CN103374078A - Homopolysaccharide in ganoderma sinensis submerged fermentation mycelium, as well as preparation method and applications thereof - Google Patents
Homopolysaccharide in ganoderma sinensis submerged fermentation mycelium, as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of homopolysaccharide which exists in ganoderma sinensis submerged fermentation mycelium and has a novel structure, and applications of the homopolysaccharide in the preparation of antineoplastic drugs and immune adjustment drugs. The homopolysaccharide is characterized in that the total polysaccharide content of the homopolysaccharide is more than 95%, the molecular weight of the homopolysaccharide is 10000-30000 Daltons, and preferentially is 21190 Daltons, and the homopolysaccharide has the characteristics of high efficiency, low toxicity and immunity enhancement in the antineoplastic application. The homopolysaccharide in the ganoderma sinensis submerged fermentation mycelium, provided by the invention, is high in polysaccharide content and purity, and is excellent in administration dosage, and the preparation method is suitable for industrial production.
Description
Technical field
The present invention relates to purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1, its preparation method and the purposes in preparation antitumor drug and immunomodulator thereof.
Background technology
Tumour is common, the most serious a kind of disease that the world today directly jeopardizes the human life, thereby how effectively to prevent and treat the focus that tumour has become medical field research.Traditional operation, chemicotherapy method can produce the side effects such as internal organs damage, immunologic function inhibition when prolonging tumour patient life, patient's quality of life is obviously descended.At present, fungus polysaccharide is usually used in the assisting therapy of tumour because having the advantages such as toxic side effect is little, safe, the inhibition tumor effect is good, and it has become the important means of oncotherapy as a kind of immunostimulant adjuvant radiotherapy, chemotherapy.
The multiple efficacies such as edible and medicinal fungi has anti-tumor virus, reducing blood-fat, delays senility, protecting liver and expelling toxin, promotion nucleic acid and protein biosynthesizing, in state-owned long use historical.Studies show that in a large number that both at home and abroad fungus polysaccharide separates from fungus sporophore, mycelium, fermented liquid and obtains, and is the active component of fungal drug, is to control the cell fission differentiation, regulates a class active polysaccharide of Growth of Cells aging.
Purple sesame (Ganoderma sinense) is as Ganoderma fungi (2010 editions Chinese Pharmacopoeias), and it has significantly antitumor, the effect that improves immunizing power.Because its preparation technology is indefinite, polysaccharide content lower (general＜60% weight).Other purple sesame Crude polysaccharides complicated components, main pharmacodynamics position and component are not yet illustrated.In addition because purple sesame sporophore resource scarcity, rare purple sesame product on the market.
Purple sesame Crude polysaccharides solubleness is very poor, and viscosity is larger, and color is dark, and is highly seasoned, and complicated component, molecular weight are from several thousand to up to ten million, and difficulty finds suitable quality controlling means, and activeconstituents is indefinite, and structure activity relationship and mechanism of action also have no way of investigating.If the Crude polysaccharides separation and purification therefrom can be obtained active homogeneous polysaccharide, can not only improve the drug effect level, and quality control, structure activity relationship and mechanism of action investigation are very helpful.
Chinese patent 200710045369.4 " a kind of purple sesame fermentation process and the purple camphorata mycelium that makes " discloses a kind of method of purple sesame liquid submerged fermentation aerial mycelium, this mycelium extracts the Crude polysaccharides content obtain in 39～75% weight with water extraction and alcohol precipitation method, in the pharmacological evaluation, oral dosage has certain anti-tumor activity when being 40mg/kg/ days in its body.Chinese patent 200710045368.x " intra-polysaccharides from mycelia of ganoderma sinensis and its preparation method and application " discloses and has a kind ofly extracted the method for preparing Crude polysaccharides from the mycelium that purple sesame liquid submerged fermentation produces.The water extraction and alcohol precipitation method that the method relates to extracts and obtains Crude polysaccharides content in 39～75% weight.The dosage of its pharmacological evaluation also is 40mg/kg/ days (administering modes: oral medicine feed).Yang Guohong, Yang Yifang, Jin Juandi (the antitumor active site research [J] of purple sesame liquid submerged fermentation. herbal medicine, 2008,39 (6): 877-880) the anti-tumor activity polysaccharide of purple sesame liquid submerged fermentation is studied, extract from purple camphorata mycelium and obtain intracellular polyse, the dosage of its pharmacological evaluation also is 40mg/kg/ days (administering modes: oral medicine feed).Yet above patent application and article all do not relate to the research of the homogeneous polysaccharide of tool anti-tumor activity, and Crude polysaccharides exists that polysaccharide content is not high, dosage is unexcellent, activeconstituents is clear and definite not and Anticancer Effect and Mechanism is had no way of deficiencies such as investigation.Seek that polysaccharide content is high, dosage is more excellent, no matter the clear and definite single antitumor homogeneous polysaccharide of activeconstituents is to study on mechanism, or uses all significant in antitumor drug.
