CN1448409A - Chi lucid ganoderma polysaccharide and prep. and use thereof - Google Patents
Chi lucid ganoderma polysaccharide and prep. and use thereof Download PDFInfo
- Publication number
- CN1448409A CN1448409A CN 03113450 CN03113450A CN1448409A CN 1448409 A CN1448409 A CN 1448409A CN 03113450 CN03113450 CN 03113450 CN 03113450 A CN03113450 A CN 03113450A CN 1448409 A CN1448409 A CN 1448409A
- Authority
- CN
- China
- Prior art keywords
- red ganoderma
- glp
- polysaccharide
- water
- volume
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention obtains one kind of coarse red glossy ganoderma polysaccharide product and two kinds of single red glossy ganoderma polysaccharide products. Red glossy ganoderma sporophore powder is processed through the steps of water extraction, concentration, centrifugating after adding ethanol to obtain supernatant liquid, adding ethanol and centrifugating to obtain the precipitate as the coarse red glossy ganoderma polysaccharide product with molecular weight 4000-6000. The coarse product is dissolved in water and eluted in boric acid type ion exchange column with distilled water and 0.05 mol/L boric acid solution successively and the first effluent and the second effluent are collected separately, dialyzed and freeze dried to obtain single red glossy ganoderma polysaccharide products GLPL1 and GLPL2 of average molecular weight 4100 and 5700 separately. All the products has tumor growth inhibiting effect and may be used in preparing antitumor medicine.
Description
One, technical field
The present invention relates to red ganoderma polysaccharide that extracts from red ganoderma [Ganoderma Lucidum (Leys.Fr.) Karst] fruit body powder and preparation method thereof, also relate to antitumor drug.
Two, background technology
Because tumour cell and some normal cell of human body have the similarity of height, the chemotherapeutic of energy direct killing cancer cells also can be killed and wounded those normal cells mostly, and these most of just chemotherapeutic produce the reason place of toxic side effect.Because the generation of tumour and development and body immunity are closely related, therefore, there is the expert to propose the notion of ' tumour immunotherapy ': in the normal human, to exist the panimmunity factor, they can implement effectively to attack to cancer cells, for example, lentinan, krestin have extensive immunomodulatory and anti-tumor activity, and existing approved becomes clinical cancer therapy drug commonly used.This by the therapy that improves autoimmunity one of the effective way for the treatment of tumour of can yet be regarded as, tumour immunotherapy truly has its unique potentiality and practical value.
Studies show that in a large number: the polysaccharide (as lentinan, krestin, Schizophyllum commune Fr polysaccharides etc.) that derives from some fungi has antitumor action.Polysaccharide is as a kind of wide spectrum immunomodulator, can pass through multipath, too many levels, many target spots adjusting body's immunological function, by host intermediary, stimulate maturation, differentiation and the breeding of the various immunologically competent cells of body, promote the secretion of various lymphokines, regulate the formation of antibody and complement, the balance of adjusting nerve-endocrine-immunomodulatory network (NIM) etc. restores balance body immune system, engulfs the expeling tumour cell by the resistibility of body own.On the other hand, some polysaccharide also have direct inhibition and lethal effect to cancer cells.Compare with the traditional antineoplastic thing: the toxic side effect of polysaccharide medicine is minimum, to the immunity of organisms not damaged and booster action is arranged, be difficult for causing put, multiple complications that chemotherapy inevitably descends and causes because of immunity of organisms.To should not accept because of health is weak to put, the patient of chemotherapy radiotherapy is especially suitable.Share with other drug, existing anti-tumor activity also has the attenuation of immune pharmacology.Therefore be considered to extremely rising a kind of cancer therapy drug always.But because the structure function research of polysaccharide relatively lags behind, present clinical antitumor polyose medicament only two kinds of lentinan and the krestins of formally being used for, proportion is very few in the colony of antitumor drug, and vast market and industry space are still arranged.
Glossy ganoderma is the treasure in the traditional Chinese medicine and pharmacy treasure-house, belongs to Basidiomycetes, polyporaceae, and Ganoderma has the medicinal history in more than 2,000 year in China, and The experimental results both domestic and external proves that glossy ganoderma has pharmacological action widely, and toxicity is extremely low.The glossy ganoderma complex chemical composition mainly contains polyose, ucleosides, furans, triterpenes, alkaloid, amino acid and trace element.Wherein ganoderan is its effective constituent of strengthening the body resistance to consolidate the constitution, and has antitumor action.Miyazaki etc. isolate a kind of molecular weight from Ganoderma sporophore be 40000 water-soluble polysaccharide, Mizuno etc. extract from Ganoderma sporophore and have obtained molecular weight and be respectively two kinds of polysaccharide of 1050000 and 450000, behind intraperitoneal injection, three kinds of polysaccharide all have remarkable restraining effect to murine sarcoma S180; Li Rongzhi, He Yunqing etc. have been separated to molecular weight and have been respectively 16200,24500,35000 and 40,000 four kinds of polysaccharide from red sesame, 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 in addition, increases the secretion of TNF-α, TNF-γ.The ganoderan molecular weight of having reported at present with antitumor action is most for high molecular (molecular weight be several ten thousand to hundreds of thousands of) polysaccharide, and is less to the antitumor research of lower molecular weight ganoderan.Studies show that both at home and abroad: the activity of polysaccharide is relevant with many factors, and is as molecular weight, solvability, viscosity etc., particularly closely related with its space structure.In general, solvability is tending towards Yi Rong, its better effects if that can be water-soluble; The relative molecular weight of polysaccharide is big more, its viscosity is also big more, and the viscosity of polysaccharide also can influence its practical application effect, is very promising antitumor drug as schizophan, but it is originally too big because of viscosity, can't clinically use, the back reduces molecular weight by the part degraded, viscosity also reduces, but because its basic repeating structure is constant, also keep anti-tumor activity, now in clinical use and obtained good effect.Recent study finds that many micromolecular polysaccharides and oligosaccharides have good biological activity and pharmacological action, and as medicine, micromolecular polysaccharide good water solubility, viscosity are low, be easy to make various preparations, convenient drug administration has more superiority in making, store and using.
