CN102040749B - Ganoderma sinense mycelium polysaccharide refined substance as well as preparation method and use thereof - Google Patents
Ganoderma sinense mycelium polysaccharide refined substance as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a ganoderma sinense mycelium polysaccharide refined substance containing polysaccharide and monosaccharide; the polysaccharide refined substance is characterized in that the content of the polysaccharide is greater than 90% and the content of the monosaccharide is smaller than 5%. The invention also provides a preparation method and use of the ganoderma sinense mycelium polysaccharide refined substance. The ganoderma sinense mycelium polysaccharide refined substance provided by the invention has the advantages of higher polysaccharide content, lower administration dose,centralized molecular weight range and higher purity; and the preparation technology of the ganoderma sinense mycelium polysaccharide refined substance is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of purple camphorata mycelium refinishing polyose thing, its preparation method and its purposes at field of medicaments.
Background technology
Multiple efficacies such as edible and medicinal fungi has anti-tumor virus, reducing blood-fat, delays senility, protecting liver and expelling toxin, promotion nucleic acid and protein biosynthesizing, in state-owned long use history.Studies show that in a large number that both at home and abroad fungus polysaccharide separates from fungus sporophore, mycelium, fermented liquid and obtains, and is the drug effect position of fungal drug, is to control the cell fission differentiation, regulates the old and feeble class active polysaccharide of cell growth.
Purple sesame (Ganoderma Sinense Zhao, Xu et Zhang) is a kind of higher fungi, belongs to Basidiomycetes, Aphyllophorales, polyporaceae, Ganoderma.God is protected in favourable joint, beneficial vital essence, hard muscles and bones, good color, the effect of strengthenings by means of tonics such as treatment consumptive disease.More to red sesame (often the doing glossy ganoderma) research that belongs to xenogenesis together both at home and abroad, but less to the research of purple sesame.Purple sesame contains macromolecular substance such as polysaccharide, protein, amino acid, also contains small-molecule substances such as alkaloid, triterpenes, sterol, tonka bean camphor, phenols.Discover that at present that purple sesame extract (based on polysaccharide) has is antitumor, the effect of radioprotective, leukocyte increasing.
Purple sesame polysaccharide research still is in the starting stage, the general content of purple sesame Crude polysaccharides lower (being lower than 40%), and its component complexity, and since purple sesame sporophore resource scarcity, rare purple sesame product on the market.
Purple sesame wild resource poorness, the restriction that artificial culture is subjected to natural cause, management factors causes raw materials quality uncontrollable, at present also can not large-scale production, thus influenced the exploitation of purple sesame from resource view.The liquid submerged fermentation technology has comparatively ripe application at biological field at present, and it is produced mycelium and is not affected by the external environment, and output is big, and quality and natural suitable even because natural.Utilize submerged fermentation to produce purple camphorata mycelium technology and can solve its resource problem, and reach quality controllable, realize suitability for industrialized production at last.
From the mycelium that purple sesame liquid submerged fermentation obtains, extract and obtain polysaccharide, prove that through pharmacological evaluation it has obvious antineoplastic.Chinese patent 200710045369.4 " a kind of purple sesame fermentation process and the purple camphorata mycelium that makes " discloses a kind of method of purple sesame liquid submerged fermentation aerial mycelium, and Chinese patent 200710045368.x " intra-polysaccharides from mycelia of ganoderma sinensis and its preparation method and application " discloses and a kind ofly extracted the method for preparing Crude polysaccharides from the mycelium that purple sesame liquid submerged fermentation produces.This mycelium that above-mentioned patent relates to extracts the Crude polysaccharides content obtain 39～75% with water extraction and alcohol precipitation method.The dosage of its pharmacological evaluation be 40mg/kg/day (administering mode: oral medicine feed), to the inhibiting rate of mouse H22 liver cancer and Lewis lung cancer respectively 45～55% and 40～50%.Yang Guohong, Yang Yifang, Jin Juandi (the antitumor active site research [J] of purple sesame liquid submerged fermentation. herbal medicine, 2008,39 (6): 877-880) the anti-tumor activity polysaccharide of purple sesame liquid submerged fermentation is studied, extract from purple camphorata mycelium and obtain intracellular polyse, the dosage of its pharmacological evaluation also is 40mg/kg/day (administering mode: oral medicine feed), show tangible anti-tumor activity.Above patent and article all do not relate to the research of polysaccharide molecular weight and polysaccharide monose composition.
Tradition extraction method of polysaccharides water extraction and alcohol precipitation method, the Crude polysaccharides content that this method obtains is low, the polysaccharide fraction complexity, and contain macromolecular components such as protein pigment simultaneously.