Summary of the invention
The technical problem to be solved in the present invention provides the antitumor homogeneous polysaccharide of purple sesame liquid submerged fermentation mycelium that a kind of polysaccharide content is high, can use in preparation antitumor drug and immunoregulation druge.
As used in the present invention, " liquid submerged fermentation " is a kind of fermentation process commonly used on the fermentation industry, and its substratum is liquid.
As used in the present invention, " mycelium " is that mycelia gathers together and consists of certain macrostructure.
As used in the present invention, " homogeneous polysaccharide " is the polysaccharide of monose by being polymerized, and the sugar of polymerization has specific structural unit.Homogeneous polysaccharide can be divided into the glycan of identical monose formation and the mixed polysaccharide that different monose form.
The purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide that provides a kind of structure brand-new is provided first purpose of the present invention, with its called after GS-A-1, it is characterized in that the total polysaccharides content of described homogeneous polysaccharide is greater than 95% weight, is preferably more than 98.5% weight; And the molecular weight of described homogeneous polysaccharide GS-A-1 is 10000-30000 dalton, is preferably 21190 dalton.
According to of the present invention one preferred embodiment, analyze and to learn through TLC (thin-layer chromatographic analysis) and HPLC (high performance liquid chromatography), polysaccharide among the described homogeneous polysaccharide GS-A-1 mainly is comprised of semi-lactosi and seminose, the mol ratio of described semi-lactosi, seminose is 1.2-2.2: 1, and preferred 1.7: 1.
Further, learn through periodate oxidation and Smith degradation analysis that described homogeneous polysaccharide GS-A-1 passes through (1 → 2) by semi-lactosi and seminose, the assorted poly-polysaccharide that (1 → 6) connects.
Second purpose of the present invention is to provide the preparation method of above-mentioned purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1, it is characterized in that, comprise the steps: that purple sesame liquid submerged fermentation mycelium obtains Crude polysaccharides through water extract-alcohol precipitation, Crude polysaccharides obtains purple sesame deep liquid mycelium homogeneous polysaccharide GS-A-1 through ultrafiltration, concentrated and column chromatography.
Above-mentioned preparation method is simple, with short production cycle, lower cost.
According to of the present invention one preferred embodiment, ultrafiltration step is undertaken by ultra-filtration membrane.The selective retention molecular weight is the ultra-filtration membrane of 1-5 ten thousand.More preferably, the molecular weight cut-off of described ultra-filtration membrane is 30,000.
According to of the present invention one preferred embodiment, described ultra-filtration membrane can be made by polymeric amide poly (ether sulfone) film or cellulose acetate.
According to of the present invention one preferred embodiment, enrichment step is undertaken by nanofiltration membrane.The nanofiltration membrane that is preferably consisted of by polyamide compoiste material.Preferred molecular weight cut-off is the nanofiltration membrane of 150-300.
According to of the present invention one preferred embodiment, the column chromatography step is undertaken by adding negative pressure.The plant and instrument of taking is peristaltic pump, automatic collection instrument.
According to of the present invention one preferred embodiment, the selected filler of the present invention is anionite-exchange resin.
According to a particularly preferred embodiment of the present invention, the filler anionite-exchange resin that column chromatography is selected is DEAE-cellulose 52.
The concrete steps that the present invention prepares homogeneous polysaccharide GS-A-1 are as follows: purple camphorata mycelium (" purple camphorata mycelium " of the present invention is according to disclosed method gained in the Chinese patent 200710045369.4 " a kind of purple sesame fermentation process and the purple camphorata mycelium that makes ") extracting in water, behind the extracting solution concentrating under reduced pressure, alcohol precipitation (usually using ethanol), after the precipitation part is dried to constant weight, be dissolved in again water, filter, ultra-filtration membrane ultrafiltration with certain molecular weight, obtain trapped fluid and see through liquid with the nanofiltration membrane of certain molecular weight is concentrated respectively again, see through the liquid lyophilize with above-mentioned, obtain molecular weight less than 30,000 purple camphorata mycelium polysaccharide fraction GS-A; Obtain purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1 through anion-exchange resin column again.
The present invention utilizes membrane technique with column chromatography purple sesame liquid submerged fermentation mycelium Crude polysaccharides purifying to be become homogeneous polysaccharide, and homogeneous polysaccharide GS-A-1 had obvious activity after pharmacological evaluation showed purifying in the application of antitumor drug.
The purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1 content that the inventive method obtains is preferably 98.5% weight (detection of phenolsulfuric acid method) for greater than 95% weight, and its molecular weight is 21190Da.Pharmacology activity research is carried out at this homogeneous polysaccharide position, and the result shows the effect that this homogeneous polysaccharide and endoxan (CTX) coupling have efficacy enhancing and toxicity reducing and improve immunizing power.
Therefore, the 3rd purpose of the present invention is to provide the application of above-mentioned purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1 in preparation antitumor drug and immunity regulation medicine.
Compared with prior art, the polysaccharide content of the purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1 that present method provides is higher, purity is higher, and activeconstituents is single clear and definite, and dosage is more excellent, and its preparation technology also is suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is the HPGPC collection of illustrative plates of the purple sesame liquid submerged fermentation of the embodiment of the invention 1 gained mycelium homogeneous polysaccharide GS-A-1.
Fig. 2 is embodiment 1 step 3) the middle elution curve of GS-A in DEAE.
Fig. 3 is the glucose typical curve.
Fig. 4 is the TLC result after the GS-A-1 hydrolysis, wherein 1 fructose, 2 Fucoses, 3 pectinoses, 4 seminoses, 5 glucose sugar, 6GS-A-1 hydrolyzed solution, 7 wood sugars, 8 semi-lactosis, 9 rhamnosyls, 10 ribose.
Fig. 5 A-5D is respectively the HPLC collection of illustrative plates of the GS-A-1 that water, semi-lactosi, seminose, embodiment 1 makes.
Fig. 6 is the Periodic acid typical curve of GS-A-1.
Fig. 7 A-7C is respectively the HPLC collection of illustrative plates of the GS-A-1 degraded sample that water, glycerine, embodiment 1 makes.
Fig. 8 is the uv-spectrogram of the GS-A-1 that makes of embodiment 1.
Fig. 9 is the infared spectrum of the GS-A-1 that makes of embodiment 1.
Figure 10 is the GS-A-1's that makes of embodiment 1
1The H-NMR collection of illustrative plates.
Figure 11 is the GS-A-1's that makes of embodiment 1
13C NMR collection of illustrative plates.
Embodiment
In order to understand better the present invention, illustrate by following examples, but these embodiment are not construed as limiting the invention.
Embodiment 1: the preparation of purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1:
1) preparation of Crude polysaccharides:
Purple sesame liquid submerged fermentation mycelium (described purple sesame liquid submerged fermentation liquid obtains by method described in the Chinese patent 200710045369.4 " a kind of purple sesame fermentation process and the purple camphorata mycelium that makes ") concrete grammar is as follows: separate obtaining purple sesame bacterial classification (Ganoderma sinense) from the purple sesame that Shandong is buied, in sterilisable chamber, be inoculated in through 30 minutes plate culture medium of 121 ℃ of sterilizations, 28 ℃ of cultivations.After 4-6 days in sterilisable chamber with bacterial classification inoculation in through 30 minutes shake-flask seed substratum of 121 ℃ of sterilizations, 28 ± 1 ℃ of concussions were cultivated 5-7 days on the shaking table, selected preferably bacterial classification and cultivated in fermentor tank 5-6 days, obtained purple sesame liquid fermenting mycelium.
The mycelium water is extracted 1～3h at 60～90 ℃, and the extracting solution concentrating under reduced pressure adds the ethanol precipitation, and the precipitation part is dried to constant weight, gets Crude polysaccharides.
2) ultra-filtration and separation:
With the water dissolution Crude polysaccharides of 200 times of volumes, filter, water intaking solution carry out the external-compression type uf processing (ultra-filtration membrane can be by polymeric amide make, molecular weight cut-off is 30,000 ultra-filtration membrane; Or by poly (ether sulfone) film make, molecular weight cut-off is 10,000 ultra-filtration membrane; Or by cellulose acetate make, molecular weight cut-off is 50,000 ultra-filtration membrane), sample introduction pressure=0.2Mpa, strong solution outflow pressure=0.15Mpa is divided into the Crude polysaccharides aqueous solution trapped fluid and sees through liquid.To concentrate (nanofiltration membrane is that polyamide compoiste material consists of, and molecular weight cut-off is 150,200 or 250) with nanofiltration membrane through liquid, and lyophilize, obtain seeing through liquid part GS-A.
3) column chromatography for separation:
Get 2g GS-A, with a small amount of deionized water solution dissolving, add in DEAE-cellulose 52 cellulose columns that balance is good, use the deionized water wash-out, collect 2 column volumes of elutriant with the 10mL/min pipe.Measure its absorption value every 5～10 effective phenolsulfuric acid methods, and do elution curve.Elution curve is seen accompanying drawing 2.
