CN100348619C - Ganoderrma tsugae-proteoglycan and its preparing method and use - Google Patents
Ganoderrma tsugae-proteoglycan and its preparing method and use Download PDFInfo
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- CN100348619C CN100348619C CNB2004100501514A CN200410050151A CN100348619C CN 100348619 C CN100348619 C CN 100348619C CN B2004100501514 A CNB2004100501514 A CN B2004100501514A CN 200410050151 A CN200410050151 A CN 200410050151A CN 100348619 C CN100348619 C CN 100348619C
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Abstract
The present invention discloses a ganoderrma tsugae polysaccharide, a preparing method and the application thereof. The ganoderma tsugae polysaccharide is extracted from ganoderma fruiting bodies, and the average molecular weight is between 5 and 16Kda. The ganoderma tsugae polysaccharide is composed of proteins and polysaccharides, and the components have the following proportion by weight: 15.5 to 19.5 shares of the proteins and 80 to 86 shares of the polysaccharides are composed of galactose, dextrose, mannose and fructose, the components have the following proportion by weight: 1.60 to 1.75 portions of the galactose, 1.00 portionof the dextrose, 0.15 to 0.22 portionof the mannose, and 0.10 to 0.20 portionof the fructose, and the main chain is connected by beta (1 to 3). The ganoderma tsugae polysaccharide has obvious effects of resisting tumors and improving immunity functions, and has an extensive medicinal value.
Description
Technical field
The present invention relates to a kind of protein-polysaccharide and preparation method thereof and application, particularly relate to a kind of protein-polysaccharide and the extracting method and of the application of this protein-polysaccharide that from Ganoderma tsugae (Ganoderma tsugae Murrill.), obtain as the activeconstituents of medicine.
Background technology
Treat tumor disease at present clinically, mainly adopt three kinds of modes: operation, radiation and chemotherapy, but radiotherapy, chemotherapy seriously kill and wound human normal cell and tissue, and many patient's physique are descended rapidly, even can not continued treatment.For a long time, people seek characteristics ground cancer therapy drugs such as new having toxicity is little, tumour inhibiting rate height constantly.Because the generation of tumour and the immunologic function of diffusion and human body have substantial connection, so produced the immunotherapy of tumors method.The twentieth century middle and later periods, found that some derive from the polysaccharide medicine of Mycophyta, as rainbow conk polysaccharide, lentinan, Schizophyllum commune Fr polysaccharides etc., they all have enhance immunity and resist the effect of tumour ground strenuously.These medicine ground mechanism of action are by host intermediary on the one hand, activate the various immunologically competent cells of body, promote the secretion of various lymphokines, improve the resistibility of body, reach the effect of engulfing and eliminating tumour cell, some polysaccharide on the other hand, particularly albumen-polysaccharide has direct killing effect to cancer cells, and tumour inhibiting rate is very high, be characterized in that effective dose is little, the tumor killing effect height, toxicity is very little, even nontoxic.Compare with chemotherapy, do not have leukopenia, vomiting, toxic side effect such as immunity degradation.The common use of such natural drug and chemotherapeutics can also reduce the toxic side effect of chemotherapeutics.Albumen-polysaccharide antitumor drug is and the new cancer therapy drug of the diverse class of chemotherapeutics.
Ganoderma tsugae (Gamoderma tsugae Murrill.) belongs to Basidiomycetes, polyporaceae, and a kind of treasure of Ganoderma has long medication history among the people, and pharmacologically active is extensive, and toxicity is minimum.Sporophore is its over-ground part, comprises cap and stem, is used as medicine together, and the complex chemical composition of Ganoderma tsugae sporophore has triterpenes, ucleosides, alkaloids, furans, amino acids and trace element or the like.Water soluble component mainly contains polyose, sugar-protein, albumen-polyose or the like.Studies show that at present polysaccharide and sugar-albumen or albumen-polyose mixture has enhance immunity function and antitumous effect.Since the source difference of polysaccharide, molecular weight size and space three-dimensional spirane structure difference, particularly different with the protein bound mode, aspect pharmacologically active, show than big-difference.People such as MIZUNO obtain molecular weight from red sesame sporophore be 1,000,000 and 450,000 two kind of water-soluble polysaccharide, through the mouse peritoneal drug administration by injection, does experiment with the strain of S180 knurl, has significant tumor-inhibiting action.Li Rongzhi, He Yunqing etc. obtain molecular weight and are respectively 16200,24500 from red sesame, 35000 and 40,000 four kinds of polysaccharide all can significantly increase the generation of normal mouse splenocyte IL-2.The isolated molecular weight of College of Pharmaceuticals, Beijing Medical University is the expression that the ganoderan of 7100-9300 can promote TNF α, TNF γ mRNA, increases secretion of TNF α, TNF γ mRNA or the like.Many to the research of red sesame (Ganoderma Lucidum) and purple sesame (Ganoderma Sinersis) at present.The rare report of research to Ganoderma tsugae (Ganoderma tsugae Murrill.), wherein only there is the world, Taiwan in 2003 university of becoming famous to declare name in the U.S. and be called " Polysaccharide-based extract from ganoderma, pharmaceutical use thereof, and process for preparing the same " patent, a kind of technology that obtains the water-soluble crude extract of Ganoderma tsugae is disclosed, but this technology just relates to steps such as water extract-alcohol precipitation and deproteinated, and the product that obtains is the elementary raw product of Ganoderma tsugae.