In view of the situation of prior art, need to seek polysaccharide content height, molecular weight ranges and concentrate, remove impurity such as protein, have tangible anti-tumor activity and dosage extract the Crude polysaccharides that obtains less than ordinary method purple camphorata mycelium refinishing polyose thing.
Summary of the invention
Technical problem to be solved by this invention provides a kind of purple camphorata mycelium refinishing polyose thing, and its polysaccharide content height, molecular weight ranges be little, remove impurity such as protein, have tangible anti-tumor activity and dosage extracts the Crude polysaccharides that obtains less than ordinary method.
First purpose of the present invention provides a kind of purple camphorata mycelium refinishing polyose thing, and described refinishing polyose thing contains polysaccharide and monose, it is characterized in that, the content of described polysaccharide is greater than 90%, and the content of described monose is less than 5%.
Further, described refinishing polyose thing does not contain protein.
Further, the molecular weight of polysaccharide is 100,000-2,000,000 in the described refinishing polyose thing.
Structure research to refinishing polyose thing of the present invention learns that its polysaccharide that contains is made up of semi-lactosi and rhamnosyl, and its mol ratio is 15: 1～17.4: 1, preferred 15: 1.
The present invention adopts membrane technique and quaternary salt deposit method to dwindle the molecular weight ranges of purple camphorata mycelium polysaccharide, and removes impurity such as protein, thereby obtains the refinishing polyose thing.
Second purpose of the present invention provides the preparation method of above-mentioned purple camphorata mycelium refinishing polyose thing, comprises the steps: that purple camphorata mycelium obtains Crude polysaccharides through water extract-alcohol precipitation, Crude polysaccharides through ultrafiltration, concentrate and saltout, obtain purple camphorata mycelium refinishing polyose thing.
Polysaccharide content does not contain protein greater than 90% in the purple camphorata mycelium refinishing polyose of the gained thing.Its production technique is simple, and is feasible.
As present invention further optimization, described ultrafiltration step is that the ultra-filtration membrane of 1-5 ten thousand carries out by molecular weight cut-off.More preferably, the molecular weight cut-off of described ultra-filtration membrane is 30,000.
As present invention further optimization, described ultra-filtration membrane is made by polymeric amide, poly (ether sulfone) film or cellulose acetate.
As present invention further optimization, described enrichment step is undertaken by nanofiltration membrane.Preferred molecular weight cut-off is the nanofiltration membrane of 150-300.The preferred nanofiltration membrane of being made by polymeric amide.
As present invention further optimization, the described step of saltouing is undertaken by quaternary salt deposit.Described quaternary ammonium salt can be selected from cetyl trimethylammonium bromide, hexadecyl pyridine hydrochloride or chitosan hydroxypropyl-trimethyl ammonium chloride, and the working concentration of described quaternary ammonium salt is 0.5%-8%, and preferred concentration is 2%.When concentration less than 0.5% the time, the alkaline polysaccharide precipitation is not exclusively; When concentration greater than 8% the time, easy bubble in the treating processes easily has residual.
Be studies show that by purple camphorata mycelium polysaccharide anti-tumor activity molecular weight is significantly higher than position anti-tumor activity less than 50,000 greater than 50,000 position anti-tumor activity.Quaternary salt deposit method (precipitation method) can be passed through regulator solution pH value, adopts the pH gradient method, CTAB is added in turn in the sugar soln of the pH that changes successively, just can be in acid, neutrality and basic solution the fractionation precipitation polysaccharide.
The concrete steps that the present invention prepares purple camphorata mycelium refinishing polyose thing are as follows: adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, obtain mycelium and wash with water, filter press.Water is proposed heating and is extracted 1-3h, adds 1-4 times of alcohol precipitation after the water extract concentrates and spends the night, and precipitation is taken out and dried, and obtains Crude polysaccharides.With the Crude polysaccharides water dissolution, utilize membrane technique to separate Crude polysaccharides, obtain trapped fluid (A) and see through liquid (B), the nanofiltration membrane with certain molecular weight concentrates A and B respectively again; Will be through liquid (B) lyophilize, obtain molecular weight less than 50,000 the antitumor polysaccharide of purple camphorata mycelium position GS-B; Continue lyophilize A, obtain molecular weight greater than 50,000 the antitumor polysaccharide of purple camphorata mycelium position GS-A, use further refining polysaccharide position GS-A of precipitation method (preferred quaternary salt deposit method) again, obtain purple camphorata mycelium refinishing polyose thing.(above-mentioned polysaccharide position GS-B and polysaccharide position GS-A will specifically see the following form 7 and 8 for Comparative Examples)
The concrete steps of quaternary salt deposit method are as follows:
Get dried polysaccharide position, be dissolved in the deionized water, centrifugation is got supernatant liquor and is added quaternary ammonium salt, mixing, and centrifugation is divided into throw out (1) and supernatant liquor (2).With throw out (1) with the dissolving of NaCL salts solution after, alcohol precipitation is got the precipitation dialysis tubing flowing water dialysis desalination of packing into after with the less water dissolving, liquid in the bag concentrates, lyophilize obtains acidic polysaccharose; Other gets supernatant liquor (2) and adds boric acid, transfers pH to 11 with NaOH solution, continues to transfer pH to 7 with acetic acid, and centrifugation is divided into throw out (3) and supernatant liquor (4).With throw out (3) with the dissolving of NaCL salts solution after, alcohol precipitation is got the precipitation dialysis tubing flowing water dialysis desalination of packing into after with the less water dissolving, liquid in the bag concentrates, lyophilize obtains neutral polysaccharide; Get supernatant liquor (4) and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrate in the taking-up dialysis tubing, lyophilize namely gets the purple camphorata mycelium refinishing polyose thing (alkaline polysaccharide) of the present invention.