Three, summary of the invention
The objective of the invention is to:
A kind of red ganoderma polysaccharide crude and method for making thereof are provided, and the red ganoderma polysaccharide crude is used to prepare antitumor drug.Two kinds of red ganoderma single polysaccharides and method for making thereof are provided.Two kinds of red ganoderma single polysaccharides are used to prepare antitumor drug.
Technical scheme of the present invention is as follows:
A kind of ganoderan crude product, it is by red ganoderma fruit body powder water extraction, extracting solution is concentrated, add chloroform and propyl carbinol, concuss leaves standstill, remove sub-cloud organic layer and middle layer, water layer is dialysed to flowing water, concentrate dialysate, 3/4 volume of ethanol of adding dialysis concentrated solution volume, 4 ℃ of placements, the centrifugal precipitation of removing to remove the high-molecular weight polysaccharide, keeps supernatant liquor, add its 3 times of volume of ethanol in the supernatant liquor, 4 ℃ of placements, centrifugal, the molecular-weight average that collecting precipitation obtains is 4000 ~ 6000 red ganoderma polysaccharide crude.
A kind of method for preparing above-mentioned red ganoderma polysaccharide crude, it is made up of the following step:
Step 1, get the red ganoderma fruit body powder, the water that adds 10-30 times of weight, 90-100 ℃ was extracted 8-12 hour, intermittently stirred the centrifugal precipitation of going of 3000rpm, get extracting solution, residue after centrifugal adds the water of 5-15 times of red ganoderma fruit body powder weight again, extracts 4-8 hour the centrifugal residue of removing again at 90-100 ℃, the supernatant liquor that merges extracted twice
Step 2, with the supernatant concentration of step 1 gained to red ganoderma fruit body powder 8-12 weight doubly, the propyl carbinol of the chloroform of adding concentrated solution 1/5 volume and 1/5 volume of chloroform, concuss, standing demix, remove sub-cloud organic solvent layer and middle layer, keep water layer, repeat 4-5 time, thoroughly to remove deproteinize, to flowing water dialysis 48 hours, to remove small-molecule substance, dialyzate was concentrated into red ganoderma fruit body powder 8-12 weight doubly with gained solution, in dialyzate, slowly add dialyzate 3/4 volume of ethanol, 4 ℃ of placements, the centrifugal precipitation of removing is to remove the high-molecular weight polysaccharide, keep supernatant liquor
Step 3, in the supernatant liquor of step 2 gained, add its 3 times of volume of ethanol, 4 ℃ of placements, centrifugal, must precipitate, will precipitate water-soluble, the dehydrated alcohol that adds its 4 times of volumes, 4 ℃ of placements, the centrifugal supernatant liquor that goes is successively with dehydrated alcohol, the washing with acetone precipitation, vacuum-drying obtains product red ganoderma polysaccharide crude (GLP of the present invention
L).
The red ganoderma polysaccharide crude also can prepare with following method.
A kind of method for preparing above-mentioned red ganoderma polysaccharide crude is characterized in that being made up of the following step:
Step 1, get the red ganoderma fruit body powder, add the doubly deionized water of its weight of 20-25, under agitation 90-100 ℃ was extracted 8-12 hour, the deionized water that centrifugal back residue adds 10 times of red ganoderma fruit body powder weight again extracted 6-8 hour in 100 ℃, abandon residue after centrifugal, merge the supernatant liquor of extracted twice
Step 2, the supernatant liquor of step 1 gained is concentrated into 200L for 100 ℃, adds the propyl carbinol of 1/5 volume of the chloroform of 1/5 volume of concentrated solution volume and chloroform volume in concentrated solution, stir, standing demix is got supernatant liquor, and so the Deproteinization operation repeats 3 times,
Step 3, the supernatant liquor behind the Deproteinization put in people's vacuum tank vacuumize, took out 30 minutes, slowly add the industrial alcohol of 3/4 volume of supernatant liquor volume behind the Deproteinization at 60 ℃, 4 ℃ of placements, centrifugal, keep supernatant liquor, with the industrial alcohol of 3 times of volumes of supernatant liquor, 4 ℃ of placements, centrifugal, must precipitate, will precipitate water-solublely, add the dehydrated alcohol of its 4 times of volumes, 4 ℃ of placements, the centrifugal supernatant liquor that goes is successively with dehydrated alcohol, washing with acetone precipitation, vacuum-drying gets red ganoderma polysaccharide crude of the present invention.
The application of above-mentioned red ganoderma polysaccharide crude in the preparation antitumor drug.
