CN100347196C - Ganodema tsugae-proteoglycan and its preparing method and use - Google Patents
Ganodema tsugae-proteoglycan and its preparing method and use Download PDFInfo
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Abstract
The present invention relates to a crude product of ganoderma proteoglycan, three types of pure products of proteoglycan, a preparation method and the application thereof in preparing anti-tumor drugs. The crude product of ganoderma proteoglycan is a mixture containing three types of single proteoglycan of P1, P2 and P3, wherein the product comprises the proteoglycan of the following proportion by weight: 1.50 to 3.00% of proteoglycan P1, 52 to 58% of proteoglycan P2 and 20 to 25% of proteoglycan P3, and polysaccharide parts of the polysaccharides are all uniform glucan with the glycosidic bonds of beta (1 to 3) configuration, wherein the n value of P1 is 500 to 611, and the molecular weight is 180 to 220Kda. The n value of P2 is 42 to 69, and the molecular weight is 15 to 25Kda. The n value of P3 is 17 to 34, and the molecular weight is 6 to 12Kda. The protein part is combined to the reduction end of the polysaccharide part in integral form.
Description
Technical field
The present invention relates to proteoglycan that extracts from Ganoderma tsugae (Ganoderma tsugae Murrill.) sporophore and preparation method thereof, also relate to the application of proteoglycan in antitumor drug.
Background technology
Treat tumor disease at present clinically, main operation, radiotherapy, the chemotherapy of adopting.Because radiotherapy, chemotherapy are are seriously killed and wounded human normal cell and tissue, and many patient's physique are descended rapidly, even can not continued treatment.People are at the cancer therapy drug of constantly seeking characteristics such as new having toxicity is little, tumour inhibiting rate height for this reason.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, derive from the polysaccharide medicine of Mycophyta, krestin is arranged, lentinan, Schizophyllum commune Fr polysaccharides, all have enhance immunity power and antineoplastic action, its mechanism of action is by host intermediary, activates the various immunologically competent cells of body, promotes the secretion of various lymphokines, improve the resistibility of body, reach the effect of engulfing and eliminating tumour cell.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 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 mouse boosting cell 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.Research to Ganoderma tsugae (Ganoderma tsugae Murill.) is fewer.
International Yang Ming Mr.'s Wu of university in Taiwan United States Patent (USP) (US 6613754), " Polysaccharide-based extract from ganoderma; pharmaceuticaluse thereof; and process for preparing the same ", the technology that is adopted and the position of extract are all different with the present invention with character.
Contain several bioactive macromolecule materials in the Ganoderma tsugae sporophore, this patent relates generally to albumen dextran of alkali water lift dissolubility and preparation method thereof and the application in the preparation antitumor drug.
Summary of the invention
The object of the present invention is to provide a kind of effective dose little, the tumor killing effect height, toxicity is very little, even nontoxic Ganoderma tsugae proteoglycan and preparation method thereof and the purposes in the preparation antitumor drug.
For realizing above-mentioned purpose of the present invention, technical scheme of the present invention is as follows:
A kind of Ganoderma tsugae proteoglycan crude product GTP, it is to comprise three kinds of single proteoglycan P 1, the mixture of P2 and P3, wherein, their weight percent is respectively: P11.50～3.00%, P2 52～58%, P3 20～25%, and their polysaccharide partly is the dextran of homogeneous, and glycosidic link is the configuration of β-(1-3), its structural formula is as shown in the formula expression
Wherein, the n value of P1 is 500～611, and molecular weight is 18～220,000; The n value of P2 is 42～69, and molecular weight is 1.5～2.5 ten thousand; The n value of P3 is 17～34, and molecular weight is 0.6～1.2 ten thousand; Their protein portion is formed by 14 seed amino acids based on Serine, and being combined in the reduction end of polysaccharide part with integral form, 14 seed amino acids are respectively aspartic acid, Threonine, Serine, L-glutamic acid, glycine, L-Ala, halfcystine, methionine(Met), phenylalanine, Methionin, Histidine, arginine, proline(Pro), tyrosine.
Ganoderma tsugae proteoglycan crude product GTP prepares by the following method, the Ganoderma tsugae sporophore is pulverized the back and is carried out degreasing with organic solvent, after hot water extraction is filtered then, perhaps directly also filter with hot water extraction without degreasing, obtain the dregs of a decoction, the dregs of a decoction are extracted proteoglycan with the dilute alkaline soln that contains sodium tetrahydroborate, it is neutral regulating pH, get supernatant liquor and add the absolute alcohol precipitation, resolution of precipitate is in water, behind micro-filtration, ultrafiltration and concentration, and lyophilize, obtain Ganoderma tsugae proteoglycan crude product GTP of the present invention, its molecular-weight average is between 0.6～220,000.Further behind the separation and purification, can obtain pure product P1, P2, P3.
