduction 10 μ L.
2.2 monose compositional analysis
Precision takes by weighing polysaccharide sample 5mg in ampoule, with the sulphuric acid soln dissolving of 2mol/L, seals, and behind the heating hydrolysis 8h, hydrolyzed solution adds barium carbonate and is neutralized to neutrality in 100 ℃ of baking ovens, with the centrifugal BaSO that removes of 4000r/min
4Deposition, the supernatant lyophilize.Hydrolysate after acetylize with Tianjin, island GC 2010 gas chromatography system analyses.
2.3IR measure
Get 1mg through exsiccant polysaccharide sample, in agate mortar, grind evenly (operation under ir lamp) gently through exsiccant KBr powder with 100～200mg.Be pressed into thin slice through tabletting machine, measure in NEXUS 670 intelligent Fourier infrared spectrographs and obtain infrared spectrogram.
2.4NMR measure
Get polysaccharide sample 20mg through D
2After the O exchange 3 times, be dissolved in 0.5mLD
2Among the O, with BRUKER400 type NMR,
13C-NMR (100MHz, temperature 673.2K) (referring to Fig. 2).
2.5 Congo red experiment
Take by weighing shizandra berry polysaccharide fraction 5mg; The Congo red reagent that adds 2mL zero(ppm) water and 2mL80 μ mol/L adds the NaOH solution of 1mol/L afterwards gradually, and the concentration that makes mixed solution is brought up to 0.5mol/L (0,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5mol/L) gradually from 0; And scan with ultraviolet-visual spectrometer; Measure maximum light absorption value under each alkaline gradient (reference is the Congo red reagent of 2mL zero(ppm) water and 2mL80 μ mol/L, adds the NaOH solution of 1mol/L gradually, makes the concentration of mixed solution progressively bring up to 0.5mol/L from 0); With NaOH concentration is X-coordinate, and maximum absorption wavelength is that ordinate zou is drawn.
3, experimental result
The SCPP11 molecular weight is 3.4 * 10
3Da.SCPP11 is by seminose, and glucose and semi-lactosi are formed, and its mol ratio is a seminose: glucose: semi-lactosi=1: 11.38: 3.55.The shizandra berry polysaccharide is at 3395cm
-1, 2927cm
-1, 1384cm
-1And 1025-1152cm
-1Strong absorption peak all appears in the place, (is the charateristic avsorption band of sugar, wherein 3395cm
-1Be-OH stretching vibration peak, 2927cm
-1And 1384cm
-1Be respectively C-H stretching vibration peak and C-H flexural vibration peak, the 1025-1152cm of methylene radical
-1Vibration peak for pyranose ring); 852cm
-1And 893cm
-1Charateristic avsorption band shows that SCPP11 has α-glycosidic link and β-glycosidic link simultaneously; 855cm
-1And 931cm
-1Charateristic avsorption band shows that SCPP11 has (1 → 3)-α-glucan.Fig. 2 is the shizandra berry polysaccharide
13The C-NMR spectrogram, δ 104ppm, δ 102ppm, δ 99ppm, δ 95ppm and δ 91ppm are the chemical shift of anomeric carbon, show that SCPP11 has α-glycosidic link and β-glycosidic link simultaneously; Substituted C for not taking place in the peak between δ 77～70ppm
2, C
3, C
4, C
5Chemical shift; Substituted C for not taking place in δ 61 and δ 60ppm
6Chemical shift.In addition, 170～180ppm does not see carbon signal, shows that it does not contain uronic acid, with infrared consistent with the compositional analysis result.Fig. 3 is SCPP11 and the Congo red maximum absorption wavelength variation that has formed complex compound, the maximum absorption wavelength red shift that the maximum absorption wavelength of complex compound is Congo redder.When NaOH concentration raise, the maximum absorption wavelength of complex compound sharply descended, and explained that the NaOH of high density has destroyed the spirane structure of polysaccharide, it is disintegrated be a sub-thread ball of string, and cause maximum absorption wavelength to descend, and inferred that SCPP11 has triple-helix structure.