A kind of red ganoderma single polysaccharide, it is to be dissolved in water by above-mentioned red ganoderma polysaccharide crude, last borate type ion exchange column, use distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of first effluent, the red ganoderma single polysaccharide GLP of the present invention that obtains through the dialysis postlyophilization
L1, its molecular-weight average is 4,100, and it is made up of glucose, and glycosidic link is a beta configuration.A kind ofly prepare above-mentioned red ganoderma single polysaccharide GLP
L1Method, it is made up of the following step:
Step 1, with red ganoderma polysaccharide crude GLP
LBe dissolved in the distilled water of 50 times of weight, on balance is good borate type ion exchange column,
Step 2, above-mentioned ion exchange column used successively the 0.05mol/L borax soln wash-out of the distilled water of 2.6 times of column volumes, 1 times of column volume, substep is collected elutriant, sulfuric acid anthrone colorimetry is followed the tracks of and is detected sugared content, collect the elutriant of first effluent, after dialysis, lyophilize obtains red ganoderma single polysaccharide GLP of the present invention
L1
Above-mentioned red ganoderma single polysaccharide GLP
L1Application in the preparation antitumor drug.
A kind of red ganoderma single polysaccharide, it is to be dissolved in water by above-mentioned red ganoderma polysaccharide crude, last borate type ion exchange column, use distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of second effluent, the red ganoderma single polysaccharide GLP of the present invention that obtains through the dialysis postlyophilization
L2, its molecular-weight average is 5,700, and it is made up of glucose and semi-lactosi, and glycosidic link is a beta configuration.A kind ofly prepare above-mentioned red ganoderma single polysaccharide GLP
L2Method, it is made up of the following step:
Step 1, with red ganoderma polysaccharide crude GLP
LBe dissolved in the distilled water of 50 times of weight, on balance is good borate type ion exchange column,
Step 2, above-mentioned ion exchange column used successively the 0.05mol/L borax soln wash-out of the distilled water of 2.6 times of column volumes, 1 times of column volume, substep is collected elutriant, sulfuric acid anthrone colorimetry is followed the tracks of and is detected sugared content, collect the elutriant of second effluent, after dialysis, lyophilize obtains red ganoderma single polysaccharide GLP of the present invention
L2
Above-mentioned red ganoderma single polysaccharide GLP
L3Application in the preparation antitumor drug.
The resulting low-molecular-weight red ganoderma polysaccharide crude GLP of the present invention
LMolecular weight is below 6000, mainly by GLP
L1And GLP
L2Form single red ganoderma polysaccharide GLP
L1And GLP
L3Molecular weight be respectively 4,100 and 5,700, molecular weight is less, good water solubility, viscosity is low, is easy to make various formulations, convenient drug administration.Preliminary drug efficacy study result shows: GLP
LEntity tumor-bearing mice tumour (B16BL6) growth and ascitic type tumor-bearing mice tumour (S180) growth had the obvious suppression effect.GLP
L1KB cell proliferation to vitro culture has restraining effect, and this effect is certain dose-dependently.GLP
L2To the KB of vitro culture, BGC, Caco cell proliferation has significant inhibitory effect, and this effect also is certain dose-dependently, so they are very potential antitumor drugs.
Four, embodiment
Embodiment 1. red ganoderma polysaccharide crude GLP
LPreparation
Take by weighing 50 gram red ganoderma fruit body powder adding distil water 1000mL, boiling water bath extracted 10 hours, intermittently stirred.The centrifugal precipitation of going of 3000rpm gets extracting solution.Centrifugal back residue adds 500mL distilled water, and boiling water bath extracts 6h, removes residue after centrifugal.The supernatant liquor that merges extracted twice.With supernatant concentration to 500mL.Add the chloroform of concentrated solution 1/5 volume (100mL) and the propyl carbinol of chloroform 1/5 volume (20mL), concuss, standing demix, till not having precipitation to the interface of chloroform and water, remove sub-cloud organic solvent layer and middle layer, keep water layer, to remove deproteinize, 5 times repeatedly, gained solution is dialysed 48 hours to remove small-molecule substance to flowing water, dialyzate is concentrated into 500mL.The industrial alcohol that slowly adds 375mL in dialyzate is placed 4h for 4 ℃, and is centrifugal, and (volume is v to keep supernatant liquor
1), get deposited components A.In supernatant liquor, add 3 times of v
1The industrial alcohol of volume is placed 4h for 4 ℃, and is centrifugal, gets deposited components B.B is water-soluble with deposited components, adds the dehydrated alcohol of 4 times of volumes, places 4h for 4 ℃, the centrifugal supernatant liquor that goes, and successively with dehydrated alcohol, the washing with acetone precipitation, vacuum-drying obtains product one red ganoderma polysaccharide crude (code name GLP of the present invention
L).
Change the distilled water that extracts for the first time among the embodiment 1 into 500mL or 1500mL, extracting temperature is 90 ℃, extracts 8 or 12 hours, for the second time the distilled water that extracts changes 250 or 1000mL into, and extracting temperature is 90 ℃, extracts 4 hours or 8 hours, other condition is constant, obtains red ganoderma polysaccharide GLP equally
L, yield is also basic identical.
With isolating protein operation among the embodiment 14 times repeatedly, dialyzate is concentrated into 400mL or 600mL, and other condition is constant, obtains red ganoderma polysaccharide GLP equally
L, yield is also basic identical.