Specifically, prepare the method for above-mentioned Ganoderma tsugae proteoglycan crude product GTP, form by the following step:
(1) get the Ganoderma tsugae fruit body powder and mince, add 10～40 times of deionized waters of its weight, heated and stirred was extracted 2～3 hours at every turn, filtered, and discarded filtrate, repeated 3～6 times, and it is standby to obtain the dregs of a decoction.
(2) in the above-mentioned dregs of a decoction, add 2～10% dilute alkaline solns of 10～40 times of its sporophore medicinal powder weight, contain sodium tetrahydroborate 0.04～0.12% in this solution, after fully stirring,, filter in 20～40 ℃ of lixiviates 25～30 hours.It is neutral regulating filtrate pH, places clarification, centrifugal, collect supernatant liquor, the absolute alcohol that adds 2～4 times of its volumes, the limit edged stirs, and places clarification, centrifugal, precipitation is dissolved in the deionized water, and through 20 μ m～1 μ m micro-filtrate membrane filtration, filtrate concentrates through 3～5KD ultra-filtration and separation again, lyophilize obtains Ganoderma tsugae proteoglycan crude product GTP.
Used dilute alkaline soln can be NaOH, KOH, LiOH, Na in the aforesaid method
2CO
3Perhaps NaHCO
3Deng dilute solution; Above-mentioned absolute alcohol can be anhydrous methanol or dehydrated alcohol.
Ganoderma tsugae proteoglycan crude product GTP also can prepare with following method:
(1) get the Ganoderma tsugae fruit body powder and mince, add its weight 5-8 70～99% organic solvents doubly and carry out degreasing, be heated to 70-80 ℃, refluxing extraction, each 3-6 hour, repeat 3-6 time, filter, merging filtrate reclaims organic solvent, and the dregs of a decoction are standby.
(2) 10～40 times of deionized waters of adding sporophore medicinal powder weight in step (1) the gained dregs of a decoction are heated to 98-100 ℃, extract 3～4 hours at every turn, repeat 4～6 times, filter, and the dregs of a decoction are standby.
(3) in the dregs of a decoction of step (2) gained, 2～10% the dilute alkaline soln that adds 10～40 times of its sporophore medicinal powder weight, contain sodium tetrahydroborate 0.04～0.12% in this solution, after fully stirring evenly, filtered in 25～30 hours in 20～40 ℃ of lixiviates, it is neutral regulating filtrate pH, places clarification, centrifugal collection supernatant liquor, the absolute alcohol that adds 2～4 times of its volumes, the limit edged stirs, and places clarification, after centrifugal, precipitation is dissolved in the deionized water, behind 3～1 μ m micro-filtrations, again through 3～5KD ultrafiltration and concentration, lyophilize obtains Ganoderma tsugae proteoglycan crude product GTP.
Used organic solvent can be methyl alcohol, ethanol, propyl alcohol, propyl carbinol, amylalcohol, isopropylcarbinol, acetone, sherwood oil, ether, chloroform or tetracol phenixin etc. in the aforesaid method; Used dilute alkaline soln can be NaOH, KOH, LiOH, Na
2CO
3Perhaps NaHCO
3Deng dilute solution; Above-mentioned absolute alcohol can be anhydrous methanol or dehydrated alcohol.
Above-mentioned Ganoderma tsugae proteoglycan crude product GTP can use in the preparation antitumor drug, and they can be used as injection (powder pin, liquid drugs injection), tablet, capsule, electuary, powder or oral liquid and use, and its zooperal effective dose is 5mg/kg.
Three kinds of single proteoglycan P1 of Ganoderma tsugae, P2, P3, their polysaccharide partly is the dextran of homogeneous, and glycosidic link is beta configuration, and its structural formula is as shown in the formula expression,
Their protein portion is formed by 14 seed amino acids based on Serine, and being combined in the reduction end of polysaccharide part with integral form, 14 seed amino acids are respectively aspartic acid, Threonine, Serine, L-glutamic acid, glycine, L-Ala, halfcystine, methionine(Met), phenylalanine, Methionin, Histidine, arginine, proline(Pro), tyrosine.