Respectively with absorbance larger 45,50,60, the sample of No. 65 pipes carries out efficient liquid phase chromatographic analysis, it is unimodal to find that No. 45 pipe samples show as, and respectively near the samples No. 45 pipes is carried out efficient liquid phase chromatographic analysis, finds that the sample chromatogram figure of 42～47 pipes is consistent.The sample that merges 42～47 pipes, concentrated, lyophilize.With this sample called after GS-A-1.
Embodiment 2: the phenol sulfuric acid process is measured the polysaccharide content of the purple sesame liquid submerged fermentation of the present invention mycelium homogeneous polysaccharide GS-A-1
Concrete steps are as follows:
Standard substance preparation: the glucose 25mg of accurately weighed drying, with water dissolution and be settled to 100ml, be made into the 250ug/ml standard solution, get respectively 0.05,0.1,0.15,0.2,0.25,0.3mL in test tube, add deionized water and complement to 0.3mL, add respectively 5% phenol 0.7mL and fully shake up, add fast vitriol oil 4mL, 50 ℃ of water-bath 40min are cooled to room temperature, in contrast product.
The preparation of blank product: get the 0.6ml deionized water, add 5% phenol 1.4ml, vitriol oil 8ml, 50 ℃ of heating in water bath 40min are cooled to room temperature, as blank solution.
Specification Curve of Increasing: standard solution and blank product solution are surveyed its absorption value with ultraviolet spectrophotometry in the 488nm place, and do typical curve.Typical curve is seen accompanying drawing 3..
Sample preparation: get the accurately weighed 5mg of GS-A-1 sample, with deionized water dissolving and be settled to 10ml, preparation 0.5mg/ml solution, get 0.05ml in test tube, add water and complement to 0.3ml, add successively phenol, the vitriol oil with the standard substance compound method, 50 ℃ of heating in water bath 40min, be cooled to room temperature, as sample liquid.
Detect: testing sample is surveyed its absorption value with ultraviolet spectrophotometry in the 488nm place, the polysaccharide content that records GS-A-1 with typical curve is 98.5% weight.
Embodiment 3: the molecular weight of demarcating polysaccharide among the purple sesame liquid submerged fermentation of the present invention mycelium homogeneous polysaccharide GS-A-1 with dextran standard Dextron series
Concrete grammar is as follows:
Chromatographic condition: TSK-GEL GMPWxl chromatographic column; Moving phase is water; Flow velocity: 0.3mL/min; 35 ℃ of column temperatures; Detector is: the differential detector.
Mark product Dextron, GS-A-1 sample are used respectively water dissolution, sample introduction.
Measure the retention time of dextran Dextron series and GS-A-1, molecular weight logarithmic value with the retention time of Dextron-Dextron series is done typical curve, according to retention time and the typical curve of GS-A-1, the GS-A-1 peak molecular weight is 21190 dalton as can be known.
Embodiment 4: the structure elucidation of purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide GS-A-1
1) fully acid hydrolysis:
Get the GS-A-1 that 5mg embodiment 1 makes, put into ampoule, add 2mL, the trifluoracetic acid of 2mol/L (TFA) fills N
2Rear envelope bottle is at 110 ℃ of lower hydrolysis 4h.With solution decompression evaporate to dryness in the bottle, then add methyl alcohol 1～2mL evaporate to dryness, the triplicate operation is to remove TFA fully.The water that adds again 0.5mL in the sample makes sample dissolution.
1.1GS-A-1 the TLC result after the hydrolysis:
In high-efficient silica gel plate (model: put respectively polysaccharide hydrolysis sample and monose HSGF254), respectively with acetone-water (24: 1); The up second outspread of propyl carbinol-ethyl acetate-Virahol-Acetic Acid-Water-pyridine (35: 100: 60: 35: 30: 35), taking-up is dried, and heats 10min with aniline-phthalic acid in 110 ℃.There are semi-lactosi and two spots of seminose in TLC show sample corresponding position, sees accompanying drawing 4.
As can be seen from Figure 4, a kind of mixed polysaccharide of being formed by semi-lactosi, seminose of GS-A-1 of the present invention.
1.2HPLC detect the polysaccharide monosaccharide component
The chromatographic column condition:
Liquid-phase chromatographic column: Kromasil 100-5NH
2(250 * 4.6mm)
Detector: differential refraction detector
Moving phase: acetonitrile: water=3: 1
Column oven: 35 ℃ of detector temperatures: 35 ℃
Flow velocity: 1mL/min
The HPLC detected result is seen accompanying drawing 5A-5L.
According to the molar weight that mark is tasted with discrimination semi-lactosi and seminose, in conjunction with the peak area ratio of polysaccharide sample HPLC, can infer semi-lactosi in the polysaccharide GS-A-1 monose: seminose=1.7: 1 (mol ratio).Can prove further that TLC selects exists semi-lactosi and seminose monose in the plate.