Summary of the invention
The antineoplaston medicine and the antitumor auxiliary therapeutic agent that the purpose of this invention is to provide pure product of a kind of Ganoderma tsugae protein-polysaccharide and preparation method thereof and be activeconstituents with this Ganoderma tsugae protein-polysaccharide.
A kind of Ganoderma tsugae protein-polysaccharide extracts from the Ganoderma tsugae sporophore, and its molecular-weight average is 0.5-1.6 ten thousand dalton.Be made up of albumen and polysaccharide, the weight ratio of described albumen and polysaccharide is an albumen: polysaccharide=(15.5-19.5): (80-86); Described polysaccharide is made up of semi-lactosi, glucose, seminose and fructose, and their weight ratio is a semi-lactosi: glucose: seminose: fructose=(1.60-1.75): 1.00: (0.15-0.22): (0.10-0.20), main chain connects with β (1 → 3).
The method for preparing the single protein-polysaccharide of Ganoderma tsugae may further comprise the steps: 1) the Ganoderma tsugae fruit body powder minces and uses pure degreasing, obtains the dregs of a decoction;
2) dregs of a decoction deionized water extraction is collected extracting solution;
3) extracting solution is precipitated with alcohol;
4) the throw out laggard capable micro-filtration of water dissolution;
5) filtrate is used the 3-5KD membrane ultrafiltration, the trapped substance on the collection membrane through gained filtrate behind the ultra-filtration membrane of 80-100KD;
6) DEAE-Sepharose (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl-) chromatography column on the trapped substance are used the deionized water wash-out, collect elutriant, through the 3-5KD film, and the trapped substance on the collection membrane;
7) gel column on the trapped substance is collected first-class part in the elutriant;
8) concentrate through the 3-5KD membrane ultrafiltration, lyophilize obtains product.
In above-mentioned preparation method, the described alcohol that is used for the degreasing of Ganoderma tsugae sporophore is ethanol or the methyl alcohol of 75-95%, the weight of ethanol or methyl alcohol is the mince 6-12 doubly (8-10 is doubly best) of weight of described Ganoderma tsugae fruit body powder, degreasing is carried out under 75-80 ℃ of condition, refluxing extraction 2-3 hour, repeat 3-6 time; Described dregs of a decoction deionized water extraction, collect in the extracting solution, the weight of deionized water is the mince 10-30 doubly (15-25 is doubly best) of weight of described Ganoderma tsugae fruit body powder, in 90-100 ℃ (98-100 ℃ best), intermittently stir, extracted 2-3 hour, and repeated 3-7 time, extracting solution be concentrated into described Ganoderma tsugae fruit body powder mince weight 7-12 doubly; Described with extracting solution with alcohol precipitation, be in extracting solution, to add ethanol or methyl alcohol makes its final concentration reach 75-85%, the limit edged stirs, and places under 4-10 ℃ of condition, it is centrifugal that (8000rpm 15min) obtains precipitation; Described micro-filtration carries out secondary at least, and the aperture of filter membrane is 10 μ m for the first time, if carry out secondary, the aperture of filter membrane is respectively that 10 μ m, 3 μ m are advisable, if carry out three times, the aperture of filter membrane is respectively that 10 μ m, 5 μ m and 1 μ m are advisable; DEAE-Sepharose on the described trapped substance (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl
-) in the chromatography column, if adopt DEAE-Sepharose (F.F.) chromatography column to carry out chromatography, generally select the chromatography column of 3 * 80cm specification for use, last sample speed should be 0.2-0.5ml/min, should be 2-4ml/min with the speed of deionized water wash-out, if adopt the SepharoseCL-6B chromatography column to carry out chromatography, generally select the chromatography column of 3 * 70cm specification for use, last sample speed should be 0.2-0.8ml/min, should be 1-2ml/min with the speed of deionized water wash-out, if adopt DEAE-Cellulose (Cl
-) chromatography column carries out chromatography, generally selects the chromatography column of 3 * 75cm specification for use, last sample speed should be 0.25ml/min, makes moving phase wash-out pillar with deionized water, speed should be 4ml/min; Described gel column can be selected existing gel column for use, and for example (3 * 80cm), with 0.1～0.4mol/L NaOH wash-out, flow velocity is generally 1～5ml/min to the Sephadex-G100 gel column.
