CN1303099C - Ganoderma tsugae-proteoglycan mixture and its preparing method and use - Google Patents
Ganoderma tsugae-proteoglycan mixture and its preparing method and use Download PDFInfo
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- CN1303099C CN1303099C CNB200410050150XA CN200410050150A CN1303099C CN 1303099 C CN1303099 C CN 1303099C CN B200410050150X A CNB200410050150X A CN B200410050150XA CN 200410050150 A CN200410050150 A CN 200410050150A CN 1303099 C CN1303099 C CN 1303099C
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Abstract
The present invention discloses a ganoderma tsugae-proteoglycan mixture, a preparation method and the application thereof. The ganoderma tsugae-proteoglycan mixture is a substance with the molecular weight of 6 to 12KDa. The substance is obtained by using a method comprising the following procedures: carrying out degreasing on ganoderma fruiting body crushing objects by using alcohols to obtain dregs of decoction, extracting the dregs of decoction by ionized water and collecting extract, precipitating the extract by alcohols, carrying out micro filtration after dissolving sediment by water, carrying out ultrafiltration on obtained filtrate after the filtrate passes through an ultrafiltration film of 50 to 80KD by using a film of 3 to 5KD, and obtaining refined products of tsugae-proteoglycan mixtures after carrying out chromatographic separation of DEAE-Cellulose on trapped substances on the film. The ganoderma tsugae-proteoglycan mixture has obvious functions of restraining tumors and improving immunity functions of bodies, and has an extensive medicinal value.
Description
Technical field
The present invention relates to a kind of protein-polysaccharide mixture and preparation method thereof and application, particularly relate to a kind of protein-polysaccharide mixture and the extracting method and of the application of this protein-polysaccharide mixture that from Ganoderma tsugae (Ganoderma tsugae Murrill.), obtain as injection or oral pharmaceutical activeconstituents.
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 are constantly seeking the new cancer therapy drug that toxicity is little, tumour inhibiting rate is high, and have obtained certain effect.Because the generation of tumour and the immunologic function of diffusion and human body have substantial connection, therefore 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 power and antineoplastic action.The mechanism of action of these medicines is 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, is characterized in that effective dose is little, the tumour inhibiting rate height, toxicity is very little, even nontoxic, can also reduce the toxic side effect of chemotherapeutics with the common use of chemotherapeutics.Compare with chemotherapy, immunotherapy does not have leukopenia, vomiting, toxic side effect such as immunity degradation.
Ganoderma tsugae (Gamoderma tsugae Murrill.) is a Basidiomycetes, polyporaceae, and a kind of fungi 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 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, and these 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 fromganoderma, 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 purpose of this invention is to provide a kind of Ganoderma tsugae protein-polysaccharide mixture and preparation method thereof and be the activeconstituents of injection, oral or external curing tumour medicine with this Ganoderma tsugae protein-polysaccharide mixture.
The molecular weight of Ganoderma tsugae protein-polysaccharide mixture provided by the present invention is 6000-12000 dalton, 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) gained filtrate is used the 3-5KD membrane ultrafiltration behind the ultra-filtration membrane of filtrate process 50-80KD (50,000-80,000 dalton), and the trapped substance on the film is the refining preceding crude product of upper prop.
6) in order to obtain smart product, after the ultrafiltration, DEAE-Sepharose (F.F.), SepharoseCL-6B or DEAE-Cellulose (Cl on the last trapped substance
-) chromatography column, use the deionized water wash-out, collect elutriant, through the 3-5KD film, the concentrated frozen drying obtains protein-polysaccharide mixture highly finished product.In this treating process, 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, and the speed of making moving phase wash-out pillar with deionized water should be 2-4ml/min.
In the process of extracting Ganoderma tsugae protein-polysaccharide mixture, 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 70-80 ℃ 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 low temperature, 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.
Experiment showed, that Ganoderma tsugae protein-polysaccharide mixture with method of the present invention preparation is that the medicine of activeconstituents has the effect of obvious suppression tumour and improves immunity function.
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.
Ganoderma tsugae protein-polysaccharide mixture 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 125mg/kg (mouse), nontoxic substantially, have broad application prospects.
Embodiment
Embodiment 1, preparation Ganoderma tsugae albumen-polysaccharide mixture
Take by weighing Ganoderma tsugae fruit body powder 100 gram, add 1 liter of 80% ethanol, 75-78 ℃ 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, carry out ultrafiltration with the 3KD film again, the gained trapped substance concentrates, and lyophilize obtains Ganoderma tsugae albumen-polysaccharide mixture.
Embodiment 2, preparation Ganoderma tsugae albumen-polysaccharide mixture
Take by weighing Ganoderma tsugae fruit body powder 100 gram, 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, be concentrated into 1 liter of volume, cooling adds 4 liters of anhydrous methanols, the limit edged stirs, and places clarification, centrifugal (7000-8000rpm for 4 ℃, 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, the gained trapped substance concentrates, lyophilize obtains Ganoderma tsugae albumen-polysaccharide mixture.
Embodiment 3, preparation Ganoderma tsugae albumen-polysaccharide mixture
Take by weighing 50kg Ganoderma tsugae sporophore flour in extractor, add 450 liter of 90% ethanol, stir down, in 78 ℃ 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, and repeated 5 times, filter, filtrate merges, and being concentrated into volume is 250 liters, under agitation, add 5 times of volume dehydrated alcohols, put 4 ℃ of low temperature places, clarification, centrifugal (7000-8000rpm, 15min), the precipitate with deionized water dissolving is by 10 μ m micro-filtrations, 1 μ m micro-filtration, and then through after the 80KD ultrafiltration, use the 3KD membrane concentration, lyophilize gets Ganoderma tsugae albumen-polysaccharide mixture.