Embodiment 2. red ganoderma polysaccharide crude GLP
LThe pilot scale extraction process
Take by weighing the red ganoderma fruit body powder of 20kg, add the deionized water of 400L, drop in the extractor, stir following 100 ℃ and extract 10h.Centrifugal back residue adds the 200L deionized water, and 100 ℃ are extracted 6h, removes residue after centrifugal.The supernatant liquor that merges extracted twice.Supernatant liquor is concentrated into 200L for 100 ℃.In concentrated solution, add the chloroform (40L) of concentrated solution 1/5 volume and the propyl carbinol (8L) of 1/5 chloroform volume, stir 30min, venting, standing demix, siphon gets supernatant liquor.The Deproteinization operation repeats 3 times.With Deproteinization liquid (volume v
0) return in the vacuum tank and vacuumize (60 ℃ 30min), slowly add 3/4 times of v
0The industrial alcohol of volume is placed 4h for 4 ℃, and is centrifugal, and (volume is v to keep supernatant liquor
1), remove deposited components A.In supernatant, add 3 times of v
1The industrial alcohol of volume is placed 4h for 4 ℃, and is centrifugal, gets deposited components B.B is water-soluble with deposited components, adds the dehydrated alcohol of 4 times of volumes of the aqueous solution, places 4h, the centrifugal supernatant liquor that goes for 4 ℃.Precipitate successively with dehydrated alcohol, washing with acetone, vacuum-drying obtains product one red ganoderma polysaccharide crude GLP of the present invention
L
Embodiment 3. single polysaccharide GLP
L1And GLP
L2Preparation
With 100mg red ganoderma polysaccharide crude GLP
LFully be dissolved in 5mL distilled water, on balance is good borate type DE-32 ion exchange column (1.9 * 20cm), last sample flow velocity is 0.27mL/min, uses distilled water 146mL respectively, 0.05mol/L borax soln 56mL wash-out, flow velocity 20mL/h, substep is collected elutriant, and sulfuric acid anthrone colorimetry is followed the tracks of and is detected sugared content, merge same composition, with the elutriant dialysis of first, second part effluent, lyophilize obtains red ganoderma single polysaccharide finished product of the present invention: GLP
L1And GLP
L2
Embodiment 4. red ganoderma single polysaccharide GLP
L1And GLP
L2Physico-chemical property
1. spectroscopic analysis
UV spectrum: get GLP
L1And GLP
L2In right amount, the dissolving of 0.9%NaCl solution is configured to the solution that concentration is 1mg/mL, in the interscan of 200～400nm scope.GLP
L1And GLP
L2UV spectrum at the no absorption peak of 280nm and 260nm place, show not contain protein and nucleic acid.
Infrared spectra: get GLP
L1And GLP
L2In right amount, pressing potassium bromide troche, 4000～400cm
-1Interval scanning.The IR spectrum resolution is as follows: 3400 (O-H), 2900 (C-H), 1640 (C=O), 1400 (C-O).GLP
L1And GLP
L2Infrared spectra 890
-1There is absorption at the place, and GLP is described
L1And GLP
L2Glycosidic link be beta configuration.
2. purity testing
Gel column chromatography: get GLP
L1And GLP
L2In right amount, through Sephadex G-100 gel filtration chromatography, 0.1mol/L NaCl wash-out, flow velocity 18mL/h, every pipe is collected 2mL, and sulfuric acid anthrone colorimetry is followed the tracks of and is detected sugared content, merges same composition.Draw the relation curve of wash-out pipe number and absorbancy.GLP
L1And GLP
L2Sephadex G-100 gel filtration chromatography elution curve be single symmetrical peak, show that this sample purity is higher.
3. mean molecule flow measurement
Adopt gel filtration method: (65cm * 12mm), sample is gone up sample to Sephadex G-100 gel dress post in succession, with 0.1mol/L NaCl wash-out.Measure each standard substance Dextran T-10 earlier, Dextran T-40, the elution volume Ve of Dextran T-70 and the void volume Vo of blue dextran draw Ve/Vo and each molecular weight logarithmic relationship typical curve, survey GLP again
L1And GLP
L2Ve, try to achieve GLP by typical curve
L1And GLP
L2Molecular-weight average be respectively 4,100 and 5,700.
4. monose composition measuring
4.1 thin-layer chromatography
Hydrolysis: get GLP
L1And GLP
L2In right amount, respectively with 2M trifluoroacetic acid (TFA) hydrolysis.TFA is taken out in the decompression of cooling back, adds small amount of methanol, and decompressing and extracting repeatedly several times, is dissolved in distilled water with hydrolysate at last.
Thin-layer chromatography: silica gel G bed board, CMC are tackiness agent.With the above-mentioned aqueous solution and monose standard model point sample, developping agent is an ethyl acetate: Virahol: water=65: 23.5: 11.5.Thin layer plate dries after launching, and sprays the aniline-phthalic acid test solution, heating 10min colour developing in 110 ℃ of baking ovens.Thin-layer chromatography colour developing back pectinose is the reddish-brown spot, and other standard monose are the brown spot.The result shows, GLP
L1Only show a glucose spot; GLP
L2Show glucose spot and very light semi-lactosi spot.
4.2 gas chromatographic analysis
Hydrolysate carry out alditol acetateization.Hydrolysate and standard monose sodium borohydride reduction splash into acetic acid decomposing excessive sodium borohydride, add methyl alcohol repeated treatments evaporate to dryness after the evaporated under reduced pressure.Add aceticanhydride: anhydrous pyridine (1: 1) heavily is dissolved in chloroform and can carries out gas phase analysis in 100 ℃ of water-bath acetylize 20min.
Analytical conditions for gas chromatography: Hewlett-Packard's 6890 gas chromatographs, the HP-5 capillary column (30.0m * 0.25mm * 0.25um).260 ℃ of gasification temperatures.110 ℃ to 190 ℃ of temperature programmings: 3 ℃/min; 190 ℃ to 206 ℃: 2 ℃/min; 206 ℃ to 280 ℃: 20 ℃/min, keep 2min.Fid detector, carrier gas are nitrogen, flow velocity 25.0mL/min, 300 ℃ of detector temperatures.