Experiment showed, that Ganoderma tsugae protein-polysaccharide with method of the present invention preparation is the effect that the medicine of activeconstituents has obvious suppression and treatment tumour, immunizing power that simultaneously can also enhancing body.When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention is made generally in powder injection, also can make various ways such as injection liquid, tablet, granula, capsule, oral liquid, paste, creme.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally 1-2mg Ganoderma tsugae protein-polysaccharide/kg body weight/day.
Ganoderma tsugae protein-polysaccharide with method preparation of the present invention is the flap of a kind of creamy white or yellow-white, and light weight is soluble in water, the purity height, and viscosity is less, is easy to select for use various formulations, convenient drug administration.
Experiment shows, medicine tumour inhibiting rate height of the present invention, and toxicity is minimum, shows acute toxicology LD through the integral animal experimental study
50Do not measure, record maximum (drug administration by injection) dosis tolerata 150mg/kg (mouse), nontoxic substantially, have broad application prospects.
Embodiment
Embodiment 1, preparation Ganoderma tsugae albumen-polysaccharide
Take by weighing Ganoderma tsugae fruit body powder 100 gram, add 1 liter of 85% ethanol, 75 ℃ of reflux 2 hours, repeat 3 times, filter, in the dregs of a decoction, add 2 liters of deionized waters, heated and boiled stirs at interval, extracts 3 hours at every turn, repeat 5 times, filter united extraction liquid, being concentrated into volume is the 0.5-1 liter, is chilled to room temperature, adds 2.5 liters of dehydrated alcohols, the limit edged stirs, and places clarification, centrifugal (7000-8000rpm for 4 ℃, 15min), get the precipitation be dissolved in water after, 10 μ m and 1 μ m micro-filtration, get filtrate, ultra filtration 80KD gets filtrate then, carries out ultrafiltration with the 3KD film again, after the gained trapped substance is dissolved in deionized water, on balance is good DEAE-Cellulose (Cl
-) post (3 * 75cm), last sample flow velocity is 0.25ml/min, make moving phase wash-out pillar with deionized water then, elution speed 4ml/min collects effluent liquid, by the 3KD film, concentrate postlyophilization, obtain Ganoderma tsugae albumen-polysaccharide mixture, get this Ganoderma tsugae albumen-polysaccharide mixture 200mg and be dissolved in the 10ml distilled water last Sephadex-G100 gel column (3 * 80cm), with 0.2mol/L NaOH wash-out, flow velocity 1ml/min detects with UV-detector, registering instrument record elution curve, collect the first component stream fluid, after process 3KD ultra-filtration membrane concentrated, lyophilize obtained Ganoderma tsugae albumen-polysaccharide.
Embodiment 2, preparation Ganoderma tsugae albumen-polysaccharide
Take by weighing Ganoderma tsugae fruit body powder 100 grams, add 1.5 liters of 80% methyl alcohol, 80 ℃ of reflux 3 hours, repeat 5 times, filter, in the dregs of a decoction, add 4 liters of deionized waters, heated and boiled stirs at interval, extracts 3 hours at every turn, repeat 6 times, filter, merging filtrate is concentrated into 1 liter of volume, cooling, add 4 liters of anhydrous methanols, the limit edged stirs, and places clarification for 4 ℃, centrifugal (8000rpm, 15min), precipitation is dissolved in water, through 10 μ m, 5 μ m and 1 μ m micro-filtration, get filtrate through ultra-filtration membrane 80KD, get filtrate, carry out ultrafiltration with the 3KD film again, after the gained trapped substance is dissolved in deionized water, on balance is good Sepharose-CL6B post (3 * 70cm), last sample flow velocity is 0.6ml/min, makes moving phase wash-out pillar, elution speed 1.5ml/min with deionized water then, collect effluent liquid, by the 3KD film, concentrate postlyophilization, get Ganoderma tsugae albumen-polysaccharide mixture, getting this Ganoderma tsugae albumen-polysaccharide mixture 200mg is dissolved in the 10ml distilled water, last Sephadex-G100 gel column (3 * 80cm), with 0.4mol/L NaOH wash-out, flow velocity 5ml/min, detect with UV-detector, registering instrument record elution curve is collected the first component stream fluid, after concentrating through the 3KD ultra-filtration membrane, lyophilize obtains Ganoderma tsugae albumen-polysaccharide.