Polysaccharide content is greater than 90% in the purple camphorata mycelium refinishing polyose of the gained thing after testing, and contents of monosaccharides is less than 5%, and does not contain protein, and molecular weight ranges is 100,000-2,000,000.Its structure research is learnt that its monose consists of semi-lactosi and rhamnosyl.
Pharmacological evaluation is carried out at this polysaccharide position, when dosage is 2.223mg/kg/day, intraperitoneal injection is 55～60% to the inhibiting rate of H22 rat liver cancer, is 45～50% to the inhibiting rate of Mice Bearing Lewis Lung Cancer, has remarkable antitumor effect and dosage is little.This polysaccharide pH is 7.20-7.35.
The present invention has determined monose composition and the molecular weight ranges in the purple camphorata mycelium refinishing polyose thing, and prove that by pharmacological evaluation this purple camphorata mycelium refinishing polyose thing has remarkable antitumor effect, and the little (dosage: 2.223mg/kg/day of dosage, administering mode: abdominal injection), toxicity is little, can be used for antitumor drug or adjuvant, be suitable as particularly injection formulations of preparation that raw material is used for anti-tumor agent.
Therefore, the 3rd purpose of the present invention provides the application of above-mentioned purple camphorata mycelium refinishing polyose thing in preparation antitumor drug and raising immunizing power medicine.
Compare with the purple camphorata mycelium polysaccharide of prior art, the polysaccharide content of purple camphorata mycelium refinishing polyose thing provided by the invention is higher, molecular weight ranges is little, removed impurity such as protein, have tangible anti-tumor activity, dosage is littler and toxicity is littler.
Description of drawings
Fig. 1 is the HPGPC collection of illustrative plates of purple camphorata mycelium refinishing polyose thing of the present invention.As seen from Figure 1 and calculate, this refinishing polyose thing is made up of two peaks, and molecular weight is respectively greater than 2,000,000 and 170,000, and proportion of composing is 64: 36.
Embodiment
The concrete steps that the phenol sulfuric acid process is measured polysaccharide (and monose) content are as follows:
Mark product preparation: the fixed dry good glucose 25mg of accurate title, fixed molten to 100ml, be made into 250ug/ml mark product solution, get 0.1,0.2,0.4,0.5,0.7 respectively, 0.80.ml is in different test tubes, add water and supply 1ml, add 5% phenol 0.5ml respectively, fully shake up, add vitriol oil 3.5ml fast, put 40 ℃ of water-baths, reacted 30 minutes, cold water is placed 5min again, makes mark product liquid.
Blank preparation: get the 2ml deionized water, add 5% phenol 1ml, vitriol oil 7ml, heating in water bath, blank solution is made in cooling.
Typical curve is drawn: standard liquid to be measured and blank solution are surveyed its absorption value with ultraviolet spectrophotometry in the 490nm place, and do typical curve.
The sample preparation: get the accurate title of Crude polysaccharides sample and decide 20mg, preparation 2mg/ml solution is got 0.2ml in test tube in the deionized water of adding 10ml, adds water and supplies 1ml, adds phenol, the vitriol oil successively by mark product liquid, heating in water bath, and sample liquid is made in cooling.
Detect: testing sample is surveyed its absorption value with ultraviolet spectrophotometry in the 490nm place, survey its polysaccharide content with typical curve.