2) periodate oxidation:
2.1 Periodic acid typical curve:
30mmol/L Periodic acid liquid preparation: precision take by weighing sodium periodate 322.08mg with water dissolution and constant volume in the 50mL volumetric flask.
The drafting of typical curve: get 8 250mL volumetric flasks and add successively respectively sodium periodate solution 0.2,0.4,0.6,0.8,1,1.5,2mL adds the deionized water constant volume.After placing 10min, as blank, use spectrophotometry with water, survey optical density at the wavelength place of 223nm, take sodium periodate concentration (μ mol/L) as X-coordinate, optical density value is that ordinate zou is made typical curve.See Fig. 6.
2.2 periodic acid oxidation:
Precision takes by weighing GS-A-1 10mg in the 10mL volumetric flask, then adds 15mmol/L NaIO4 and is settled to scale.Be placed on dark place reaction (4 ℃), sampling pitch time (0h, 2h, 24h, 48h, 72h......) 0.1mL is with 250 times of distilled water dilutings, use ultraviolet spectrophotometer, make blank with distilled water, survey absorbance at 223nm wavelength place, until absorbance is constant.Add ethylene glycol termination reaction (remaining Periodic acid neutralizes), periodate oxidation is finished.
2.3 the growing amount with NaOH titration formic acid:
Phenolphthalein indicator preparation: take by weighing 1g phenolphthalein, add 95% ethanol 100ml dissolving evenly, and get final product.
0.1mol/L the NaOH titrating solution is demarcated: the Potassium Hydrogen Phthalate 0.6g that precision takes by weighing 105 ℃ of constant weights is dissolved in the cold water that 50mL newly boiled, phenolphthalein is titrated to the aobvious pink of solution as indicating liquid.
Finally recording the NaOH strength of solution is 0.0986mol/L.
Methyl red indicator preparation: precision takes by weighing 0.1g methyl red solid, adds 0.05mol/L NaOH solution 7.4mL and makes it dissolving, and thin up is to 200mL again, and get final product.
Get the above-mentioned oxidation solution of 2mL, add 1 methyl red and make indicator, be titrated to oxidation solution by red yellowing with 0.0986mol/L NaOH (demarcating with Potassium Hydrogen Phthalate), calculate the formic acid growing amount.
2.4 the result shows:
2.4.1 recording the 223nm wavelength A of place value when light absorption value is constant is 0.562.Periodic acid liquid absorption value behind 24h is unchanged.So can draw the consumption of Periodic acid and example reaction is 0.127mol.
2.4.2 calculating at last the formic acid growing amount according to titration results is 0.0493mmol.
2.4.3 and the Periodic acid consumption is higher than two times of the formic acid growing amount, illustrates to have the hexose residue 1 → 2,1 → 2 that is not produced formic acid by periodate oxidation, 6,1 → 4,1 → 4,6 of bondings; Have a small amount of formic acid to generate explanation to have 1 →, the hexose residue of 1 → 6 of bonding, Periodic acid consumption and formic acid growing amount show that the saccharide residue in the molecule may be all oxidized.
3) Smith DeR
The solution dialysis (Mw=3500) of GS-A-1 after ethylene glycol processed after with periodate oxidation, distill water dialysis 48 hours.Concentrated, add the NaBH4 reduction and spend the night.Being neutralized to pH with Glacial acetic acid is 6～7, dialysis, and decompression is steamed to the 2mL, carries out lyophilize.The sample of reduction is put into ampoule, add 2mL 2mol/L TFA, fill N
2Rear tube sealing, in 110 ℃ of lower hydrolysis 4h, the solution decompression evaporate to dryness in the reaction flask adds the methyl alcohol of 1～2mL again, evaporate to dryness, triplicate is to eliminate excessive TFA, and the 1mL water dissolution of the sample after the hydrolysis is carried out the HPLC analysis.
3.1 chromatographic condition:
Liquid-phase chromatographic column: Kromasil 100-5NH
2(250 * 4.6mm)
Detector: differential refraction detector
Moving phase: acetonitrile: water=3: 1
Column oven: 35 ℃ of detector temperatures: 35 ℃
Flow velocity: 1mL/min
3.2 result: see accompanying drawing 7A-7C.
GS-A-1 degraded product HPLC detects a large amount of glycerine and shows that polysaccharide GS-A-1 contains the of bonding that can produce glycerine, namely 1 →, 1 → 2,1 → 6,1 → 2,6, the glycosidic link of bonding that GS-A-1 is described is 1 →, 1 → 2,1 → 6,1 → 2,6.
In sum: the primary structure of GS-A-1 is:
4) uv-spectrogram: see accompanying drawing 8.
Get the GS-A-1 sample and carry out the ultraviolet full wavelength scanner, have no absorption at 260nm and 280nm place, prove not contain nucleic acid and protein.