Embodiment 3, preparation Ganoderma tsugae albumen-polysaccharide
Take by weighing 50kg Ganoderma tsugae sporophore flour in extractor, add 450 liter of 85% ethanol, stir down, in 75-80 ℃ of refluxing extraction, each 2 hours, repeat 3 times, reclaim ethanol, the dregs of a decoction add 1000 liters in water, are heated to 100 ℃, extracted 3 hours, repeat 5 times, filter, filtrate merges, being concentrated into volume is 250 liters, under agitation, add 5 times of volume dehydrated alcohols, put 4 ℃ of low temperature places, clarification, centrifugal (8000rpm, 15min), the precipitate with deionized water dissolving, by 10 μ m micro-filtrations, 1 μ m micro-filtration, and then, use the 3KD membrane concentration, after the gained trapped substance is dissolved in deionized water through after the 80KD ultrafiltration, on balance is good DEAE-Sepharose (F.F.) post (3 * 80cm), last sample flow velocity is 0.4ml/min, makes moving phase wash-out pillar, elution speed 3ml/min with deionized water then, collect effluent liquid, by the 3KD film, concentrate postlyophilization, get Ganoderma tsugae albumen-polysaccharide mixture, getting this Ganoderma tsugae albumen-polysaccharide mixture 200mg is dissolved in the 10ml distilled water, last Sephadex-G100 gel column (3 * 80cm), with 0.1mol/L NaOH wash-out, flow velocity 3ml/min, detect with UV-detector, registering instrument record elution curve is collected the first component stream fluid, after concentrating through the 3KD ultra-filtration membrane, lyophilize obtains Ganoderma tsugae albumen-polysaccharide.
The detection of embodiment 4, Ganoderma tsugae albumen-polysaccharide
Getting Ganoderma tsugae albumen-polysaccharide that embodiment 1 obtains detects it
One, purity testing
Adopt gel column Sephadex-G100 post (3 * 70cm).It is an amount of to get Ganoderma tsugae albumen-polysaccharide, soluble in water, behind the upper prop with 0.2mol/L NaOH wash-out, flow velocity 0.2ml/min collects effluent liquid with the substep collector, and every pipe meets 2ml, collect 180 pipes, carry out determination of polysaccharide, draw the elution curve between absorbance and the pipe number with the phenol sulfuric acid process.The result is single symmetrical peak, shows that the purity of Ganoderma tsugae albumen-polysaccharide is very high.
Two, the mensuration of molecular-weight average
Adopt gel filtration method, (3 * 32cm), it is an amount of to take by weighing Ganoderma tsugae albumen-polysaccharide with TOYOPERAL HW65F post, be dissolved in the NaOH solution of 0.2mol/L, same processing standard polysaccharide T-10, T-40, T-70, T-110, T-150, T-200 with 0.2mol/L NaOH wash-out, collect V respectively
0And V
e, draw V
e/ V
0With each standard sugar molecular weight logarithmic relationship typical curve, the molecular-weight average of finding Ganoderma tsugae albumen-polysaccharide on typical curve is 10,000.
Three, monose compositional analysis
Take by weighing an amount of Ganoderma tsugae albumen-polysaccharide; use the dilute sulphuric acid hydrolysis; analyse gas chromatographic analysis with its derivative-acetylate with ply of paper respectively after the hydrolysis, repeatedly experimental result proves that all Ganoderma tsugae albumen-polysaccharide is made up of semi-lactosi, glucose, seminose and fructose.With glucose is 100%, and then ratio is: (1.60-1.75): 1.00: (0.15-0.22): (0.10-0.20).
Four, spectroscopic analysis
1, Infrared spectroscopy
Get an amount of Ganoderma tsugae albumen-polysaccharide, pellet technique is at 2200～400cm
-1The scope interscan is in 890cm
-1There is absorption peak at the place, shows to have β-conformation.