Contents of monosaccharides is measured
3,5-edlefsen's reagent: precision takes by weighing 1.000g 3, and the 5-dinitrosalicylic acid adds earlier the 20mL2mol/L sodium hydroxide solution in the 100mL beaker, add the 30g Seignette salt again, adds water and fully dissolves and be settled to 500mL
The glucose solution preparation: precision takes by weighing dry good glucose 200mg, fixed moltenly be mixed with 2mg/ml solution to 100ml, get 0,0.4,0.8,1.2,1.6 respectively, 2.0ml in the 25ml volumetric flask, add water respectively and supply 2ml, each adds 3,5-dinitrosalicylic acid 2ml, boiling water boils 5min, immediately cooling, be diluted to scale with deionized water, shake up, survey its ultraviolet absorption value at the 550nm place, do typical curve.
Sample detection: sample thief 12mg adds deionized water 2ml and makes it dissolving in the 10ml volumetric flask, adds 3,5-dinitrosalicylic acid 2ml again, and boiling water boils 5min, cooling immediately, and thin up is to scale.Survey its uv-absorbing at 550 places, bring typical curve into, calculate contents of monosaccharides.
The purity of the purple camphorata mycelium refinishing polyose of efficient gel chromatography determination the present invention thing and the concrete grammar of polysaccharide molecular weight are as follows:
Polysaccharide molecular weight: chromatographic condition: TSK-GEL GMPWxl chromatographic column; Moving phase is water; Flow velocity: 0.3mL/min; 35 ℃ of column temperatures; Detector is: the differential detector.
Mark product Dextron, polysaccharide sample are used water dissolution, sample introduction respectively.
Measure dextran Dextron series and sample retention time, do typical curve with the molecular weight logarithmic value of retention time-Dextron series of Dextron, bring the retention time of polysaccharide sample into typical curve, can know the molecular weight at each polysaccharide peak by inference.
Different molecular weight polysaccharide ratio: the peak area ratio by each polysaccharide peak is different molecular weight polysaccharide ratio.
The concrete steps of protein negative reaction are as follows:
Ninhydrin reaction (protein discriminating)
The ninhydrin solution compound method: the 0.2g triketohydrindene hydrate is dissolved in 100mL95% ethanol, and concentration is 0.2% ninhydrin reaction (protein discriminating): sample solution is dripped on white plaque (furnace pot), add a triketohydrindene hydrate liquid, whether qualitative detection has protein.
The concrete steps of proximate analysis are as follows:
The monose compositional analysis
1 complete acid hydrolysis and tlc analysis
Get 10mg polysaccharide sample and put into the 2ml ampoule, add the 2ml2mol/L trifluoroacetic acid, inflated with nitrogen sealing, 110 ℃ of oil baths 6 hours.After oil bath finishes, cooling, it is yellow that solution is, and pours the 25ml heart into and revolve in the steaming bottle, clean ampoule with ultrapure water, pour into to revolve and steam in the bottle, add 5ml methyl alcohol, 50 ℃ revolve steaming to doing, add 5ml methyl alcohol again, revolve and steam to doing, so repeat 3 times, get false add 1ml ultrapure water dissolving after being spin-dried at last.Get 5 μ L and on cellulose plate, carry out tlc analysis with the contrast of standard monose, measure the monosaccharide residue kind of polysaccharide sample.Developping agent is: one: acetone: water=24: 1 is made into 200ml solution.Two: propyl carbinol: ethyl acetate: isopropylcarbinol: acetic acid: water: pyridine=35ml: 100: 60: 35: 30: 35.Developer is aniline-phthalic acid, sprays the back in 100 ℃ of heated baking 10min colour developings.
2HPLC measures the monose ratio
Chromatographic condition: Kromasil 100-5NH
2Moving phase: acetonitrile: water=75: 25; Column temperature: 35 ℃, flow velocity: 1mL/min; Detector: differential detector
Precision takes by weighing monose mark product and second half polysaccharide hydrolysis sample dissolves with moving phase, is mixed with sample introduction, measures retention time and the peak area of mark product and sample.
Retention time and monose mark product retention time by hydrolyzation sample contrast the kind of determining contained monose in the hydrolyzation sample.
According in monose mark product peak area and the hydrolyzation sample the ratio of the content of contained monose in the ratio calculating hydrolyzation sample of peak area at corresponding monose peak.Formula is as follows:
Embodiment 1
Crude polysaccharides extracts:
Adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, get fermented liquid 1300L, obtain mycelium and wash with water, filter press.Get mycelium and propose heating extraction 2 times with 3 times of water, each 3h, extracting temperature is 90 ℃.The water extract merges and to be concentrated into the mycelia scale of construction and the amount of liquid medicine ratio is 1: 2, after add 4 times of 95% alcohol precipitation and spend the night, precipitation is taken out and is dried, and obtains Crude polysaccharides 119.22g.
The polysaccharide content that the phenol sulfuric acid process records this Crude polysaccharides is 34.9%.