Wherein, P1 is light brown solid substance, and its n value is 500～611, and molecular weight is 18～220,000, and specific rotatory power is [α]
D 26(10%NaOH)=and 38-45.2, c=0.5%, the weight content of polysaccharide part is 60.4～71.2%, the weight content of protein portion is 39.6～28.8%;
P2 is light brown solid substance, and its n value is 42～69, and molecular weight is 1.5～2.5 ten thousand, and specific rotatory power is [α]
D 26(10%NaOH)=12.6-16.2, c=0.5%, the weight content of polysaccharide part is 65～76%, the weight content of protein portion is 35～24%, and the content (μ g/mg) of its 14 seed amino acid in P2 is: aspartic acid Asp (0.20-0.4), Threonine Thr (0.10-0.2), Serine Ser (0.3-0.4), L-glutamic acid Glu (0.05-0.15), glycine Gly (0.4-0.55), L-Ala Ala (0.03-0.16), halfcystine Cys (0.08-0.22), methionine(Met) Met (0.20-0.30), phenylalanine Phe (0.10-0.22), Methionin Lys (0.10-0.25), Histidine His (0.06-0.20), arginine Arg (3.0-5.5), proline(Pro) Pro (0.1-0.23), tyrosine Tyr (0.01-0.07);
P3 is light brown solid substance, and its n value is 17～34, and molecular weight is 0.6～1.2 ten thousand, and specific rotatory power is [α]
D 26(10%NaOH)=185-199, c=0.5%, the weight content of polysaccharide part is 56.6～78%, the weight content of protein portion is 43.4～22%, and the content (μ g/mg) of its 14 seed amino acid in P3 is: aspartic acid Asp (3.4-.48), Threonine Thr (1.7-2.2), Serine Ser (4.8-5.6), L-glutamic acid Glu (0.81-1.2), glycine Gly (4.8-6.2), L-Ala Ala (1.0-2.23), halfcystine Cys (2.0-3.01), methionine(Met) Met (3-4.35), phenylalanine Phe (0.51-1.8), Methionin Lys (1.07-2.3), Histidine His (1.02-2.34), arginine Arg (0.9-1.5), proline(Pro) Pro (1.1-1.94), tyrosine Tyr (1.6-2.90).
Three kinds of single Ganoderma tsugae proteoglycan P1, P2, the preparation method of P3 is, by above-mentioned Ganoderma tsugae proteoglycan crude product GTP water-soluble after, last gel column carries out chromatographic separation, with 0.2～0.4mol/L NaOH eluant solution of 2～4 times of column volumes, collect first-class fluid, second effluent liquid and the 3rd effluent liquid respectively, concentrate through 3～5KD ultra-filtration membrane respectively, after the desalination, lyophilize gets the single proteoglycan P1 of thing Ganoderma tsugae of the present invention, P2, P3 respectively again.
Specifically, prepare the method for the single proteoglycan P1 of Ganoderma tsugae, P2, P3, form by the following step:
(1) above-mentioned Ganoderma tsugae proteoglycan crude product is dissolved in 20～60 times of distilled water after, last gel column carries out chromatographic separation, last sample speed 0.2～0.8ml/min;
(2) after upward sample finishes, with 0.1～0.5mol/L NaOH eluant solution, elution speed 0.4～8ml/min, collect first component, second component, the 3rd component stream fluid respectively, and respectively after 3～5KD ultra-filtration membrane desalination and concentration, lyophilize obtains the single proteoglycan P1 of the present invention, three pure product of P2, P3.
Above-mentioned gel column can be Sephadex-G150 post, Sepharose F.F. post, Sephadex G-50F post, Sephadex G-50C post, Sephadex G-75 post or Sephadex G-100 post.
The single proteoglycan P1 of gained of the present invention, three pure product of P2, P3 can be used in the preparation cancer therapy drug respectively, they can be used as injection (powder pin, liquid drugs injection), tablet, capsule, electuary, powder or oral liquid and use, and its zooperal effective dose is 5mg/kg.
Ganoderma tsugae proteoglycan crude product GTP of the present invention and three pure product P1 of proteoglycan, P2, the preparation method of P3 can be used to prepare the proteoglycan of other glossy ganoderma equally, for example red sesame, purple sesame, Tibetan glossy ganoderma etc.
Proteoglycan of the present invention shows that through pharmacological experiment study it has enhance immunity function, anti-cell mutation effect, reducing blood-fat, hypoglycemic, many-sided physiological action such as the protection liver is calmed the nerves, calmness, and is antitumor.Through molecular biology and cellular pharmacology research, it can divide, break up by regulating cell, regulates cell growth and decline.Fungus polysaccharide is very fast as the progress of medicine, has been used for the immunotherapy of cancer clinically, has showed good antineoplastic activity.Experiment is proof also, share with cytotoxic drug, has efficacy enhancing and toxicity reducing, improves the patient immune function, prolongs life and basic bright prospect of curing.
Proteoglycan tumour inhibiting rate of the present invention is higher, and the dosage of 5mg/kg is just near the tumour inhibiting rate of the endoxan (CTX) of 30mg/kg dosage.Compare with the chemotherapeutic of using in the background technology, radiotherapy medicine, proteoglycan toxicity of the present invention is minimum, immunocompetence and increase in life span height.Its toxicity is, oral experimentation on animals medium lethal dose LD50 is 32.50g/kg, and the injection maximum tolerated dose is 150mg/kg.GTP, P1, P2, toxicity, tumour inhibiting rate, increase in life span and the immunocompetence of P3 and chemotherapeutics endoxan (CTX) are compared as follows: (CTX 30mg/kg abdominal injection, 10 days; GTP and P1, P2, P3, intravenous injection, 10 days)
Table 1
| Sample | GTP (5mg/kg) | P1 (5mg/kg) | P2 (5mg/kg) | P3 (5mg/kg) | CTX (30mg/kg) | |
| Tumour inhibiting rate (%) | S180 | 69.5 | 73.2 | 64 | 67.4 | 80.90 |
| H22 | 66.7 | 76 | 74 | 68 | 82.56 | |
| Liweis | 72.8 | 78.6 | 80 | 79 | 82.27 | |
| Toxicity | Substantially nontoxic | Nontoxic | Nontoxic | Nontoxic | Toxicity is big | |
| Immunocompetence | High | High | High | High | Do not have | |
| Increase in life span (%) | 252.1 | 243.9 | 237.4 | 220.6 | 0 | |
Increase in life span=administration group average the survival of number of days/blank group number of days * 100% of on average surviving
The tumour inhibiting rate of proteoglycan of the present invention is compared all high than close medicine, for example, Shandong Normal University's journal the 13rd volume P174, the strain of lentinan abdominal injection 24mg/kg S180 knurl, tumour inhibiting rate is 52.8%; Research and development of natural products Vol.12No.6P74, Lu petrel etc.Krestin: to the strain of S180 knurl, 10mg/kg abdominal injection, inhibitory rate 60～70%.Lentinan is 43% to the tumour inhibiting rate of S180, is 47% tumour inhibiting rate to hepatoma H22, and strain is a tumour inhibiting rate more than 30% to polyporusum bellatus to the S180 knurl.