Gas chromatographic analysis: GLP
L1And GLP
L2Gas chromatographic analysis the results are shown in Table 1.Contrast monose standard substance color atlas as can be known, GLP
L1Form by glucose; GLP
L2Form by glucose and a small amount of semi-lactosi.
Table 1.GLP
L1And GLP
L2Gas chromatograph results
Group retention time (min)
23.981 24.358 31.807 32.128 32.339
D-rhamnose +
D-arabinose +
D-mannose +
D-glucose +
D-galactose +
GLP
L1 +
GLP
L2 + +
Embodiment 5. red ganoderma polysaccharide crude GLP of the present invention
LAnd red ganoderma single polysaccharide GLP
L1And GLP
L2Activity research
Experimental study proves that polysaccharide of the present invention has certain antitumor action, and main experimental result is as follows:
1.GLP
LRestraining effect to entity tumor-bearing mice tumour (B16BL6) growth
Get 60 of C57BL6 mouse, male and female half and half are divided into 6 groups at random, 10 every group, are respectively GLP
LGroup (0.1,0.2,0.4g/kg), krestin group (1.65g/kg), 5-FU organizes (0.02g/kg), model group (equal-volume NS), and except that 5-FU organized intraperitoneal injection, all the other respectively organized equal gastric infusion.With the good B16BL6 melanoma cell of 0.25% trysinization adherent growth, add the centrifugal supernatant of abandoning of nutrient solution 800rpm, again with serum free medium suspendible tumour cell, adjust cell count.Then in every inoculated tumour cell suspension 0.2mL of the subcutaneous place of right side of mice inguinal region (about 10
6Individual), inoculation begins gastric infusion next day, and continuous 20 days, the subcutaneous place of observed and recorded mouse inguinal region every day tumor growth situation, mouse active state and dead mouse situation.Administration finishes the back and puts to death mouse, strips the separation tumor tissue, and with scales/electronic balance weighing, the record knurl is heavy.Calculate tumour inhibiting rate.
Tumour inhibiting rate=(1-administration group knurl weight/model group knurl is heavy) * 100%
Table 2.GLP
LTo solid-type (B16BL
6) restraining effect (x ± SD) of tumor-bearing mice tumor growth
Heavy (weight in wet base) tumour inhibiting rate (%) of group dosage (g/kg) solid tumor
Model group-5.419 ± 1.201 100
5-Fu 0.02 1.278±0.439** 76.4
Krestin 1.65 4.296 ± 1.180 20.7
GLP
L 0.40 3.098±1.003** 42.8
0.20 3.611±1.092* 33.4
0.10 4.108±0.810* 24.2
Annotate: * P＜0.05 * * P＜0.01
Behind inguinal region place inoculation B16BL6 tumour cell about 6 days, model group began that the lump that can touch is arranged, and after this lump increases gradually, and greatly as peach-pit, tumour was good at bulk-growth to 20 days model group lumps.Each administration group mouse inguinal region place lump speed of growth is slower, shows as GLP
LEach dosage knurl weight average is little than model group, and three kinds of dosage group knurls weigh with model group relatively significant difference (P＜0.01, P＜0.05), and GLP is described
LSuppressing aspect the transplantability solid-type tumour remarkable effect is arranged all.
2.GLP
LInfluence to ascitic type tumor-bearing mice tumour (S180) growth
Get the S180 cell that is in logarithmic phase, with 0.25% trysinization, be diluted to cell suspension with serum free medium again, this cell suspension abdominal injection is inoculated in the ICR mouse body, breeding observing in the laminar-flow rack, when treating that mouse web portion swells (mouse interior tumor is bred rapidly), prepare mouse inoculation for the second time.
Get 60 of ICR mouse, male and female half and half are divided into 6 groups at random, 10 every group, are respectively GLP
LGroup (0.1,0.2,0.4g/kg), krestin group (0.4g/kg), 5-FU group, model group (equal-volume NS), except that 5-FU group intraperitoneal injection, all the other respectively organize equal gastric infusion.
Get that above-mentioned inoculation is crossed and the normal ICR mouse of tumor growth, extract ascites,, make the tumour cell suspension and adjust cell count, every mouse peritoneal injection inoculation tumour cell suspension 0.2mL (about 10 with the physiological saline dilution
6Individual), inoculation begins administration next day, continuous 20 days, every day observed and recorded mouse peritoneal situation, activity situation and dead mouse situation.Adopt the chi square test statistics according to mouse death rate.
The restraining effect that table 3. polysaccharide is grown to ascitic type tumor-bearing mice tumour (S180) (x ± SD, n=10)
Group dosage (g/kg) mortality ratio (%) X
2
Model group-100-
5-Fu 0.02 20 13.33**
Krestin 1.65 100-
GLP
L 0.40 30 11.00**
0.20 50 6.67**
0.10 60 5.00**
The mouse of inoculation ascitic type S180 tumour cell showed as the belly enlargement after ten days, One's spirits are drooping, the movable minimizing, and begin to occur death gradually, all dead 20 days left and right sides model group mouse, above-mentioned performance also appears in administration group mouse, but its mortality ratio descends to some extent, with model group notable difference is arranged relatively, show that it can delay the death time of mouse, has restraining effect to tumour.