Crude polysaccharides is refining:
Getting above-mentioned Crude polysaccharides with 20 times of water dissolution, is that 30,000 ultra-filtration membrane separates Crude polysaccharides liquid with molecular weight, and it is concentrated to get the trapped fluid molecular weight cut-off and be 1,500,000 nanofiltration membrane, gets the concentrated solution freeze-drying.
Get dried polysaccharide position (be example with 1g), be dissolved in the 100mL deionized water, centrifugation is got supernatant liquor and is added 2% trimethylammonium hexadecyl brometo de amonio, mixing, centrifugation is got supernatant liquor and is added 1% boric acid 50mL, transfer pH to 11 with 1MNaOH solution, continue to transfer pH to 7, centrifugation with acetic acid.Get supernatant liquor and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrates in the taking-up dialysis tubing, and lyophilize namely gets the purple camphorata mycelium refinishing polyose of the present invention thing.
The yield of this refinishing polyose thing is 64.5%, and polysaccharide content is 92.5%, and contents of monosaccharides is 1.7%, and proteins react is negative.Its pH value is 7.12.When it was 2.712mg/kg/day at dosage, intraperitoneal injection was 54.25% to the inhibiting rate of H22 rat liver cancer, was 49.14% to the inhibiting rate of Mice Bearing Lewis Lung Cancer.
Embodiment 2:
Crude polysaccharides extracts:
Adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, get fermented liquid 1300L, obtain mycelium and wash with water, filter press.Get mycelium and propose heating extraction 3 times with 3 times of water, each 2h, extracting temperature is 80 ℃.The water extract merges and to be concentrated into the mycelia scale of construction and the amount of liquid medicine ratio is 2: 1, after add 3 times of 85% alcohol precipitation and spend the night, precipitation is taken out and is dried, and obtains Crude polysaccharides 144.46g.
The polysaccharide content that the phenol sulfuric acid process records this Crude polysaccharides is 42.8%.
Crude polysaccharides is refining:
Getting above-mentioned Crude polysaccharides with 20 times of water dissolution, is that 30,000 ultra-filtration membrane separates Crude polysaccharides liquid with molecular weight, and it is concentrated to get the trapped fluid molecular weight cut-off and be 2,000,000 nanofiltration membrane, gets the concentrated solution freeze-drying.
Get dried polysaccharide position (be example with 1g), be dissolved in the 100mL deionized water, centrifugation is got supernatant liquor and is added 8% trimethylammonium hexadecyl brometo de amonio, mixing, centrifugation is got supernatant liquor and is added 1% boric acid 50mL, transfer pH to 11 with 1MNaOH solution, continue to transfer pH to 7, centrifugation with acetic acid.Get supernatant liquor and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrates in the taking-up dialysis tubing, and lyophilize namely gets the purple camphorata mycelium refinishing polyose of the present invention thing.
The yield of this refinishing polyose thing is 62.8%, and polysaccharide content is 94.3%, and contents of monosaccharides is 1.7%, and proteins react is negative.Its pH value is 7.22.When it was 2.223mg/kg/day at dosage, intraperitoneal injection was 57.73% to the inhibiting rate of H22 rat liver cancer, was 44.34% to the inhibiting rate of Mice Bearing Lewis Lung Cancer.
Embodiment 3:
Crude polysaccharides extracts:
Adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, get fermented liquid 1300L, obtain mycelium and wash with water, filter press.Get mycelium and propose heating extraction 3 times with 3 times of water, each 2h, extracting temperature is 80 ℃.The water extract merges and to be concentrated into the mycelia scale of construction and the amount of liquid medicine ratio is 2: 1, after add 3 times of 85% alcohol precipitation and spend the night, precipitation is taken out and is dried, and obtains Crude polysaccharides 144.46g.
The polysaccharide content that the phenol sulfuric acid process records this Crude polysaccharides is 42%.
Crude polysaccharides is refining:
Getting above-mentioned Crude polysaccharides with 20 times of water dissolution, is that 30,000 ultra-filtration membrane separates Crude polysaccharides liquid with molecular weight, and it is concentrated to get the trapped fluid molecular weight cut-off and be 2,500,000 nanofiltration membrane, gets the concentrated solution freeze-drying.
Get dried polysaccharide position (be example with 1g), be dissolved in the 100mL deionized water, centrifugation is got supernatant liquor and is added 5% trimethylammonium hexadecyl brometo de amonio, mixing, centrifugation is got supernatant liquor and is added 1% boric acid 50mL, transfer pH to 9 with 1MNaOH solution, continue to transfer pH to 7, centrifugation with acetic acid.Get supernatant liquor and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrate in the taking-up dialysis tubing, and lyophilize, namely.