The The pharmacological results part
1, GTP, P1, P2, the antitumor action of P3 proteoglycan
Test method
Get the ascites of the 7th day knurl source animal of inoculation under the aseptic condition, add the stroke-physiological saline solution dilution, making its concentration is 1-2 * 10
7Individual cancer cells/ml alive gets 30 kunming mices, male and female half and half, and every mouse right fore oxter subcutaneous vaccination 0.2ml contains oncocyte 0.5ml alive approximately.The inoculation back is divided into 3 groups at random with laboratory animal next day, blank group, positive controls, Ganoderma tsugae proteoglycan GTP (administration 5mg/kg) group.Blank group intraperitoneal injection of saline every day 0.2ml/10g, successive administration 10 days; Positive controls intraperitoneal injection of cyclophosphamide 30mg/kg, the next day administration; Ganoderma tsugae polysaccharide GTP intravenous injection, continuous 10 days.After the tumor-bearing mice drug withdrawal next day with sacrifice of animal half, dissect and subcutaneously get the knurl piece and weigh, average knurl of calculating each group respectively is heavy, calculates tumour inhibiting rate.Treat all dead increase in life span that calculates of blank group mouse.
Tumour inhibiting rate=(the average knurl of the average knurl weight-administration of blank group group is heavy)/average knurl of blank group heavy * 100%
Table 2 Ganoderma tsugae proteoglycan GTP is to the restraining effect of mice lung cancer Liweis (x ± SD)
| Blank group | CTX(30mg/kg，ip) | GTP(5mg/kg，iv) | |
| Knurl heavy (g) | 3.052±0.804 | 0.541±0.407 *** | 0.831±0.467 *** |
| Tumour inhibiting rate (%) | 0 | 82.27 | 72.78 |
Compare with control group,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Table 3 Ganoderma tsugae proteoglycan GTP is to the restraining effect of rat liver cancer H22 (x ± SD)
| Blank group | CTX(30mg/kg，ip) | GTP(5mg/kg，iv) | |
| Knurl heavy (g) | 4.435±1.079 | 0.773±0.545 *** | 1.476±0.658 *** |
| Tumour inhibiting rate (%) | 82.56 | 66.71 |
Compare with control group,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Table 4 Ganoderma tsugae proteoglycan GTP is to the restraining effect of mouse S180 (x ± SD)
| Blank group | CTX(30mg/kg，ip) | GTP(5mg/kg，iv) | |
| Knurl heavy (g) | 2.767±0.807 | 0.528±0.545 *** | 0.844±0.602 *** |
| Tumour inhibiting rate (%) | 80.90 | 69.50 |
Compare with control group,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
2, the efficacy enhancing and toxicity reducing effect of GTP proteoglycan
2.1, GTP merges that low dose of endoxan (CTX) obviously is better than GTP to the restraining effect of rat liver cancer H22 or low dose of chemotherapeutics uses separately, sees Fig. 1.
Indicate: GTP and merge that low dose of Rheumatrex and ametycin also obviously are better than GTP to the restraining effect of rat liver cancer H22 or low dose of chemotherapeutics uses separately.
2.2, GTP merges that radiotherapy obviously is better than GTP to the restraining effect of rat liver cancer H22 or radiotherapy is used separately, sees Fig. 2.
2.3, GTP is for the provide protection of the granulocyte poison of chemotherapy
Mouse is fed the chemotherapeutics endoxan after 48 hours or 72 hours, gives GTP96,120,144 hours, measures leukocytic quantity.The white blood cell count of chemotherapy mouse group reduces, and is restored in the mouse of hello the GTP group again.See Fig. 3.