Test anti-liver cancer (HepG-2) activity research in the shizandra berry polysaccharide fraction body
1 instrument, material and reagent
1.1 instrument
Spectra Max 190 ELIASAs, U.S. molecular device company
Sigma 1-13 high speed tabletop centrifuge, Sigma company
The digital control constant temperature water-bath, the Ying Yu of Gongyi City gives magnificent instrument plant
HSS-1 type thermostatic bath, Jintan, Jiangsu Medical Instruments factory, China
BS124S type electronic balance, Beijing Sai Duolisi instrument system ltd
Refiner, German IKA company
1.2 material and reagent
1.2.1 laboratory animal
The ICR mouse, female, body weight 20 ± 2g, the cleaning level is purchased in Yangzhou University comparative medicine center, conformity certification numbering: SCXK (Soviet Union) 2007-0001.Quarantine 3d before the experiment, raising temperature is 21 ± 1 ℃, humidity is 60 ± 5%.
1.2.2 experiment reagent and medicine
Shizandra berry polysaccharide (self-control); 5-fluor-uracil; Saline water; TNP;
The IL-2 enzyme linked immunological kit; TNF-α enzyme linked immunological kit; Mda (MDA) test kit; Superoxide dismutase (SOD) test kit; Bio-engineering research institute is built up in Nanjing;
Glacial acetic acid min. 99.5, CaCl
2Deng being commercially available analytical pure all.
1.3 statistical procedures
Data are handled with the SPSS15.0 statistical software.The result is expression with
, and two group differences adopt the t-check; Many group differences adopt one-way analysis of variance (One-way ANOVA).There is statistical significance P＜0.05.
2 experimental techniques
2.1 guarantor's kind of tumor-bearing mice knurl strain, go down to posterity and the transplanted tumor modelling
(1) guarantor's kind of knurl strain
Get 2～3 of the kunming mices of body weight 20 ± 2g, every meets 0.2ml, and the strain of abdominal cavity inoculation Heps ascitic tumor knurl passed a generation in regularly per 7～9 days.
(2) foundation of oxter transplanted tumor model
Get that went down to posterity 7 days in the abdominal cavity and well-grown Heps ascitic tumor mouse; Take off cervical vertebra and put to death, 75% ethanol disinfection animal skin is under aseptic condition; Cut off mouse peritoneal; Draw well-grown ascites, observe the ascites color (ascites be the oyster white person that do not have the flocculent precipitate for good, bloody ascites is then unavailable) and with trypan blue staining observation viable count (viable count＞98%); Ascites is diluted with saline water, the piping and druming mixing, and the cell counting count board counting, the adjustment cell count is 2 * 10
7Individual viable cell/mL; Get Kunming mouse, armpit skin conventional alcohol disinfecting in right side is held the 1mL syringe in the right oxter subcutaneous injection 0.2mL of mouse tumor cell suspension, processes focal tumor model.
2.2 experiment is divided into groups and administration
50 female mices were fed 3 days in the experiment preadaptation, and the inoculation back is divided into 5 groups with it next day at random, 10 every group, establishes normal group, model group, positive controls (5-Fu), high and low two dose groups of SCPP11; Begin administration behind the inoculation 24h, every day 1 time, 10d continuously.(irritate the stomach amount and only be 0.2mL/, abdominal injection 0.1ml/ is only) grouping, dosage and route of administration are following:
Normal group: healthy mice, irritate every day the stomach same dose saline water (0.2mL/10g/d, i.g)+intraperitoneal injection of saline (0.1mL/10g/d, i.p), ad lib and drinking-water;
Model group: the inoculation mouse, irritate every day the stomach same dose saline water (0.2mL/10g/d, i.g)+intraperitoneal injection of saline (0.1mL/10g/d, i.p), ad lib and drinking-water;
The 5 FU 5 fluorouracil group: the inoculation mouse, irritate every day the stomach same dose saline water (0.2mL/10g/d, i.g)+5-Fu of abdominal injection same dose (25mg/kg/d, i.p), ad lib and drinking-water;
Shizandra berry polysaccharide administration group: the inoculation mouse, divide high and low two dose groups,
1. SCPP11-H irritate respectively every day stomach same dose shizandra berry polysaccharide (200mg/kg/d, i.g/), ad lib and drinking-water;
2. SCPP11-L irritate respectively every day stomach same dose shizandra berry polysaccharide (50mg/kg/d, i.g/), ad lib and drinking-water;
2.3 the relevant index of investigating:
2.3.1, the growth of animal situation
Observe animal every day once, observing animal mental status, behavior and hair color has no abnormally, weighs and writes down to understand the weight of animals changing conditions (weighing in 0,14 day).