3.GLP
L1And GLP
L2Restraining effect to the cancer cell multiplication of vitro culture
[test used cell is respectively the various tumour cells of taking the logarithm vegetative period: human cervical carcinoma cell (Hela), human erythroleukemia cell (K562), gastric carcinoma cells (BGC), people's rhinitis cancer cells (KB), human colon cancer cell (Caco), human liver cancer cell (SMMC-7721)] with being inoculated in 96 orifice plates behind the tryptic digestion, it is 400 that each hole of experimental group adds final concentration, 200,100, the GLP of 50ug/mL
L1Or GLP
L2, it is the IFN-α of 1ug/mL that each hole of positive controls adds final concentration, and the every hole of negative control adds 1640 substratum with volume, and each group is all established 3 multiple holes, in 37 ℃, 5%CO
2After incubator is cultivated 48h, add MTT, measure OD after continuing to cultivate 4h
570, and according to following formula calculating inhibitory rate of cell growth (IR).The results are shown in Table 4-7.
Inhibitory rate of cell growth (IR)=(1-experimental group OD value/control group OD value) * 100%
Table 4.GLP
L1Restraining effect to the KB cell proliferation of vitro culture
Group dosage (ug/mL) IR (%)
IFN-α 1 32.7
GLP
1 50 11.5
100 17.3
200 25.0
400 42.3
Table 5.GLP
L2Restraining effect to the KB cell proliferation of vitro culture
Group dosage (ug/mL) IR (%)
IFN-α 1 32.7
GLP
L 100 17.3
200 19.2
400 44.2
Table 6.GLP
L2Restraining effect to the BGC cell proliferation of vitro culture
Group dosage (ug/mL) IR (%)
IFN-α 1 32.7
GLP
L3 100 22.7
200 22.7
400 27.3
Table 7.GLP
L2Restraining effect to the Caco cell proliferation of vitro culture
Group dosage (ug/mL) IR (%)
IFN-α 1 32.7
GLP
L3 100 13.3
200 20.0
400 26.7
By table 4-7 as can be known, GLP
L1, GLP
L2KB cell proliferation is all had significant inhibitory effect, and this effect is dose-dependently, and GLP
L2BGC, Caco cell proliferation also there is certain restraining effect.
Claims (10)
1. ganoderan crude product, it is characterized in that: it is by red ganoderma fruit body powder water extraction, extracting solution concentrates, add chloroform and propyl carbinol, concuss leaves standstill, and removes sub-cloud organic layer and middle layer, water layer is dialysed to flowing water, concentrate dialysate, 3/4 volume of ethanol of adding dialysis concentrated solution volume, 4 ℃ of placements, the centrifugal precipitation of removing, keep supernatant liquor, add its 3 times of volume of ethanol, 4 ℃ of placements in the supernatant liquor, centrifugal, the molecular-weight average that collecting precipitation obtains is 4000～6000 red ganoderma polysaccharide crude.
2. method for preparing the described red ganoderma polysaccharide crude of claim 1 is characterized in that being made up of the following step:
Step 1, get the red ganoderma fruit body powder, the water that adds 10-30 times of weight, 90-100 ℃ was extracted 8-12 hour, intermittently stirred the centrifugal precipitation of going of 3000rpm, get extracting solution, centrifugal back residue adds the water of 5-15 times of red ganoderma fruit body powder weight again, extracts 4-8 hour the centrifugal residue of removing again at 90-100 ℃, the supernatant liquor that merges extracted twice
Step 2, with supernatant concentration to red ganoderma fruit body powder 8-12 weight doubly, add the chloroform of concentrated solution 1/5 volume and the propyl carbinol of chloroform 1/5 volume, concuss, standing demix removes sub-cloud organic solvent layer and middle layer, keeps water layer, repeat 4-5 time, to flowing water dialysis 48 hours, dialyzate was concentrated into red ganoderma fruit body powder 8-12 weight doubly with gained solution, slowly added dialyzate 3/4 volume of ethanol in dialyzate, 4 ℃ of placements, the centrifugal precipitation of removing keeps supernatant liquor
Step 3, in supernatant liquor, add 3 times of volume of ethanol, 4 ℃ of placements, centrifugal, must precipitate, will precipitate water-soluble, the dehydrated alcohol that adds its 4 times of volumes, 4 ℃ of placements, the centrifugal supernatant liquor that goes is successively with dehydrated alcohol, the washing with acetone precipitation, vacuum-drying obtains product red ganoderma polysaccharide crude GLP of the present invention
L
3. method for preparing the described red ganoderma polysaccharide crude of claim 1 is characterized in that being made up of the following step:
Step 1, get the red ganoderma fruit body powder, add the deionized water of 20-25 times of weight, under agitation 90-100 ℃ was extracted 8-12 hour, centrifugal back residue adds the deionized water of 10 times of weight of red ganoderma fruit body powder again, and 100 ℃ were extracted 6-8 hour, abandons residue after centrifugal, the supernatant liquor that merges extracted twice
Step 2, the supernatant liquor of step 1 gained is concentrated into 200L for 100 ℃, adds the chloroform of 1/5 volume of concentrated solution volume and the propyl carbinol of chloroform volume 1/5 volume and stir in concentrated solution, standing demix is got supernatant liquor, and so the Deproteinization operation repeats 3 times,
Step 3, the supernatant liquor behind the Deproteinization put in people's vacuum tank vacuumize, took out 30 minutes at 60 ℃,
The industrial alcohol of 3/4 volume of supernatant liquor volume behind the slow adding Deproteinization, 4 ℃ of placements, centrifugal, keep supernatant liquor, with the industrial alcohol of 3 times of volumes of supernatant liquor, 4 ℃ of placements, centrifugal, must precipitate, will precipitate water-soluble, the dehydrated alcohol that adds its 4 times of volumes, 4 ℃ of placements, the centrifugal supernatant liquor that goes is successively with dehydrated alcohol, washing with acetone precipitation, vacuum-drying gets red ganoderma polysaccharide crude of the present invention.