3GTP, P1, P2, the raising immunity of P3 proteoglycan
3.1, GTP, P1, P2, P3 is to the influence of lotus Lewis lung cancer cell mouse spleen lymphocyte proliferation
Test method
36 of C57BL/6 mouse, every mouse armpit subcutaneous vaccination Lewis lung cancer cell is divided into 6 groups at random, and 6 every group, i.e. GTP, P1, P2, P3 (5mg/kg) group, lotus knurl control group (physiological saline control group), other establishes one group of normal control group.Administration finishes the back and puts to death animal next day, gets spleen under the aseptic condition, the counting splenocyte, and the adjustment cell concn is 1 * 10
7Individual/ml, every hole adds cell suspension 100ul on 96 well culture plates, ConA 50ul and nutrient solution, and every animal is established two multiple holes, 37 ℃, 5%CO
2Cultivated 18 hours under the condition, add the 3H-TdR0.5uci/ hole, continue to cultivate 18 hours.With bull cell harvestor collecting cell, on liquid scintillation instrument, survey the CPM value, and compare with lotus knurl control group.
Table 5GTP, P1, P2, P3 are to the influence of tumor-bearing mice spleen lymphocyte proliferation
| Group | Dosage | Number of animals | CPM( x±SD) |
| Normal control | 6 | 3355±676 ** | |
| Lotus knurl contrast (NS) | 6 | 460±99 | |
| GTP | 5mg/kg | 6 | 2638±809 ** |
| P1 | 5mg/kg | 6 | 3011±507 ** |
| P2 | 5mg/kg | 6 | 2733±830 ** |
| P3 | 5mg/kg | 6 | 2300±469 ** |
Compare with lotus knurl group:
*P＜0.01.
3.2, GTP, P1, P2, P3 is to the enhancement of tumor-bearing mice NK cytoactive
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, i.e. GTP, P1, P2, the P35mg/kg group, lotus knurl control group (physiological saline control group), other establishes one group of 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.
Table 6GTP, P1, P2, P3 are to the active influence of tumor-bearing mice NK
| Group | Dosage | Number of animals | CPM( x±SD) | Pi(％) |
| Normal control | 6 | 5118±846 ** | ||
| Lotus knurl contrast (NS) | 6 | 14553±2440 | ||
| GTP | 5mg/kg | 6 | 11859±1014 ** | 18.52 |
| P1 | 5mg/kg | 6 | 13389±2131 ** | 8.00 |
| P2 | 5mg/kg | 6 | 12877±1011 ** | 11.52 |
| P3 | 5mg/kg | 6 | 11098±2487 ** | 23.74 |
Compare with the physiological saline group:
*P＜0.05,
*P＜0.01.
3.3, GTP, P1, P2, the enhancement that P3 produces tumor-bearing mice IL-2
36 of C57BL/6 mouse, every mouse armpit subcutaneous vaccination Lewis lung cancer cell is divided into 6 groups at random, and 6 every group, promptly GTP 5mg/kg organizes, lotus knurl control group (physiological saline control group), other establishes one group of normal control group.Administration finishes the back and puts to death animal next day, gets spleen under the aseptic condition, the preparation splenocyte suspension, and adjusting cell concn is 1 * 10
7Individual/ml, every hole adds 2ml cell and ConA 5ug/ml, 37 ℃, 5%CO on 24 orifice plates
2Cultivate under the condition and collected supernatant liquor in 24 hours, with normal C57BL/6 mouse chest cell, with
3H-TdR mixes method and measures the IL-2 activity, and compares with control group and administration group.
Table 7GTP, P1, P2, P3 produce the influence of IL-2 to tumor-bearing mice
| Group | Dosage | Number of animals | CPM( x±SD) |
| Normal control | 6 | 711±115 | |
| Lotus knurl contrast (NS) | 6 | 460±150 | |
| GTP | 5mg/kg | 6 | 1172±765 ** |
| P1 | 5mg/kg | 6 | 2044±890 ** |
| P2 | 5mg/kg | 6 | 1607±211 ** |
| P3 | 5mg/kg | 6 | 1090±556 ** |
Compare with the physiological saline group:
*P＜0.05,
*P＜0.01.
Description of drawings
Fig. 1 GTP proteoglycan is to the efficacy enhancing and toxicity reducing effect of chemotherapy;
Fig. 2 GTP proteoglycan is to the efficacy enhancing and toxicity reducing effect of radiotherapy;
Fig. 3 is the provide protection of proteoglycan crude product GTP for the granulocyte poison of chemotherapy.
Embodiment
The preparation of embodiment 1 Ganoderma tsugae proteoglycan crude product GTP
Take by weighing the Ganoderma tsugae fruit body powder 1kg that minces, add 80% methyl alcohol 8L, be heated to 75 ℃, refluxed 3 hours, and repeated 3 times, filter, merging filtrate reclaims methyl alcohol, adds the deionized water 15L of suitable sporophore medicinal powder weight in the dregs of a decoction, heated and boiled is extracted, and each 2 hours, repeats 6 times, filter, get the dregs of a decoction, in the dregs of a decoction, add 8% (containing 0.10% sodium tetrahydroborate) sodium carbonate solution 10L, after fully stirring, 40 ℃ were soaked 30 hours, filtered, filtrate is regulated pH6～7 with acetic acid, places clarification, and is centrifugal, collect supernatant liquor, add the anhydrous methanol of 2 times of its volumes, the limit edged stirs, place clarification, after centrifugal, precipitation is dissolved in the deionized water, behind 1 μ m micro-filtration, again through the 5KD ultrafiltration, after concentrating, lyophilize obtains the present invention-Ganoderma tsugae proteoglycan crude product GTP.