2, ultraviolet spectral analysis
It is an amount of to get Ganoderma tsugae albumen-polysaccharide, soluble in water, scans between 200～400nm, at the 280nm place absorption peak is arranged, and shows that albumen exists.Owing at substep collector same pipe number interval, single albumen absorption peak and polysaccharide absorption curve (phenolsulfuric acid method) are arranged, judge that this albumen and polysaccharide are that intramolecularly combines.
3,
1H and
13The C nucleus magnetic resonance
Get an amount of Ganoderma tsugae albumen-polysaccharide, be dissolved in D
2Among the O, survey
1H and
13The C spectrum.With
13The C spectrum is main, on this spectrum, has nuclear magnetic resonance peaks such as chemical shift 100.7,78.5,76.1,73.7,72.4,71.5,71.1,69.4,63.9; With
1H information is reference, and judge that the chemical structure of Ganoderma tsugae albumen-polysaccharide is: contain 17% proteic protein-polysaccharide conjugate based on semi-lactosi and glucose, its backbone structure is: β (1 → 3) semi-lactosi.
Embodiment 5, tumor-inhibiting action experiment
With different cancer cells is experimental subjects, and with the positive contrast of endoxan, with the negative contrast of physiological saline, Ganoderma tsugae albumen-polysaccharide that the research embodiment of the invention 1～5 obtains is to the restraining effect of cancer cells, and wherein the method for calculation of tumour inhibiting rate are:
Experimental result is shown in table 1, table 2, and from experimental result as can be seen, Ganoderma tsugae protein-polysaccharide of the present invention has stronger restraining effect to cancer cells.Ganoderma tsugae albumen-polysaccharide all has in various degree restraining effect to the growth of mouse S180 sarcoma, rat liver cancer H22 and Mice Bearing Lewis Lung Cancer, to transplanting human body intestinal cancer LOVO, cancer of the stomach MKN45, liver cancer QGY in nude mice, all have obvious antineoplastic, dose-effect relationship is obvious.
Table 1, Ganoderma tsugae albumen-polysaccharide are to the restraining effect of the tumor growth of mouse
Table 2, Ganoderma tsugae albumen-polysaccharide are to transplanting in the restraining effect of the human tumor growth of nude mice
The synergism of embodiment 7, Ganoderma tsugae albumen-polysaccharide
With rat liver cancer H22 is experimental subjects, and research Ganoderma tsugae albumen-polysaccharide merges the synergy of low dose of endoxan, Rheumatrex and ametycin.
Calculate tumour inhibiting rate according to following formula:
The effect of drug combination is calculated according to WebbShi mark product method:
(fa)
1，2＝1-[1-(fa)
1][1-(fa)
2]
(fa)
1(fa)
2Be respectively the tumour inhibiting rate of two medicines, (fa)
1,2Be the theoretical additive effect of calculating.If the effect of drug combination is greater than theoretical additive effect, then expression is collaborative.
Test-results is as shown in table 3, data from table as can be seen, after Ganoderma tsugae albumen-polysaccharide merges low dose of endoxan, Rheumatrex and ametycin, the restraining effect of rat liver cancer H22 obviously is better than Ganoderma tsugae albumen-polysaccharide or low dose of chemotherapeutics uses separately, can draws low dose of Ganoderma tsugae albumen-polysaccharide and merge low dose of chemotherapy rat liver cancer H22 is had certain synergistic conclusion.Ganoderma tsugae albumen-polysaccharide and low dose of chemotherapeutics endoxan, methotrexate and mitomycin merge to be used, calculate through WebbShi mark product method, Ganoderma tsugae albumen-polysaccharide and above-mentioned three kinds of structures are totally different, the diverse chemotherapeutics of pharmacological mechanism all has synergy, simultaneously, with the radiotherapy combined utilization, tumor-inhibiting action also obviously is better than independent radiotherapy.
Table 3 Ganoderma tsugae albumen-polysaccharide merging CTX, MTX, MMC are to the effect of rat liver cancer H22
| Sample | Tumour inhibiting rate (%) | (fa) 1，2(％) | Merge effect |
| Ganoderma tsugae albumen-polysaccharide | 45.10 | ||
| CTX | 40.11 | ||
| Ganoderma tsugae albumen-polysaccharide+CTX | 68.40 | 72.9 | Collaborative |
| MIX | 26.44 | ||
| Ganoderma tsugae albumen-polysaccharide+MTX | 49.60 | 59.59 | |
| MMC | 44.99 | ||
| Ganoderma tsugae albumen-polysaccharide+MMC | 65.31 | 69.75 | Collaborative |
Compare with the physiological saline group: * P＜0.05, * * P＜0.01.