5) infared spectrum:
Get 1mg GS-A-1 sample, carry out IR with pressing potassium bromide troche and detect.The results are shown in accompanying drawing 9.
The GS-A-1 infrared spectrogram has polysaccharide characteristic absorbance, 3427.3cm
-1Broad peak be-stretching vibration of OH.2924.8cm
-1The peak is the C-H stretching vibration of sugar, 1640.1cm
-1Be the characteristic peak of syrup compound, 1400cm
-1Near the peak that occurs is because the angle vibration of C-H causes.1147.5cm
-1The peak be the stretching vibration of C-O.GS-A-1 is at 1074.2cm
-1The peak be the absorption peak that the absorption of common pyranose ring lactone and hydroxyl produces, be because the asymmetrical stretching vibration of the upper C-O-C ehter bond of sugar ring.At 874.7cm
-1There is absorption peak at the place, and it may be the absorption peak of semi-lactosi, contains the α glycosidic link in the structure.At 799.5cm
-1There is absorption peak at the place, and it may be the absorption peak of seminose, contains the α glycosidic link in the structure.Illustrate that GS-A-1 is pyranose, its glycosidic link type is α-configuration.
6) nuclear magnetic spectrum:
Nuclear magnetic spectrum (NMR): get the polysaccharide sample 10mg nuclear magnetic tube of packing into, be dissolved in the heavy water, carry out in nuclear magnetic resonance analyser
1H-NMR,
13The spectroscopic analysis such as C-NMR.
1The H-NMR atlas analysis: such as Figure 10, mainly solve glycosidic link configuration problem in the polysaccharide structures, because the signal of polysaccharide is accumulated in the narrow range of chemical shift δ 3.3～5.2 mostly, δ 3.3～4.3 is sugar ring proton signal.The overlapped intersection of proton signal of other C2～C6 is resolved difficulty except the upper proton signal of C1 is easily resolved.Visible C1 chemical shift of proton is positioned at 4.8～5.2 of δ from accompanying drawing 2, proves that then GS-A-1 contains α type pyranohexose, conforms to infrared result.
13C-NMR atlas analysis: such as Figure 11, all show multiplet, the complicacy of expression polysaccharide structures.The substituted monosaccharide or the simple oligosaccharides that have belonged to by the comparative chemistry displacement belong to unknown polysaccharide, with GS-A-1's
13C-NMR figure spectrum signal ownership is as follows:
δ 104.3 and δ 100.6 are the end group carbon of sugar, and these two carbon signals illustrate that this homogeneous polysaccharide is comprised of two monose, and their carbon signal chemical displacement value is all greater than δ 100, in conjunction with
1The H-NMR collection of illustrative plates illustrates the α-L-type that is configured as of homogeneous polysaccharide GS-A-1 glycosidic link.δ 63.8 is unsubstituted C6, and supposition may be the C6 of the α of different mode of connection-L-semi-lactosi and α-L-seminose.
Embodiment 5: the pharmacologically active of the purple sesame liquid submerged fermentation homogeneous polysaccharide GS-A-1 that embodiment 1 makes
1) to the efficacy enhancing and toxicity reducing effect of H22 liver cancer kunming mice:
With 2 H22 liver cancer mouses, to put to death and be placed on Bechtop, the belly unhairing is cut off cortex, extract ascites with asepsis injector, exhaust and wash again the abdominal cavity one time with 0.5ml physiological saline afterwards, merge ascites and washing lotion, dilution, the microscopically cell counting makes liver cancer cell concentration become 2 * 10
7Individual/ml.Then in the right oxter of Kunming mouse, with 0.2ml/ amount only, inoculate liver cancer cell.
Normal Kunming mouse according to the body weight random packet, is divided into Normal group, model group, endoxan group, endoxan and lentinan Combined Preparation group, endoxan and GS-A-1 Combined Preparation group.Except Normal group, other is respectively organized mouse and all inoculates the ascites lung carcinoma cell in right oxter with 0.2ml/ amount only.Begin administration from inoculating rear second day, successive administration 7 days.Wherein endoxan is with the dosed administration of 25mg/kg, and the polysaccharide group is all with the dosed administration of 1.5mg/kg.Vena ophthalmica is got blood after the administration in the 9th day, and separation of serum is surveyed routine blood test; Peel off the knurl body after the execution, and get spleen, weigh.
Table 1GS-A-1 is to the effect of kunming mice H22 liver cancer efficacy enhancing and toxicity reducing
* P＜0.5, * * P＜0.01 is than CTX
In the experiment of kunming mice H22 liver cancer model, endoxan (25mg/kg) and GS-A-1 (1.5mg/kg) associating intraperitoneal injection and endoxan (CTX, 25mg/kg) the abdominal injection tumour inhibiting rate significantly improves separately, spleen weight, white corpuscle and thrombocyte all significantly increase, and the lentinan group is then without positive effect (seeing Table 1).GS-A-1 has the effect of efficacy enhancing and toxicity reducing to CTX in kunming mice H22 liver cancer model.