This polysaccharide yield is 59.5%, and polysaccharide content is 92.38%, and contents of monosaccharides is 2.0%, and proteins react is negative.Its pH value is 7.19.When it was 3.147mg/kg/day at dosage, intraperitoneal injection was 46.44% to the inhibiting rate of H22 rat liver cancer, was 43.36% to the inhibiting rate of Mice Bearing Lewis Lung Cancer.
Embodiment 4
Crude polysaccharides extracts:
Adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, get fermented liquid 1300L, obtain mycelium and wash with water, filter press.Get mycelium and propose heating extraction 2 times with 3 times of water, each 3h, extracting temperature is 90 ℃.The water extract merges and to be concentrated into the mycelia scale of construction and the amount of liquid medicine ratio is 1: 2, after add 4 times of 95% alcohol precipitation and spend the night, precipitation is taken out and is dried, and obtains Crude polysaccharides 119.22g.
The polysaccharide content that the phenol sulfuric acid process records this Crude polysaccharides is 30%.
Crude polysaccharides is refining:
Getting above-mentioned Crude polysaccharides with 20 times of water dissolution, is that 30,000 ultra-filtration membrane separates Crude polysaccharides liquid with molecular weight, and it is concentrated to get the trapped fluid molecular weight cut-off and be 3,000,000 nanofiltration membrane, gets the concentrated solution freeze-drying.
Get dried polysaccharide position (be example with 1g), be dissolved in the 100mL deionized water, centrifugation is got supernatant liquor and is added 2% hexadecyl pyridine hydrochloride, mixing, centrifugation is got supernatant liquor and is added 1% boric acid 50mL, transfer pH to 11 with 1MNaOH solution, continue to transfer pH to 7, centrifugation with acetic acid.Get supernatant liquor and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrates in the taking-up dialysis tubing, and lyophilize namely gets the purple camphorata mycelium refinishing polyose of the present invention thing.
The yield of this refinishing polyose thing is 64.5%, and polysaccharide content is 9% (wrong), and contents of monosaccharides is 1.7%, and proteins react is negative.Its pH value is 7.25.When it was 2.712mg/kg/day at dosage, intraperitoneal injection was 54.25% to the inhibiting rate of H22 rat liver cancer, was 49.14% to the inhibiting rate of Mice Bearing Lewis Lung Cancer.
Embodiment 5:
Crude polysaccharides extracts:
Adopt the purple sesame bacterial classification in Shandong (Ganoderma sinense) to carry out liquid fermenting, get fermented liquid 1300L, obtain mycelium and wash with water, filter press.Get mycelium and propose heating extraction 3 times with 3 times of water, each 2h, extracting temperature is 80 ℃.The water extract merges and to be concentrated into the mycelia scale of construction and the amount of liquid medicine ratio is 2: 1, after add 3 times of 85% alcohol precipitation and spend the night, precipitation is taken out and is dried, and obtains Crude polysaccharides 144.46g.
The polysaccharide content that the phenol sulfuric acid process records this Crude polysaccharides is 42%.
Crude polysaccharides is refining:
Getting above-mentioned Crude polysaccharides with 20 times of water dissolution, is that 30,000 ultra-filtration membrane separates Crude polysaccharides liquid with molecular weight, and getting the trapped fluid molecular weight cut-off is that 2,000,000 nanofiltration membrane are concentrated, gets the concentrated solution freeze-drying.
Get dried polysaccharide position (be example with 1g), be dissolved in the 100mL deionized water, centrifugation, get supernatant liquor and add 2% chitosan hydroxypropyl-trimethyl ammonium chloride, mixing, centrifugation, get supernatant liquor and add 1% boric acid 50mL, transfer pH to 11 with 1MNaOH solution, continue to transfer pH to 7, centrifugation with acetic acid.Get supernatant liquor and transfer pH to 4.4 with acetic acid, add 1.5 times of ethanol alcohol precipitations and spend the night, precipitation is got in centrifugation, with dialysing with dialysis tubing flowing water after the water dissolution, behind the liquid, concentrates in the taking-up dialysis tubing, and lyophilize namely gets the purple camphorata mycelium refinishing polyose of the present invention thing.
The yield of this refinishing polyose thing is 62.8%, and polysaccharide content is 94.3%, and contents of monosaccharides is 1.7%, and proteins react is negative.Its pH value is 7.35.When it was 2.223mg/kg/day at dosage, intraperitoneal injection was 57.73% to the inhibiting rate of H22 rat liver cancer, was 44.34% to the inhibiting rate of Mice Bearing Lewis Lung Cancer.