Embodiment 4, preparation Ganoderma tsugae albumen-polysaccharide mixture elaboration
After the Ganoderma tsugae albumen-polysaccharide mixture that obtains among the embodiment 1 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, detects with UV-detector, write down elution curve with registering instrument, effluent liquid concentrates postlyophilization by the 3KD film, gets Ganoderma tsugae albumen-polysaccharide mixture.
Embodiment 5, preparation Ganoderma tsugae albumen-polysaccharide mixture elaboration
After the Ganoderma tsugae albumen-polysaccharide mixture that obtains among the embodiment 3 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, detect with UV-detector, write down elution curve with registering instrument, effluent liquid is by the 3KD film, concentrate postlyophilization, get Ganoderma tsugae albumen-polysaccharide mixture.
Embodiment 6, 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 mixture 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 as shown in Table 1 and Table 2, show that Ganoderma tsugae albumen-polysaccharide mixture all has restraining effect in various degree to begin administration to transplanting in human body intestinal cancer LOVO, cancer of the stomach MKN45, the liver cancer QGY of nude mice after seeing knurl to the growth of mouse S180 sarcoma, rat liver cancer H22 and Mice Bearing Lewis Lung Cancer, all have obvious antineoplastic, dose-effect relationship is obvious.
Table 1, Ganoderma tsugae albumen-polysaccharide mixture are to the restraining effect of the tumor growth of mouse
Table 2, Ganoderma tsugae albumen-polysaccharide mixture are to transplanting in the restraining effect of the human tumor growth of nude mice
The synergism of embodiment 7, Ganoderma tsugae albumen-polysaccharide mixture
After Ganoderma tsugae albumen-polysaccharide mixture (1,0.3mg/kg) merges low dose of endoxan (20mg/kg), Rheumatrex (2mg/kg) and ametycin (1mg/kg), the restraining effect of rat liver cancer H22 obviously is better than Ganoderma tsugae albumen-polysaccharide mixture powder injection or low dose of chemotherapeutics uses separately.According to WebbShi mark product method calculating proof Ganoderma tsugae albumen-polysaccharide mixture powder injection and said medicine synergy is arranged.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, proves that low dose of Ganoderma tsugae albumen-the polysaccharide mixture powder injection merges low dose of chemotherapy that rat liver cancer H22 is had certain synergy, simultaneously and radiotherapy merge, tumor-inhibiting action also obviously is better than independent radiotherapy.
Table 3 Ganoderma tsugae albumen-polysaccharide mixture 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 mixture | 42.71 | ||
| CTX | 40.11 | ||
| Ganoderma tsugae albumen-polysaccharide mixture+CTX | 65.82 | 65.69 | Collaborative |
| MTX | 26.44 | ||
| Ganoderma tsugae albumen-polysaccharide mixture+MTX | 52.80 | 57.86 | |
| MMC | 44.99 | ||
| Ganoderma tsugae albumen-polysaccharide mixture+MMC | 65.82 | 68.49 |
Compare with the physiological saline group:
*P＜0.05,
*P＜0.01.
Claims (9)
1, a kind of Ganoderma tsugae protein-polysaccharide mixture is that molecular weight is 6000-1.2 ten thousand dalton, and the material that the method that usefulness 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 3KD membrane ultrafiltration through gained filtrate behind the ultra-filtration membrane of 80KD, and the trapped substance on the film is a product.
2, mixture according to claim 1 is characterized in that: after the described ultrafiltration, 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.
3, mixture according to claim 1 and 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 refluxing extraction under 75-80 ℃ of condition; Described dregs of a decoction deionized water extraction is collected 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 and extract concentrated extracting solution; With the alcohol precipitation, is to add ethanol or methyl alcohol in extracting solution with described extracting solution, makes its final concentration reach 75-85%, the centrifugal precipitation that obtains; Described micro-filtration carries out twice at least, and the filter membrane aperture is respectively 10 μ m and 1 μ m.
4, a kind of method for preparing Ganoderma tsugae protein-polysaccharide mixture 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) throw out carries out micro-filtration after with deionized water dissolving;
5) filtrate is used the 3KD membrane ultrafiltration through gained filtrate behind the ultra-filtration membrane of 80KD, and the trapped substance on the film is a product.
5, method according to claim 4 is characterized in that: after the described ultrafiltration, 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 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.
7, according to claim 4 or 5 described methods, it is characterized in that: 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, 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, is 1 μ m for the second time.
8, a kind of medicine of antitumor and strengthening immunity, its activeconstituents is any described Ganoderma tsugae protein-polysaccharide mixture among the claim 1-3.
9, medicine according to claim 8 is characterized in that: described medicine is injection, oral preparation or exterior-applied formulation.
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| CN103494978B (en) * | 2013-09-28 | 2015-04-29 | 河南九福来科技集团股份有限公司 | Method for preparing freeze-dried powder injection for treating liver ascitic tumor by using aloe and lucid ganoderma |
| CN103588893B (en) * | 2013-11-28 | 2016-06-22 | 南京农业大学 | A kind of preparation method of southern Anhui Umbilicaria esculenta polysaccharide |
| 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 |
| CN110183522B (en) * | 2019-05-29 | 2020-09-18 | 北京市农林科学院 | Glycopeptide with antidepressant function and preparation method and application thereof |
| CN110724177B (en) * | 2019-09-29 | 2023-05-26 | 杨凌萃健生物工程技术有限公司 | Ganoderma lucidum glycopeptide and preparation method thereof |
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| 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 |
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