2) to the efficacy enhancing and toxicity reducing effect of Lewis lung cancer C57BL mouse:
2 lung knurl mouse, execution is placed on Bechtop, the belly unhairing is cut off cortex with 75% alcohol disinfecting, extracts ascites with asepsis injector, put in the centrifuge tube, use again abdominal cavity of 0.5ml normal saline flushing after exhausting, merge ascites and washing lotion, add a certain amount of physiological saline mixing, centrifugal, abandon supernatant.Wash 2-3 time with physiological saline, cell counting behind the dilution mixing, adjusting lung carcinoma cell concentration is 2 * 10
7Individual/ml, be inoculated in the right oxter of mouse with 0.2ml/ amount only.
Normal Kunming mouse according to the body weight random packet, is divided into Normal group, model group, endoxan group, endoxan and lentinan Combined Preparation group, endoxan and GS-A-1 Combined Preparation group.Endoxan is with the dosage of 50mg/kg, respectively the 2nd, 4, and 6 afternoon intraperitoneal injections.The polysaccharide group is with the dosage of 1.5mg/kg, and second day begins intraperitoneal injection, continuous 12 days behind the inoculated tumour cell.Vena ophthalmica was got blood in the 14th day, surveyed routine blood test; Put to death immediately and peel off the knurl body and get spleen, weigh.
Table 2GS-A-1 is to the effect of C57BL mouse Lewis lung cancer efficacy enhancing and toxicity reducing
* P＜0.01 is than CTX
In C57BL mouse Lewis lung cancer model trial, endoxan (25mg/kg) is compared with the independent abdominal injection of endoxan (25mg/kg) with GS-A-1 (15mg/kg) associating intraperitoneal injection, tumour inhibiting rate, spleen weight, white corpuscle and Platelet Index are significantly increased, and lentinan group effect is not obvious (seeing Table 2) then.GS-A-1 has the effect of efficacy enhancing and toxicity reducing to CTX in C57BL mouse Lewis lung cancer model.
3) GS-A-1 improves the active investigation of immunizing power:
The impact of purple sesame Polysaccharides on Mice lymphopoiesis ability:
Adopt the H22 liver cancer mouse, extract ascites under the aseptic condition, adjusting cell count is 2 * 10
7Individual/ml, only inoculate in the Kunming mouse oxter with 0.2ml/.
According to the body weight random packet, be divided into model group, endoxan group, endoxan and lentinan Combined Preparation group, endoxan and GS-A-1 Combined Preparation group behind the mouse inoculation.Successive administration 9d, wherein endoxan is with the dosed administration of 25mg/kg, and the polysaccharide group is all with the dosed administration of 1.5mg/kg.In last administration 0.5h, put to death animal after getting blood, cut open under the aseptic condition and get spleen.Put into 100 order Stainless Steel Cloths, reject reticular tissue and fat with eye scissors, place the culture dish that fills the cold D-Hank ' of 5-10ml s liquid, pulverize the spleen tissue with nook closing member, make cell suspension, place centrifuge tube, the centrifugal 5min of 1000r/min abandons supernatant.RBC is removed in hypotonic processing, with cold D-Hank ' s liquid washed cell 3 times, adds 10%NBS-RPMI-1640 nutrient solution 1.5ml suspension splenocyte again, counting, and adjusting cell count is 5 * 10
6Add 96 orifice plates behind individual/ml, every hole 100ul, blank well only adds nutrient solution, and other holes add ConA (50ug ml by echelon design
-1), LPS (25ug ml
-1) 20ul, stimulate respectively T, bone-marrow-derived lymphocyte.At CO
2After cultivating 48h in the incubator, add 10ulMTT, inhale behind the continuation cultivation 4h and abandon all supernatants, every hole adds the 100ulDMSO termination reaction, the 1min that vibrates on the vibrator, and microplate reader 570nm reads plate, and the result is with the OD value representation.