Change methanol concentration among the embodiment 1 into 85%, refluxed each 2 hours or 4 hours, repeat 6 times or 4 times, concentration of sodium carbonate changes 5% or 10% into, and extraction temperature is 25 ℃ or 30 ℃, and other condition is constant, obtains the GTP crude product equally, and yield is basic identical.
The preparation of embodiment 2 Ganoderma tsugae proteoglycan crude product GTP
Take by weighing the Ganoderma tsugae fruit body powder 1kg that minces, add 99% ethanol 9L, heat 80 ℃, refluxing extraction each 4 hours, repeats 5 times, filter, get the dregs of a decoction, in the dregs of a decoction, add deionized water 10L, boil extraction, each 3 hours, repeat 4 times, filter, get the dregs of a decoction, in the dregs of a decoction, add the sodium hydroxide solution 12L of 6% (containing 0.08% sodium tetrahydroborate), after fully stirring, soaked filtration 30 hours in 30 ℃, filtrate is transferred pH6～7 with hydrochloric acid, places clarification, and is centrifugal, collect supernatant liquor, add the dehydrated alcohol of 2 times of its volumes, stir while adding, place clarification, centrifugal, precipitation is dissolved in the deionized water, behind 3 μ m micro-filtrations, through the 3KD ultrafiltration, concentrate, after the lyophilize, obtain the present invention-Ganoderma tsugae proteoglycan crude product GTP.
Embodiment 3 changes alcohol concn among the embodiment 2 into 80% or 95%, and refluxing extraction each 3 hours or 6 hours repeats 6 times or 4 times, naoh concentration changes 3% or 8% into, and extraction temperature changes 25 ℃ or 40 ℃ into, and other condition is constant, can obtain the GTP crude product equally, yield is basic identical.
Embodiment 4 takes by weighing the Ganoderma tsugae fruit body powder 1kg that minces, and adds 15 times of deionized waters of its weight, and heated and boiled stirs, and extracts 2 hours at every turn, filters, and discards filtrate, repeats 3 times.In the dregs of a decoction, add the 2～8%NaOH solution 10L that contains sodium tetrahydroborate 0.04%, after fully stirring,, filter in 20 ℃ of lixiviates 25 hours.Filtrate is regulated pH neutrality with hydrochloric acid, places clarification, and is centrifugal, collect supernatant liquor, the dehydrated alcohol that adds 2 times of its volumes, the limit edged stirs, and places clarification, centrifugal, precipitation is dissolved in the deionized water, and through 3 μ m micro-filtrate membrane filtrations, filtrate concentrates through 3～5KD ultra-filtration and separation again, lyophilize obtains thing of the present invention-Ganoderma tsugae proteoglycan crude product GTP.
The preparation of the single proteoglycan P1 of embodiment 5 Ganoderma tsugae sporophores, P2, P3.
GTP200mg is dissolved in the 10ml distilled water with Ganoderma tsugae sporophore proteoglycan crude product, last Sephadex-G150 post (2 * 80cm), last sample speed 0.25ml/min, with 0.2mol/L NaOH eluant solution, elution speed 0.5ml/min, detect with UV-detector, registering instrument record elution curve, collect first component, second component, the 3rd component stream fluid respectively, repeat refining three times, after 3KD ultra-filtration membrane desalination and concentration, lyophilize respectively obtains the single proteoglycan P1 of the present invention, the pure product of P2, P3 respectively.
The preparation of the single proteoglycan P1 of embodiment 6 Ganoderma tsugae sporophores, P2, P3.
GTP100mg is dissolved in the 5ml distilled water with Ganoderma tsugae sporophore proteoglycan crude product, last Sepharose F.F. (3 * 70cm) chromatography columns, last sample speed 0.50ml/min, with 0.3mol/L NaOH eluant solution, elution speed 3ml/min, detect with UV-detector, registering instrument record elution curve, collect first component, second component, the 3rd component stream fluid respectively, respectively after 5KD ultra-filtration membrane desalination and concentration, lyophilize respectively obtains the single proteoglycan P1 of Ganoderma tsugae of the present invention, the pure product of P2, P3.
The physico-chemical property of the single proteoglycan P1 of embodiment 6 Ganoderma tsugae sporophores, P2, P3
1. purity testing (homogeneity mensuration)
Carry out purity testing with Sephadex-G150 gel filtration chromatography method, getting above-mentioned three pure product is dissolved in the 0.2mol/L NaOH solution in right amount, with 0.2ml/min speed upper prop, with 3ml/min speed 0.2mol/L NaOH eluant solution, collect effluent liquid with the substep collector, every pipe meets 2ml, collects 160 pipes, carry out determination of polysaccharide with the phenol sulfuric acid process, draw the elution curve between absorbance and the pipe number.The result is that P1, P2, P3 adopt HPLC, and gel column is measured and obtained single symmetrical peak respectively, illustrates that three single proteoglycan purity are very high.