2.3.2, organ exponential sum tumour inhibiting rate
Next day after the last administration, fasting 8h weighs, and plucks and puts to death after eyeball is got blood; Take out knurl piece, thymus gland, spleen, the heart, liver, kidney, after the saline water washing, blot, weigh with filter paper.Be calculated as follows tumour inhibiting rate and each organ index:
Tumour inhibiting rate (%)=(C-T)/C * 100%
C is that the average knurl of model control group is heavy in the formula, and T is that the average knurl of experimental group is heavy.
Organ index=organ weights (mg)/body weight (g)
2.3.3, hematological indices
Blood 0.5～1mL is got in last administration 24 as a child back eyeball, collects blood in anticoagulant tube (purple: EDTA-K2; In the routine blood test (blood cell analysis), fully behind the mixing,, subsequent use in 4 ℃ of refrigerator storage.Carry out routine analysis of blood.
2.3.4, blood parameters
Pluck eyeball behind the last administration 24h and get blood 0.5～1mL, collect blood, centrifugal behind the placement 4h, the centrifugal 15min of 3000r/min gets serum and places 4 to preserve, and is subsequent use.Measure gpt (ALT) and glutamic-oxal(o)acetic transaminase (AST).
The content of IL-2 and TNF-α in the enzyme linked immunological kit mensuration serum.
2.3.5, to the influence of SOD and MDA in the conscience nephridial tissue
The heart, liver, kidney are respectively got and are placed the 10ml centrifuge tube about 150-400mg, add 7ml saline water, process 5% tissue homogenate, after 3000rpm/min is centrifugal, get supernatant, with the content of kit measurement mda (MDA) and superoxide dismutase (SOD).
2.3.6, data statistics
Data are handled with the SPSS15.0 statistical software.Expression that the result uses
is compared the significance between each administration group and the control group with one-way ANOVA check between group.There is statistical significance P＜0.05.
3, experimental result
3.1 SCPP11 is to the influence of HepG-2 tumor-bearing mice tumor growth
Table 1 SCPP11 is to the influence of HepG-2 tumor-bearing mice anti-tumor activity
Compare with the normal control group,
aP＜0.05
Compare with negative control group,
bP＜0.05
Compare with positive controls,
cP＜0.05 or
CcP＜0.01
As shown in table 1, administration is after 10 days, and SCPP11 has reached 68.54% to inhibiting rate in the body of HepG-2 when low dosage 50mg/kg, be higher than the inhibiting rate (61.71%) of positive drug (5-Fu).
3.2 SCPP11 is to the influence of HepG-2 tumor-bearing mice immunity
Table 2 SCPP11 is to the influence of organ index
Compare with the normal control group,
aP＜0.05
Compare with negative control group,
bP＜0.05
Compare with positive controls,
cP＜0.05 or
CcP＜0.01
SCPP11 is as shown in table 2 to the influence of tumor-bearing mice index of immunity, and administration was compared with negative control group after 10 days, and positive controls (5-Fu) thymus index and index and spleen index significantly reduce (p＜0.05), and visible, immunity has restraining effect to 5-Fu to tumor-bearing mice.And the thymus index of SCPP11 administration group and spleen index all have increase, and particularly when dosage was 50mg/kg, SCPP11 had significant difference (p＜0.05) to the increase of thymus index.It is thus clear that SCPP11 can activate the immunity system of tumor-bearing mice.
SCPP11 is as shown in Figure 4 to the influence of TNF-α in the tumor-bearing mice serum and IL-2 level, compares with negative control group, and the TNF-α and the IL-2 level of positive controls significantly reduce (p＜0.05), and visible 5-Fu has restraining effect to the secretion of these two kinds of cytokines.And SCPP11 is when dosage is 50mg/kg, and the secretion of TNF-α and IL-2 in the tumor-bearing mice serum is had certain promoter action.
3.3 SCPP11 is to the toxic influence of HepG-2 tumor-bearing mice
Can find out that from table 1 administration is after 10 days, the weight increase rate of SCPP11 administration group is significantly higher than positive controls, shows that SCPP11 is little to the body toxic side effect.
Table 3 SCPP11 is to the influence of tumor-bearing mice peripheral hemogram
Compare with the blank group,
aP＜0.05
Compare with negative control group,
bP＜0.05
Compare with positive controls,
cP＜0.05 or
CcP＜0.01
Can find out from table 3; 5-Fu can reduce tumor-bearing mice blood middle leukocytes quantity, erythrocyte number and platelet content, and SCPP11 can make thrombocyte and erythrocyte number normalizing in the tumor-bearing mice peripheral blood; And leukocytic quantity in the peripheral blood that can raise; Particularly when dosage is 50mg/kg, compare with positive controls with negative control group, leukocyte count has significant difference.