Purple sesame polysaccharide is on the impact of H22 liver cancer mouse NK cell:
Adopt the H22 liver cancer mouse, extract ascites under the aseptic condition, adjusting cell count is 2 * 10
7Individual/ml, inoculate in the Kunming mouse oxter with 0.2ml/ amount only.Successive administration 9d in last administration 0.5h, puts to death animal after getting blood, cuts open under the aseptic condition and gets spleen.Put into 100 order Stainless Steel Cloths, reject reticular tissue and fat with eye scissors, place the culture dish that fills the cold D-Hank ' of 5-10ml s liquid, pulverize the spleen tissue with nook closing member, make into cell suspension, place centrifuge tube, the centrifugal 5min of 1000r/min abandons supernatant.RBC is removed in hypotonic processing, with cold D-Hank ' s liquid washed cell 3 times, adds 10%NBS-RPMI-1640 nutrient solution 1.5ml suspension splenocyte again, counting, and adjusting cell count is 5 * 10
6Add 96 orifice plates behind individual/ml, every hole 100ul, blank well only adds nutrient solution, and other holes add ConA (50ugml by echelon design
-1) 20ul, stimulate the T lymphocyte.Cultivate 48h in the CO2 incubator after, add 10ulMTT, all supernatants were abandoned in suction after 4h was cultivated in continuation, and every hole adds the 100ulDMSO termination reaction, the 1min that vibrates on the vibrator, and microplate reader 570nm reads plate, and the result is with the OD value representation.
Adjusting target cell YAC-1 cell concn is 1 * 10
5Individual/ml, target cell Spontaneous release hole adds 200ul target cell nutrient solution, and the effect control wells only adds effector cell's nutrient solution, and reacting hole adds target cell nutrient solution 100ul, stimulates the NK cell.After in the CO2 incubator, cultivating 48h, add 10ulMTT, all supernatants are abandoned in suction after continuing to cultivate 4h, every hole adds the 100ulDMSO termination reaction, the 1min that vibrates on the vibrator, microplate reader 490nm reads plate, and the result is with the OD value representation, and according to NK cell killing rate (%)=(experimental port OD value-effector cell's control wells OD value)/target cell control wells OD value * 100%, calculate the kill rate of NK cell.
Table 3GS-A-1 improves the active investigation of immunizing power
* P＜0.05, * * P＜0.01 is than CTX
In the experiment of kunming mice H22 liver cancer model, endoxan (25mg/kg) and GS-A-1 (1.5mg/kg) associating intraperitoneal injection and endoxan (25mg/kg) separately abdominal injection can significantly increase the B cell, the number of T cell and NK cell and vigor, compare with the positive control lentinan, show better raising immunity (seeing Table 3).
Claims (15)
1. purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide is characterized in that the total polysaccharides content of described homogeneous polysaccharide is greater than 95% weight, is preferably more than 98.5% weight; And the molecular weight of described homogeneous polysaccharide is 10000-30000 dalton.
2. homogeneous polysaccharide according to claim 1 is characterized in that, the molecular weight of described homogeneous polysaccharide is 21190 dalton.
3. homogeneous polysaccharide according to claim 1 is characterized in that, described homogeneous polysaccharide mainly is comprised of semi-lactosi and seminose.
4. homogeneous polysaccharide according to claim 3 is characterized in that, the mol ratio of described semi-lactosi and seminose is 1.2-2.2: 1.
5. homogeneous polysaccharide according to claim 4 is characterized in that, the mol ratio of described semi-lactosi and seminose is 1.7: 1.
6. each described homogeneous polysaccharide is characterized in that according to claim 3-5, and described homogeneous polysaccharide is to pass through (1 → 2) by α-L-galactopyranose and α-L-mannopyranose, the assorted poly-polysaccharide that (1 → 6) connects.
7. the preparation method of the arbitrary described homogeneous polysaccharide of claim 1-6 is characterized in that, comprises the steps: that purple sesame liquid submerged fermentation mycelium obtains Crude polysaccharides through water extract-alcohol precipitation, and Crude polysaccharides obtains homogeneous polysaccharide through ultrafiltration, concentrated and column chromatography again.
8. the preparation method of homogeneous polysaccharide according to claim 7 is characterized in that, described ultrafiltration step is that the ultra-filtration membrane of 1-5 ten thousand carries out by molecular weight cut-off.
9. the preparation method of homogeneous polysaccharide according to claim 8 is characterized in that, described ultrafiltration step is that 30,000 ultra-filtration membrane carries out by molecular weight cut-off.
10. the preparation method of homogeneous polysaccharide according to claim 8 is characterized in that, described ultra-filtration membrane is made by polymeric amide, poly (ether sulfone) film or cellulose acetate.
11. the preparation method of homogeneous polysaccharide according to claim 7 is characterized in that, described enrichment step is undertaken by nanofiltration membrane.
12. the preparation method of homogeneous polysaccharide according to claim 11 is characterized in that, the molecular weight cut-off of described nanofiltration membrane is 150-300.
13. the preparation method of homogeneous polysaccharide according to claim 11 is characterized in that, described nanofiltration membrane is made by polymeric amide.
14. the preparation method of homogeneous polysaccharide according to claim 7 is characterized in that, the used filler of described column chromatography step is anionite-exchange resin.
15. the application of each described homogeneous polysaccharide of claim 1-6 in preparation antitumor drug and raising immunizing power medicine.
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