Embodiment 6: proximate analysis
Get this polysaccharide 10mg, use complete acid hydrolytic reaction, analyze and can get through TLC and HPLC: polysaccharide is made up of semi-lactosi, rhamnosyl, and mol ratio is as shown in the table:
The proximate analysis of the purple camphorata mycelium refinishing polyose of the present invention of table 1: embodiment 1～5 gained thing
| Monose is formed | Ratio | |
| Embodiment 1 | Semi-lactosi, rhamnosyl | 15.2∶1 |
| Embodiment 2 | Semi-lactosi, rhamnosyl | 15.4∶1 |
| Embodiment 3 | Semi-lactosi, rhamnosyl | 15.0∶1 |
| Embodiment 4 | Semi-lactosi, rhamnosyl | 17.8∶1 |
| Embodiment 5 | Semi-lactosi, rhamnosyl | 17.4∶1 |
Embodiment 7: molecular weight analyse
Get this polysaccharide and analyze as can be known with HPGPC, refining polysaccharide is made up of two peaks, and molecular weight is respectively 2,000,000 and 170,000.
The polysaccharide fraction result of the purple camphorata mycelium refinishing polyose of the present invention of table 2: embodiment 1～5 gained thing
| Peak 1 (molecular weight) | Peak 2 (molecular weight) | Ratio | |
| Embodiment 1 | ＞200 ten thousand | 170,000 | 70∶30 |
| Embodiment 2 | ＞200 ten thousand | 170,000 | 68∶32 |
| Embodiment 3 | ＞200 ten thousand | 170,000 | 64∶36 |
| Embodiment 4 | ＞200 ten thousand | 170,000 | 45∶55 |
| Embodiment 5 | ＞200 ten thousand | 170,000 | 40∶60 |
Test example 1: the anti-tumor in vivo pharmacological evaluation of the purple camphorata mycelium refinishing polyose of the present invention thing
Adopt the mice transplanted tumor model, estimate anti-tumor activity with tumor control rate.Concrete grammar is as follows: get well-grown mouse H22 hepatic ascites (or Mice Bearing Lewis Lung Cancer knurl piece), with physiological saline dilution (the about 1-2 of cell concn * 10
7Individual/ml), every the right armpit subcutaneous vaccination of mouse 0.2ml, random packet, if: the purple camphorata mycelium refinishing polyose matter sample group that the purple camphorata mycelium Crude polysaccharides sample sets that blank group, sunrecome positive controls, Chinese patent 200710045369.4 " a kind of purple sesame fermentation process and the purple camphorata mycelium that makes " obtain and embodiment of the invention 1-5 obtain, inoculate and play administration back next day, the administration volume is the 0.5ml/20g body weight, and per os was irritated stomach 7-10 days continuously.The inoculation back is taken off neck 10-14 day and is put to death animal, dissects and gets the knurl piece, relatively the size of each dosage group knurl weight.
According to following formula result of determination:
The purple camphorata mycelium refinishing polyose thing of table 3: embodiment 1～5 gained and the purple camphorata mycelium Crude polysaccharides of prior art sample are to the tumor-inhibiting action of rat liver cancer H22
The purple camphorata mycelium refinishing polyose thing of table 4: embodiment 1～5 gained and the purple camphorata mycelium Crude polysaccharides of prior art sample are to the tumor-inhibiting action of Mice Bearing Lewis Lung Cancer
Conclusion:
By table 3 and table 4 as can be known, purple camphorata mycelium refinishing polyose thing of the present invention has tangible anti-tumor activity and dosage extracts the Crude polysaccharides that obtains less than ordinary method.
Test example 2: the polysaccharide sample of different pH values is respectively to the restraining effect of mouse H22 liver cancer and Lewis lung cancer
Adopt the mice transplanted tumor model, estimate anti-tumor activity with tumor control rate.Concrete grammar is as follows: get well-grown mouse H22 hepatic ascites (or Mice Bearing Lewis Lung Cancer knurl piece), with physiological saline dilution (the about 1-2 of cell concn * 10
7Individual/ml), every the right armpit subcutaneous vaccination of mouse 0.2ml, random packet, if: the purple camphorata mycelium refinishing polyose matter sample group (alkaline polysaccharide) that blank group, the embodiment of the invention 1 obtain, neutral polysaccharide and acidic polysaccharose are inoculated and are played administration back next day, the administration volume is the 0.5ml/20g body weight, and per os was irritated stomach 7-10 days continuously.The inoculation back is taken off neck 10-14 day and is put to death animal, dissects and gets the knurl piece, relatively the size of each dosage group knurl weight.
According to following formula result of determination:
Table 5: different acid-basicity polysaccharide samples are respectively to the restraining effect of mouse H22 liver cancer
Compare with control group:
*P＜0.05,
*P＜0.01
Table 6: different acid-basicity polysaccharide samples are respectively to the restraining effect of Mice Bearing Lewis liver cancer
Compare with control group:
*P＜0.05,
*P＜0.01
Conclusion:
By table 5 and table 6 as can be seen the antitumous effect of purple camphorata mycelium refinishing polyose thing (alkaline polysaccharide) be better than acidity and neutral polysaccharide, be the antitumor position of purple camphorata mycelium polysaccharide.