2. mean molecule flow measurement
Adopt gel filtration method, carry out on TOYOPERAL HW65F gel column, pillar Φ 3.0 * 32.50cm at first measures polysaccharide standard substance MW and is respectively 10 * 10
3, 40 * 10
3, 70 μ 10
3, 110 * 10
3, 500 * 10
3, 2000 * 10
3Elution volume, take-off rate is 38ml/hr, collects void volume V
0With elution volume V
e, single proteic polysaccharide sample is measured equally, draws V
e/ V
0With each molecular weight logarithmic relationship typical curve, the molecular weight of trying to achieve them by typical curve is respectively that P1 is about 200,000, P2 is about 20,000, P3 about 9000.
3. monose compositional analysis
The sample hydrolysis: get P1, P2, P3 is an amount of, respectively with the dilute sulphuric acid hydrolysis, 100 ℃, 8 hours, after the hydrolysis, adopt gas chromatographic analysis after adopting Paper Chromatography and acetylize, the monose standard is glucose, seminose, wood sugar, semi-lactosi, galacturonic acid.The result: the monose in three kinds of single proteoglycan is glucose to be formed, and is dextran.The result of gas-chromatography: the retention time of the acetylate of three kinds of single proteoglycan is all consistent with the retention time of the acetylate of standard substance glucose to be 32.142min, is D-glucose so the monose of polysaccharide part is formed.
4. spectroscopic analysis
4.1 Infrared spectroscopy is got single proteoglycan P1, P2, P3 are an amount of, adopts pellet technique, at 2200～400cm
-1The scope interscan is in 890cm
-1There is absorption peak at the place, shows to have β-the glycosidic link structure.
4.2 nucleus magnetic resonance
13C spectrum has chemical shift: 105.0, characteristic peaks such as 79.3,76.2,72.9,71.9,63.9.It is (1 → 3) β-D dextran that deducibility goes out the result.
1The H spectrum also provides identical information.
Claims (10)
1. a Ganoderma tsugae (Ganoderma tsugae Murrill.) proteoglycan crude product GTP, it is characterized in that it is to comprise three kinds of single proteoglycan P1, the mixture of P2 and P3, wherein, their weight percent is respectively: P1 1.50 ~ 3.00%, P252 ~ 58%, and P3 20 ~ 25%, their polysaccharide partly is the dextran of homogeneous, glycosidic link is the configuration of β-(1-3), and its structural formula is as shown in the formula expression
Wherein, the n value of P1 is 500 ~ 611, and molecular weight is 18 ~ 220,000; The n value of P2 is 42 ~ 69, and molecular weight is 1.5 ~ 2.5 ten thousand; The n value of P3 is 17 ~ 34, and molecular weight is 0.6 ~ 1.2 ten thousand; Protein portion is combined in the reduction end of polysaccharide part with integral form.
2. method for preparing the described Ganoderma tsugae proteoglycan of claim 1 crude product GTP, it comprises the following steps:
(1) get the Ganoderma tsugae fruit body powder and mince, add 10 ~ 40 times of water of its weight, heated and stirred is extracted, is filtered, and it is standby to obtain the dregs of a decoction;
(2) in the above-mentioned dregs of a decoction, add mince 2 ~ 10% dilute alkaline solns of 10 ~ 40 times of weight of its fruit body powder, contain sodium tetrahydroborate 0.04 ~ 0.12% in this solution, after fully stirring, in 20 ~ 40 ℃ of lixiviates 25 ~ 30 hours, filter, it is neutral regulating filtrate pH, places clarification, centrifugal, collect supernatant liquor, the absolute alcohol that adds 2 ~ 4 times of its volumes, the limit edged stirs, and places clarification, centrifugal, to precipitate soluble in waterly, through 20 ~ 1 μ m micro-filtrate membrane filtrations, filtrate concentrates through 3 ~ 5KD ultra-filtration and separation again, lyophilize obtains Ganoderma tsugae proteoglycan crude product GTP.
3. method for preparing the described Ganoderma tsugae proteoglycan of claim 1 crude product GTP, it comprises the following steps:
(1) get the Ganoderma tsugae fruit body powder and mince, add its weight 5-8 70 ~ 99% organic solvents doubly and carry out degreasing, be heated to 70-80 ℃, extract, filter, the dregs of a decoction are standby;
(2) add mince 10 ~ 40 times of water of weight of fruit body powder in step (1) the gained dregs of a decoction, be heated to 98-100 ℃, extract, filter, the dregs of a decoction are standby;
(3) in the dregs of a decoction of step (2) gained, add mince 10 ~ 40 times of weight 2 ~ 10% dilute alkaline soln of its fruit body powder, contain sodium tetrahydroborate 0.04 ~ 0.12% in this solution, after fully stirring evenly,, filter in 20 ~ 40 ℃ of lixiviates 25 ~ 30 hours, it is neutral regulating filtrate pH, place clarification, centrifugal collection supernatant liquor adds the absolute alcohol of 2 ~ 4 times of its volumes, the limit edged stirs, place clarification, centrifugal after, will precipitate soluble in water, behind 3 ~ 1 μ m micro-filtrations, through 3 ~ 5KD ultrafiltration and concentration, lyophilize obtains Ganoderma tsugae proteoglycan crude product GTP again.