Embodiment 8, Ganoderma tsugae albumen-polysaccharide are to the enhancement of immunologic function
1, Ganoderma tsugae albumen-polysaccharide is to the enhancement of tumor-bearing mice spleen lymphocyte proliferation
With the positive contrast of polysaccharide-peptide, research Ganoderma tsugae albumen-polysaccharide is to the enhancement of tumor-bearing mice spleen lymphocyte proliferation, and the result is as shown in table 4, shows that Ganoderma tsugae albumen-polysaccharide has stronger enhancement to the propagation of tumor-bearing mice splenic lymphocyte.
Table 4. Ganoderma tsugae albumen-polysaccharide is to the influence of tumor-bearing mice spleen lymphocyte proliferation
Compare with lotus knurl group: * * P＜0.01.
2, Ganoderma tsugae albumen-polysaccharide is to the active activation of NK cells in mice.
The mark of target cell: get the back 24 hours well-grown YAC-1 cells that go down to posterity, by 1 * 10
6Individual/ml cell suspension adds
3H-TdR 10uci, in 37 ℃, 5%CO
2Cultivated 2 hours in the incubator, vibrated once in per 30 minutes, the cell behind the mark is resuspended in the nutrient solution with nutrient solution washing 3 times, and making cell concn is 1 * 10
5/ ml.
The NK cytoactive is measured: 36 of C57BL/6 mouse, and every mouse armpit subcutaneous vaccination Lewis lung cancer cell is divided into 6 groups at random, and 6 every group, promptly Ganoderma tsugae albumen-polysaccharide is 2 groups, Polysaccharide-peptide capsule, 2 groups of lotus knurl control groups (physiological saline control group), normal control group.Administration finishes the back and puts to death animal next day, gets spleen under the aseptic condition, the preparation splenocyte, and adjusting cell concn is 1 * 10
6Individual/ml does the effector cell, and other gets and cultivates 24 hours YAC-1 cells, and adjusting cell concn is 1 * 10
4Individual/ml is as target cell, is that 1: 100 cell is added on the 96 porocyte culture plates with target cell and effector cell's ratio, adds
3H-TdR 0.5uci/ hole, every animal is established two multiple holes, 37 ℃, 5%CO
2Cultivate under the condition after 24 hours, collecting cell is surveyed the CPM value, calculates specificity and suppresses percentage (Pi) expression NK cytoactive, and the result is as shown in table 5, shows that Ganoderma tsugae albumen-polysaccharide can obviously activate the NK cells in mice activity.
Table 5 Ganoderma tsugae albumen-polysaccharide is to the active influence of tumor-bearing mice NK
Compare with the physiological saline group; * P＜0.05, * * P＜0.01.
3, Ganoderma tsugae albumen-polysaccharide is to the influence of tumor-bearing mice IL-2 generation
24 of C57BL/6 mouse, every mouse armpit subcutaneous vaccination Lewis lung cancer cell is divided into 4 groups at random, and 6 every group, i.e. Ganoderma tsugae albumen-polysaccharide, Polysaccharide-peptide capsule, lotus knurl control group (physiological saline control group), normal control group.Administration finishes the back and puts to death animal next day, measures the production of IL-2, and the result is as shown in table 6, shows that Ganoderma tsugae albumen-polysaccharide can promote the generation of IL-2, and the phagocytic function to Turnover of Mouse Peritoneal Macrophages also has obvious facilitation simultaneously.
Table 6 Ganoderma tsugae albumen-polysaccharide mixture produces the influence of IL-2 to tumor-bearing mice
Claims (10)
1, a kind of Ganoderma tsugae protein-polysaccharide extracts from the Ganoderma tsugae sporophore, and its molecular-weight average is made up of albumen and polysaccharide between 0.5-1.6 ten thousand dalton, and the weight ratio of described albumen and polysaccharide is an albumen: polysaccharide=(15.5-19.5): (80-86); Described polysaccharide is made up of semi-lactosi, glucose, seminose and fructose, and their weight ratio is a semi-lactosi: glucose: seminose: fructose=(1.60-1.75): 1.00: (0.15-0.22): (0.10-0.20), main chain connects with β (1 → 3).