Test example 3: the polysaccharide sample of different molecular weight section is respectively to the restraining effect of mouse H22 liver cancer and Lewis lung cancer
Adopt the mice transplanted tumor model, estimate anti-tumor activity with tumor control rate.Concrete grammar is as follows: get well-grown mouse H22 hepatic ascites (or Mice Bearing Lewis Lung Cancer knurl piece), with physiological saline dilution (the about 1-2 of cell concn * 10
7Individual/ml), the right armpit subcutaneous vaccination of every mouse 0.2ml, random packet, establish: blank group, molecular weight less than 50,000 part polysaccharide (GS-B) sample sets and molecular weight greater than 50,000 part polysaccharide (GS-A).Administration is played next day in the inoculation back, and the administration volume is the 0.5ml/20g body weight, abdominal injection 7-10 days continuously.The inoculation back is taken off neck 10-14 day and is put to death animal, dissects and gets the knurl piece, relatively the size of each dosage group knurl weight.
According to following formula result of determination:
Table 7: greater than 50,000 with less than the restraining effect of 50,000 molecular weight section polysaccharide samples to mouse H22 liver cancer
Compare with control group:
*P＜0.05,
*P＜0.01
Table 8: greater than 50,000 with less than the restraining effect of 50,000 molecular weight section polysaccharide samples to Mice Bearing Lewis liver cancer
Compare with control group:
*P＜0.05,
*P＜0.01
Conclusion: greater than 50,000 part polysaccharide anti-tumor activities apparently higher than less than 50,000 polysaccharide.
Claims (19)
1. purple camphorata mycelium refinishing polyose thing, described refinishing polyose thing contains polysaccharide and monose, it is characterized in that, and the content of described polysaccharide is greater than 90%, and the content of described monose is less than 5%, and described polysaccharide is made up of semi-lactosi and rhamnosyl.
2. purple camphorata mycelium refinishing polyose thing according to claim 1 is characterized in that described refinishing polyose thing does not contain protein.
3. purple camphorata mycelium refinishing polyose thing according to claim 1 and 2 is characterized in that the molecular weight of polysaccharide is 100,000-2,000,000 in the described refinishing polyose thing.
4. purple camphorata mycelium refinishing polyose thing according to claim 1 is characterized in that the mol ratio of described semi-lactosi and rhamnosyl is 15: 1～17.4: 1.
5. purple camphorata mycelium refinishing polyose thing according to claim 4 is characterized in that the mol ratio of described semi-lactosi and rhamnosyl is 15: 1.
6. the preparation method of the arbitrary described purple camphorata mycelium refinishing polyose thing of claim 1-5, it is characterized in that, comprise the steps: that purple camphorata mycelium obtains Crude polysaccharides through water extract-alcohol precipitation, Crude polysaccharides obtains purple camphorata mycelium refinishing polyose thing through ultrafiltration, concentrated and precipitating.
7. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 6 is characterized in that, described ultrafiltration step is that the ultra-filtration membrane of 1-5 ten thousand carries out by molecular weight cut-off.
8. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 7 is characterized in that, described ultrafiltration step is that 30,000 ultra-filtration membrane carries out by molecular weight cut-off.
9. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 7 is characterized in that, described ultra-filtration membrane is made by polymeric amide, poly (ether sulfone) film or cellulose acetate.
10. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 6 is characterized in that, described enrichment step is undertaken by nanofiltration membrane.
11. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 10 is characterized in that, the molecular weight cut-off of described nanofiltration membrane is 150-300.
12. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 10 is characterized in that described nanofiltration membrane is made by polymeric amide.
13. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 6 is characterized in that, described precipitating step is undertaken by quaternary salt deposit.
14. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 13 is characterized in that, described quaternary ammonium salt is selected from cetyl trimethylammonium bromide, hexadecyl pyridine hydrochloride or chitosan hydroxypropyl-trimethyl ammonium chloride.
15. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 14 is characterized in that, described quaternary ammonium salt is cetyl trimethylammonium bromide.
16. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 13 is characterized in that, the working concentration of described quaternary ammonium salt is 0.5%-8%.
17. the preparation method of purple camphorata mycelium refinishing polyose thing according to claim 16 is characterized in that, the working concentration of described quaternary ammonium salt is 2%.
18. the application of the arbitrary described purple camphorata mycelium refinishing polyose thing of claim 1-5 in the preparation antitumor drug.
19. the application of the arbitrary described purple camphorata mycelium refinishing polyose thing of claim 1-5 in preparation raising immunizing power medicine.
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