4. Ganoderma tsugae proteoglycan P1, it is characterized in that: its molecular-weight average is 18 ~ 220,000, and its polysaccharide partly is the dextran of homogeneous, and glycosidic link is the configuration of β-(1-3), and its structural formula is as shown in the formula expression,
Wherein, n is 500 ~ 611; Protein portion is combined in the reduction end of polysaccharide part with integral form; Wherein, the weight content of polysaccharide part is 60.4 ~ 71.2%, and the weight content of protein portion is 39.6 ~ 28.8%.
5. method for preparing the described Ganoderma tsugae proteoglycan of claim 4 P1, it comprises the following steps:
(1) the described Ganoderma tsugae proteoglycan of claim 1 crude product is dissolved in 20 ~ 60 times of distilled water after, last gel column carries out chromatographic separation, last sample speed 0.2 ~ 0.8ml/min;
(2) after upward sample finished, with 0.1 ~ 0.5mol/L NaOH eluant solution, elution speed 0.4 ~ 8ml/min collected first-class fluid, and after 3 ~ 5KD ultra-filtration membrane desalination and concentration, lyophilize obtains Ganoderma tsugae proteoglycan P1.
6. Ganoderma tsugae proteoglycan P2, it is characterized in that: its molecular-weight average is 1.5 ~ 2.5 ten thousand, and its polysaccharide partly is the dextran of homogeneous, and glycosidic link is the configuration of β-(1-3), and its structural formula is as shown in the formula expression,
Wherein, n is 42 ~ 69; Protein portion is combined in the reduction end of polysaccharide part with integral form; Wherein, the weight content of polysaccharide part is 65 ~ 76%, and the weight content of protein portion is 35 ~ 24%.
7. method for preparing the described Ganoderma tsugae proteoglycan of claim 6 P2, it comprises the following steps:
(1) the described Ganoderma tsugae proteoglycan of claim 1 crude product is dissolved in 20 ~ 60 times of distilled water after, last gel column carries out chromatographic separation, last sample speed 0.2 ~ 0.8ml/min;
(2) after upward sample finished, with 0.1 ~ 0.5mol/L NaOH eluant solution, elution speed 0.4 ~ 8ml/min collected second effluent liquid, and after 3 ~ 5KD ultra-filtration membrane desalination and concentration, lyophilize obtains Ganoderma tsugae proteoglycan P2.
8. Ganoderma tsugae proteoglycan P3, it is characterized in that: its molecular-weight average is 0.6 ~ 1.2 ten thousand, and its polysaccharide partly is the dextran of homogeneous, and glycosidic link is the configuration of β-(1-3), and its structural formula is as shown in the formula expression,
Wherein, n is 17 ~ 34; Protein portion is combined in the reduction end of polysaccharide part with integral form; Wherein, the weight content of polysaccharide part is 56.6 ~ 78%, and the weight content of protein portion is 43.4 ~ 22%.
9. method for preparing the described Ganoderma tsugae proteoglycan of claim 8 P3, it comprises the following steps:
(1) the described Ganoderma tsugae proteoglycan of claim 1 crude product is dissolved in 20 ~ 60 times of distilled water after, last gel column carries out chromatographic separation, last sample speed 0.2 ~ 0.8ml/min;
(2) after upward sample finished, with 0.1 ~ 0.5mol/L NaOH eluant solution, elution speed 0.4 ~ 8ml/min collected the 3rd effluent liquid, and after 3 ~ 5KD ultra-filtration membrane desalination and concentration, lyophilize obtains Ganoderma tsugae proteoglycan P3.
10. claim 1,4, the 6 or 8 described proteoglycan application in the preparation antitumor drug respectively.
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Citations (4)
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| CN1181271A (en) * | 1996-10-31 | 1998-05-13 | 高益槐 | Water extracting process for high activity glossy ganoderma holoside and high content glossy ganoderma acid |
| KR100311317B1 (en) * | 1999-07-16 | 2001-10-18 | 이신영 | Exo-polysaccharide production from submerged mycelial culture of Ganoderma lucidum |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181271A (en) * | 1996-10-31 | 1998-05-13 | 高益槐 | Water extracting process for high activity glossy ganoderma holoside and high content glossy ganoderma acid |
| KR100311317B1 (en) * | 1999-07-16 | 2001-10-18 | 이신영 | Exo-polysaccharide production from submerged mycelial culture of Ganoderma lucidum |
| 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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