Table 4 SCPP11 is to the influence of tumor-bearing mice peripheral blood biochemical indicator
Compare with the blank group,
aP＜0.05
Compare with negative control group,
bP＜0.05
Compare with positive controls,
cP＜0.05 or
CcP＜0.01
Table 5 SCPP11 is to the influence of SOD activity and MDA level in the tumor-bearing mice liver kidney heart tissue
Compare with the blank group,
aP＜0.05
Compare with negative control group,
bP＜0.05
Compare with positive controls,
cP＜0.05 or
CcP＜0.01
Can find out from table 2, compare with the normal control group, the liver index of SCPP11 administration group, kidney exponential sum cardiac index does not all have significant difference; And the liver index of positive control has certain increase.Can find out that from table 4 5-Fu can significantly increase AST and the ALT concentration in the tumor-bearing mice serum, and SCPP11 can make the horizontal normalizing of AST in the tumor-bearing mice serum.Visible from table 5,5-Fu can reduce the activity of SOD in the tumor-bearing mice liver organization and the content of MDA, and SCPP11 can significantly improve the activity (p＜0.05) of SOD in the tumor-bearing mice liver organization when 50mg/kg.Therefore the 5-Fu liver has certain toxicity, and SCPP11 does not have liver toxicity and liver is had the certain protection effect.
4, conclusion
SCPP11 has anti-significantly liver cancer (HepG-2) activity, can significantly suppress the growth of liver cancer, and compares with 5-Fu, and this polysaccharide fraction toxicity is low, can significantly strengthen anti-oxidant activity in mouse autoimmunization ability and the body.
Claims (4)
1. a lower molecular weight shizandra berry polysaccharide is characterized in that this polysaccharide molecular weight is 3.4 * 10
3Da; Monose consists of: seminose (man): glucose (glu): semi-lactosi (gal)=1: 11.38: 3.55; This polysaccharide has α-glycosidic link and β-glycosidic link; This polysaccharide has (1 → 3)-α – VISOSE (glucan); This polysaccharide has the triple helix structure.
2. the preparation method of the described a kind of lower molecular weight shizandra berry polysaccharide of claim 1 is characterized in that carrying out according to following step:
(1) process for extracting of shizandra berry Crude polysaccharides:
With the raw material washing, 60 ℃ of oven dry are pulverized, and are put in the apparatus,Soxhlet's, and boiling range 60-90 ℃ petroleum ether degreasing 15 h filter, and 60 ℃ of oven dry of filter residue are subsequent use;
The degreasing Chinese Magnolivine Fruit is with solid-liquid ratio 1:8-1:12; In temperature 80-100 ℃ water, extract 3-5h, extract 1-3 time, merge supernatant; Supernatant is after concentrating; Add the ethanol alcohol precipitation, make that alcohol concn is 80% in the liquid concentrator of polysaccharide, the gained deposition is centrifugal, behind lyophilize 24-48h the shizandra berry Crude polysaccharides; It is an amount of to get the shizandra berry Crude polysaccharides, redissolves the back to use trichloroacetic acid method to remove albumen, falls the liquid glucose dialysis freeze-drying behind the albumen, obtains deproteinated shizandra berry polysaccharide;
(2) preparation of lower molecular weight shizandra berry polysaccharide:
Get deproteinated shizandra berry Crude polysaccharides, with the suitable quantity of water dissolving, the centrifugal insolubles of removing, supernatant separates through the DEAE-52 ion exchange column; Progressively with the NaCl aqueous solution wash-out of 0-0.2 mol/L; Draw elution curve with sulfuric acid-phynol method, collect the merging elutriant respectively according to elution curve, wherein the 0 mol/L NaCl aqueous solution be water elutriant through concentrate, after the dialysis; Last sephadex column; With 0.1 mol/L NaCl aqueous solution wash-out, collect the merging elutriant according to elution curve, through concentrate, dialysis, freeze-drying make low score son amount shizandra berry polysaccharide.
3. the application of the described a kind of lower molecular weight shizandra berry polysaccharide of claim 1 in the preparation medicines resistant to liver cancer.