2, Ganoderma tsugae protein-polysaccharide according to claim 1 is characterized in that: described Ganoderma tsugae protein-polysaccharide is to obtain with the method that may further comprise the steps: 1) the Ganoderma tsugae fruit body powder minces and uses pure degreasing, obtains the dregs of a decoction;
2) dregs of a decoction deionized water extraction is collected extracting solution;
3) extracting solution is precipitated with alcohol;
4) the throw out laggard capable micro-filtration of water dissolution;
5) filtrate is used the 3-5KD membrane ultrafiltration, the trapped substance on the collection membrane through gained filtrate behind the ultra-filtration membrane of 50-80KD;
6) DEAE-Sepharose (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl-) chromatography column on the trapped substance are used the deionized water wash-out, collect elutriant, through the 3-5KD film, and the trapped substance on the collection membrane;
7) gel column on the trapped substance is collected first-class part in the elutriant;
8) concentrate through the 3-5KD membrane ultrafiltration, lyophilize obtains product.
3, Ganoderma tsugae protein-polysaccharide according to claim 2, it is characterized in that: the described alcohol that is used for the degreasing of Ganoderma tsugae sporophore is ethanol or the methyl alcohol of 75-95%, the weight of ethanol or methyl alcohol be described Ganoderma tsugae fruit body powder mince weight 6-12 doubly, degreasing is carried out under 75-80 ℃ of condition, refluxing extraction 2-3 hour, repeat 3-6 time; Described dregs of a decoction deionized water extraction, collect in the extracting solution, the weight of deionized water be described Ganoderma tsugae fruit body powder mince weight 10-30 doubly, in 90-100 ℃, intermittently stir, extracted 2-3 hour, and repeated 3-7 time, extracting solution be concentrated into described Ganoderma tsugae fruit body powder mince weight 7-12 doubly; Described with extracting solution alcohol precipitation, be in extracting solution, to add ethanol or methyl alcohol, make its final concentration reach 75-85%, the limit edged stirs, placement at low temperatures, the centrifugal precipitation that obtains; Described micro-filtration carries out secondary at least, and the aperture of filter membrane is 10 μ m for the first time; The elutriant of described gel column is the NaOH of 0.1～0.4mol/L.
4, a kind of method for preparing the described Ganoderma tsugae protein-polysaccharide of claim 1 may further comprise the steps: 1) the Ganoderma tsugae fruit body powder minces and uses pure degreasing, obtains the dregs of a decoction;
2) dregs of a decoction deionized water extraction is collected extracting solution;
3) extracting solution is precipitated with alcohol;
4) the throw out laggard capable micro-filtration of water dissolution;
5) filtrate is used the 3-5KD membrane ultrafiltration, the trapped substance on the collection membrane through gained filtrate behind the ultra-filtration membrane of 50-80KD;
6) DEAE-Sepharose (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl-) chromatography column on the trapped substance are used the deionized water wash-out, collect elutriant, through the 3-5KD film, and the trapped substance on the collection membrane;
7) gel column on the trapped substance is collected first-class part in the elutriant;
8) concentrate through the 3-5KD membrane ultrafiltration, lyophilize obtains described Ganoderma tsugae protein-polysaccharide.
5, method according to claim 4 is characterized in that: behind the ultrafiltration and concentration of described step 8), and DEAE-Sepharose on the trapped substance (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl
-) chromatography column, use the deionized water wash-out, collect elutriant, through the 3-5KD film, the concentrated frozen drying obtains product.
6, according to claim 4 or 5 described methods, it is characterized in that: the alcohol that is used for the degreasing of Ganoderma tsugae sporophore in the described step 1) is ethanol or the methyl alcohol of 75-95%, the weight of ethanol or methyl alcohol be described Ganoderma tsugae fruit body powder mince weight 6-12 doubly, degreasing is carried out under 75-80 ℃ of condition, refluxing extraction 2-3 hour, repeat 3-6 time.
7, according to claim 4 or 5 described methods, it is characterized in that: described step 2), dregs of a decoction deionized water extraction, the weight of deionized water be described Ganoderma tsugae fruit body powder mince weight 10-30 doubly, in 90-100 ℃, intermittently stir, extracted 2-3 hour, repeat 3-7 time, extracting solution be concentrated into described Ganoderma tsugae fruit body powder mince weight 7-12 doubly; Described with extracting solution alcohol precipitation, be in extracting solution, to add ethanol or methyl alcohol, make its final concentration reach 75-85%, the limit edged stirs, placement at low temperatures, the centrifugal precipitation that obtains; Described micro-filtration carries out secondary at least, and the aperture of filter membrane is 10 μ m for the first time; The elutriant of described gel column is the NaOH of 0.1～0.4mol/L.