4. the application of red ganoderma polysaccharide crude according to claim 1 in the preparation antitumor drug.
5. red ganoderma single polysaccharide, it is characterized in that: it is to be dissolved in water by the described red ganoderma polysaccharide crude of claim 1, last borate type ion exchange column, use distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of first effluent, the red ganoderma single polysaccharide GLP of the present invention that obtains through the dialysis postlyophilization
L1, its molecular-weight average is 4,100, and it is made up of glucose, and glycosidic link is a beta configuration.
6. one kind prepares the described red ganoderma single polysaccharide of claim 4 GLP
L1Method, it is characterized in that forming by the following step:
Step 1, with red ganoderma polysaccharide crude GLP
LBe dissolved in the distilled water of 50 times of weight, on balance is good borate type ion exchange column,
Step 2, above-mentioned ion exchange column is used distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of first effluent, after dialysis, lyophilize obtains red ganoderma single polysaccharide GLP of the present invention
L1
7. red ganoderma single polysaccharide GLP according to claim 5
L1Application in the preparation antitumor drug.
8. red ganoderma single polysaccharide, it is characterized in that: it is to be dissolved in water by the described red ganoderma polysaccharide crude of claim 1, last borate type ion exchange column, use distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of second effluent, the red ganoderma single polysaccharide GLP of the present invention that obtains through the dialysis postlyophilization
L2, its molecular-weight average is 5,700, and it is made up of glucose and semi-lactosi, and glycosidic link is a beta configuration.
9. one kind prepares the described red ganoderma single polysaccharide of claim 8 GLP
L2Method, it is characterized in that forming by the following step:
Step 1, with red ganoderma polysaccharide crude GLP
LBe dissolved in the distilled water of 50 times of weight, on balance is good borate type ion exchange column,
Step 2, above-mentioned ion exchange column is used distilled water, 0.05mol/L borax soln wash-out successively, collect the elutriant of second effluent, after dialysis, lyophilize obtains red ganoderma single polysaccharide GLP of the present invention
L2
10. red ganoderma single polysaccharide GLP according to claim 8
L2Application in the preparation antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03113450 CN1202135C (en) | 2003-05-12 | 2003-05-12 | Chi lucid ganoderma polysaccharide and prep. and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03113450 CN1202135C (en) | 2003-05-12 | 2003-05-12 | Chi lucid ganoderma polysaccharide and prep. and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1448409A true CN1448409A (en) | 2003-10-15 |
| CN1202135C CN1202135C (en) | 2005-05-18 |
Family
ID=28684077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03113450 Expired - Fee Related CN1202135C (en) | 2003-05-12 | 2003-05-12 | Chi lucid ganoderma polysaccharide and prep. and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1202135C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307314C (en) * | 2004-04-28 | 2007-03-28 | 中国食品发酵工业研究院 | Enzymolytic preparation method for glossy ganoderma amylose |
| CN100347196C (en) * | 2004-06-29 | 2007-11-07 | 长春市恒泰生物技术有限公司 | Ganodema tsugae-proteoglycan and its preparing method and use |
| CN100348619C (en) * | 2004-06-28 | 2007-11-14 | 费晓方 | Ganoderrma tsugae-proteoglycan and its preparing method and use |
| CN1872882B (en) * | 2005-05-30 | 2010-05-26 | 上海水产大学 | Method for preparing laver amylose |
| CN101434660B (en) * | 2008-08-01 | 2011-08-17 | 湖北省农业科学院农产品加工与核农技术研究所 | Method for extracting Ganoderma lucidum polysaccharide by ultrasonic wave-microwave combination |
| CN103837384A (en) * | 2014-01-21 | 2014-06-04 | 吉林大学 | Measuring method of soluble saccharide |
| CN107602719A (en) * | 2017-10-20 | 2018-01-19 | 广东粤微食用菌技术有限公司 | A kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application |
| CN112979840A (en) * | 2021-04-25 | 2021-06-18 | 上海市农业科学院 | Method for separating and purifying ganoderma lucidum beta-glucooligosaccharides |
| CN114031694A (en) * | 2021-12-23 | 2022-02-11 | 云南三七科技有限公司 | Extraction method of pseudo-ginseng polysaccharide |
-
2003
- 2003-05-12 CN CN 03113450 patent/CN1202135C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307314C (en) * | 2004-04-28 | 2007-03-28 | 中国食品发酵工业研究院 | Enzymolytic preparation method for glossy ganoderma amylose |
| CN100348619C (en) * | 2004-06-28 | 2007-11-14 | 费晓方 | Ganoderrma tsugae-proteoglycan and its preparing method and use |
| CN100347196C (en) * | 2004-06-29 | 2007-11-07 | 长春市恒泰生物技术有限公司 | Ganodema tsugae-proteoglycan and its preparing method and use |
| CN1872882B (en) * | 2005-05-30 | 2010-05-26 | 上海水产大学 | Method for preparing laver amylose |
| CN101434660B (en) * | 2008-08-01 | 2011-08-17 | 湖北省农业科学院农产品加工与核农技术研究所 | Method for extracting Ganoderma lucidum polysaccharide by ultrasonic wave-microwave combination |
| CN103837384A (en) * | 2014-01-21 | 2014-06-04 | 吉林大学 | Measuring method of soluble saccharide |
| CN107602719A (en) * | 2017-10-20 | 2018-01-19 | 广东粤微食用菌技术有限公司 | A kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application |
| CN112979840A (en) * | 2021-04-25 | 2021-06-18 | 上海市农业科学院 | Method for separating and purifying ganoderma lucidum beta-glucooligosaccharides |