4. the described a kind of lower molecular weight shizandra berry polysaccharide of claim 1 is used in the healthcare products of preparation enhance immunity function.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103156106A (en) * | 2013-02-04 | 2013-06-19 | 北华大学 | Lipid lowering and liver protecting drug or health food prepared by active ingredients of fructus schisandrae chinensis and preparation method |
| CN103223050A (en) * | 2013-04-16 | 2013-07-31 | 阚兆云 | Traditional Chinese medicine extract and preparation method thereof |
| CN105418789A (en) * | 2016-01-20 | 2016-03-23 | 济宁医学院 | Method for preparing low-molecular oyster polysaccharide with immune adjustment and anti-tumor activity |
| CN106632709A (en) * | 2016-05-12 | 2017-05-10 | 江苏大学 | Carboxymethylated schisandra polysaccharide and preparation method and application thereof |
| CN106967180A (en) * | 2017-04-24 | 2017-07-21 | 中国水产科学研究院黑龙江水产研究所 | A kind of Chinese magnoliavine fruit polysaccharide extract and its preparation method and application |
| CN107455741A (en) * | 2017-08-08 | 2017-12-12 | 哈尔滨师范大学 | A kind of health food with anti-oxidation function and its preparation method and application |
| CN110632223A (en) * | 2019-07-16 | 2019-12-31 | 夏永刚 | Traditional Chinese medicine polysaccharide structural feature fingerprint spectrum and construction method and application thereof |
| CN111514160A (en) * | 2020-05-20 | 2020-08-11 | 南京中医药大学 | Application of schisandra chinensis polysaccharide in preparation of medicines or health-care products for treating inflammatory bowel diseases |
| CN112457384A (en) * | 2020-12-17 | 2021-03-09 | 南京盖斯夫医药科技有限公司 | Enzymolysis polypeptide and application thereof in preparing anti-leukemia medicine |
| CN116693716A (en) * | 2023-04-28 | 2023-09-05 | 湖北医药学院 | Radix tinosporae polysaccharide and preparation method and application thereof |
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| CN103156106A (en) * | 2013-02-04 | 2013-06-19 | 北华大学 | Lipid lowering and liver protecting drug or health food prepared by active ingredients of fructus schisandrae chinensis and preparation method |
| CN103223050A (en) * | 2013-04-16 | 2013-07-31 | 阚兆云 | Traditional Chinese medicine extract and preparation method thereof |
| CN105418789B (en) * | 2016-01-20 | 2018-04-06 | 济宁医学院 | A kind of method for preparing the low molecule oyster polysaccharide with immunological regulation and antitumor activity |
| CN105418789A (en) * | 2016-01-20 | 2016-03-23 | 济宁医学院 | Method for preparing low-molecular oyster polysaccharide with immune adjustment and anti-tumor activity |
| CN106632709A (en) * | 2016-05-12 | 2017-05-10 | 江苏大学 | Carboxymethylated schisandra polysaccharide and preparation method and application thereof |
| CN106632709B (en) * | 2016-05-12 | 2019-05-31 | 江苏大学 | A kind of carboxy methylation Fructus Schisandrae Polysaccharide and its preparation method and application |
| CN106967180A (en) * | 2017-04-24 | 2017-07-21 | 中国水产科学研究院黑龙江水产研究所 | A kind of Chinese magnoliavine fruit polysaccharide extract and its preparation method and application |
| CN106967180B (en) * | 2017-04-24 | 2019-07-16 | 中国水产科学研究院黑龙江水产研究所 | A kind of Chinese magnoliavine fruit polysaccharide extract and its preparation method and application |
| CN107455741A (en) * | 2017-08-08 | 2017-12-12 | 哈尔滨师范大学 | A kind of health food with anti-oxidation function and its preparation method and application |
| CN110632223A (en) * | 2019-07-16 | 2019-12-31 | 夏永刚 | Traditional Chinese medicine polysaccharide structural feature fingerprint spectrum and construction method and application thereof |
| CN111514160A (en) * | 2020-05-20 | 2020-08-11 | 南京中医药大学 | Application of schisandra chinensis polysaccharide in preparation of medicines or health-care products for treating inflammatory bowel diseases |
| CN111514160B (en) * | 2020-05-20 | 2021-08-17 | 南京中医药大学 | Application of schisandra chinensis polysaccharide in preparation of medicines or health-care products for treating inflammatory bowel diseases |
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