8, a kind of medicine of antitumor and strengthening immunity, its activeconstituents is the Ganoderma tsugae protein-polysaccharide, this Ganoderma tsugae protein-polysaccharide extracts from the Ganoderma tsugae sporophore, its molecular-weight average is between 0.5-1.6 ten thousand dalton, be made up of albumen and polysaccharide, the weight ratio of described albumen and polysaccharide is an albumen: polysaccharide=(15.5-19.5): (80-86); Described polysaccharide is made up of semi-lactosi, glucose, seminose and fructose, and their weight ratio is a semi-lactosi: glucose: seminose: fructose=(1.60-1.75): 1.00: (0.15-0.22): (0.10-0.20), main chain connects with β (1 → 3).
9, medicine according to claim 8 is characterized in that: described Ganoderma tsugae protein-polysaccharide is that the method that may further comprise the steps obtains: 1) the Ganoderma tsugae fruit body powder minces and uses pure degreasing, obtains the dregs of a decoction;
2) dregs of a decoction deionized water extraction is collected extracting solution;
3) extracting solution is precipitated with alcohol;
4) the throw out laggard capable micro-filtration of water dissolution;
5) filtrate is used the 3-5KD membrane ultrafiltration, the trapped substance on the collection membrane through gained filtrate behind the ultra-filtration membrane of 50-80KD;
6) DEAE-Sepharose (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl-) chromatography column on the trapped substance are used the deionized water wash-out, collect elutriant, through the 3-5KD film, and the trapped substance on the collection membrane;
7) gel column on the trapped substance is collected first-class part in the elutriant;
8) concentrate through the 3-5KD membrane ultrafiltration, lyophilize obtains product.
10, medicine according to claim 9, it is characterized in that: the alcohol that is used for the degreasing of Ganoderma tsugae sporophore in the described step 1) is ethanol or the methyl alcohol of 75-95%, the weight of ethanol or methyl alcohol be described Ganoderma tsugae fruit body powder mince weight 6-12 doubly, degreasing is carried out under 75-80 ℃ of condition, refluxing extraction 2-3 hour, repeat 3-6 time; Described step 2) Chinese medicine slag deionized water extraction, collect in the extracting solution, the weight of deionized water be described Ganoderma tsugae fruit body powder mince weight 10-30 doubly, in 90-100 ℃, intermittently stir, extracted 2-3 hour, and repeated 3-7 time, extracting solution be concentrated into described Ganoderma tsugae fruit body powder mince weight 7-12 doubly; In the described step 3) extracting solution being precipitated with alcohol, is to add ethanol or methyl alcohol in extracting solution, makes its final concentration reach 75-85%, and the limit edged stirs, and places at low temperatures, the centrifugal precipitation that obtains; Micro-filtration in the described step 4) carries out secondary at least, and the aperture of filter membrane is 10 μ m for the first time; The elutriant of gel column is the NaOH of 0.1～0.4mol/L in the described step 7).
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| SG174110A1 (en) * | 2007-07-30 | 2011-10-28 | Phyto Pharma Pte Ltd | New ganoderma tsugae var. jannieae strain tay-i and biologically active biomass and extracts therefrom |
| CN101857645B (en) * | 2010-05-20 | 2011-09-21 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN105131096A (en) * | 2015-08-10 | 2015-12-09 | 潍坊医学院 | Fructus toonae sinensis protein extraction method |
| CN107556393A (en) * | 2017-08-07 | 2018-01-09 | 华南理工大学 | A kind of hot water of the ganoderma lucidum dregs of a decoction removes sugared method and product |
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| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| CN1429837A (en) * | 2001-12-29 | 2003-07-16 | 福州绿谷生物药业技术研究所 | Grass planted ganoderma polysaccharide peptide and its extraction method |
| US6613754B1 (en) * | 2000-09-22 | 2003-09-02 | National Yang-Ming University | Polysaccharide-based extract from ganoderma, pharmaceutical use thereof, and process for preparing the same |
| CN1448409A (en) * | 2003-05-12 | 2003-10-15 | 中国药科大学 | Chi lucid ganoderma polysaccharide and prep. and use thereof |
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| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| US6613754B1 (en) * | 2000-09-22 | 2003-09-02 | National Yang-Ming University | Polysaccharide-based extract from ganoderma, pharmaceutical use thereof, and process for preparing the same |
| CN1429837A (en) * | 2001-12-29 | 2003-07-16 | 福州绿谷生物药业技术研究所 | Grass planted ganoderma polysaccharide peptide and its extraction method |
| CN1448409A (en) * | 2003-05-12 | 2003-10-15 | 中国药科大学 | Chi lucid ganoderma polysaccharide and prep. and use thereof |
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