| CN112979840B (en) * | 2021-04-25 | 2022-07-29 | 上海市农业科学院 | Method for separating and purifying ganoderma lucidum beta-glucooligosaccharides |
| CN114031694A (en) * | 2021-12-23 | 2022-02-11 | 云南三七科技有限公司 | Extraction method of pseudo-ginseng polysaccharide |
| CN114031694B (en) * | 2021-12-23 | 2023-02-03 | 云南三七科技有限公司 | Extraction method of pseudo-ginseng polysaccharide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1202135C (en) | 2005-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Rice bran polysaccharides and oligosaccharides modified by Grifola frondosa fermentation: Antioxidant activities and effects on the production of NO | |
| Yang et al. | Purification, characterization and anti-proliferation activity of polysaccharides from Flammulina velutipes | |
| Wu et al. | Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media | |
| Mizuno et al. | Antitumor-active polysaccharides isolated from the fruiting body of Hericium erinaceum, an edible and medicinal mushroom called yamabushitake or houtou | |
| CN109694416B (en) | Preparation method of high-purity, high-solubility and high-activity edible fungus polysaccharide | |
| Garcia et al. | Bioactive polysaccharides from medicinal mushrooms: A review on their isolation, structural characteristics and antitumor activity | |
| Wu et al. | Characterization of bioactive polysaccharides from Cordyceps militaris produced in China using saccharide mapping | |
| Zhuang et al. | Antitumor Activity and Immunological Property of Polysaccharides from the Mycelium of LiquidCultured Grifota frondosa | |
| Wang et al. | Mycelial polysaccharides of Lentinus edodes (shiitake mushroom) in submerged culture exert immunoenhancing effect on macrophage cells via MAPK pathway | |
| US20180193373A1 (en) | Method for Preparing Linseed Polysaccharide Having Antiviral Activity and Immunological Activity, and Use of the Linseed Polysaccharide | |
| CN1202135C (en) | Chi lucid ganoderma polysaccharide and prep. and use thereof | |
| CN1352990A (en) | Bio-active substance of antrodia camphorata mycelia, its preparing process and its composition | |
| CN104892793A (en) | Male agaric mycelium polysaccharides as well as preparation method and application thereof in resisting tumors | |
| CN101057872A (en) | Anti tumor active part of American cockroach prepared with reverse phase material and medical application | |
| Bisko et al. | Effects of cultivation parameters on intracellular polysaccharide production in submerged culture of the edible medicinal mushroom Lentinula edodes. | |
| CN111892663A (en) | Hericium erinaceus polysaccharide and preparation method and application thereof | |
| CN103304680B (en) | Beta-glucan, and extraction method and application thereof | |
| CN108752491A (en) | Ultrasound-microwave radiation technology water extraction extracts folium isatidis active polysaccharide technique | |
| CN108467438A (en) | Lucidum spore powder wall polysaccharide and its extracting method | |
| CN1273496C (en) | Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity | |
| CN104725521B (en) | A kind of Amauroderma ruda (Berk) Pat unimodal polysaccharide F212 and its preparation method and application | |
| CN114957497B (en) | Gentiana rigescens acidic polysaccharide and preparation method and application thereof | |
| CN113717296B (en) | Eucommia acidic polysaccharide, extraction method and application of eucommia acidic polysaccharide in preparation of anti-colon cancer drugs | |
| CN109232753A (en) | A kind of Boletus speciosus polysaccharide BSF-X and the preparation method and application thereof | |
| CN1123577C (en) | Ocean thalassiomycete hypoxylon polyose and its extracting process and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: CHANG AO PHARMACY TECHNOLOGY CO., LTD., NANJING C Free format text: FORMER OWNER: CHINA PHARMACY UNIVERSITY Effective date: 20060908 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20060908 Address after: 210007 Jiangsu Province, Nanjing city Qinhuai District dajiaochang Road No. 30 Patentee after: Chang'ao Science and Technology of Medical Industry Co., Ltd., Nanjing Address before: Nanjing City, Jiangsu Province, Tong Lane 210009 No. 24 Patentee before: China Pharmaceutical University |
|
| C56 | Change in the name or address of the patentee | ||
| CP02 | Change in the address of a patent holder |
Address after: No. 1, Heng Fei Road, Nanjing economic and Technological Development Zone, Jiangsu Patentee after: Chang'ao Science and Technology of Medical Industry Co., Ltd., Nanjing Address before: Jiangsu province Nanjing city Qinhuai District dajiaochang Road No. 30 Patentee before: Chang'ao Science and Technology of Medical Industry Co., Ltd., Nanjing |
|
| ASS | Succession or assignment of patent right |
Owner name: NANJING XBL-CHINA PHARMACEUTICAL TECHNOLOGY CO., L Free format text: FORMER OWNER: CHANG AO SCIENCE AND TECHNOLOGY OF MEDICAL INDUSTRY CO LTD, NANJING Effective date: 20140313 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20140313 Address after: 210038 No. 1 Heng Fei Road, Nanjing economic and Technological Development Zone, Jiangsu, China Patentee after: XENOBIOTIC LABORATORIES, INC Address before: 210038 No. 1 Heng Fei Road, Nanjing economic and Technological Development Zone, Jiangsu, China Patentee before: Chang'ao Science and Technology of Medical Industry Co., Ltd., Nanjing |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20050518 Termination date: 20140512 |