CN103193895A - Blood clam polysaccharide with alpha-1,3-glucosan main chain, and preparation method and use thereof - Google Patents
Blood clam polysaccharide with alpha-1,3-glucosan main chain, and preparation method and use thereof Download PDFInfo
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Abstract
The invention particularly relates to blood clam polysaccharide with an alpha-1,3-glucosan main chain, and a preparation method and use thereof, and belongs to the field of a natural polymer. The invention provides polysaccharide extracted from blood clam, a method for extracting the polysaccharide, and a chemical structure and use of the polysaccharide. The structure is analyzed by chemical analysis and spectroscopy; the condition that the repeated structure unit of the blood clam polysaccharide disclosed by the invention contains 1-3, 1-4 and 1-6 glucosidic bonds is found out; the main chain is formed by the 1-3 glucosidic bond; a side chain is formed by the 1-4 and 1-6 glucosidic bonds; the sugar residue is of an alpha configuration; the relative molecular weight is 8.5*10<5> Da; and the specific rotation [alpha]D<20> is +135 degrees (c0.1, H2O). The polysaccharide is glucosan with a novel structure separated from the blood clam for the first time; meanwhile, the blood clam polysaccharide disclosed by the invention can stimulate multiplication of mice splenic lymphocyte and macrophagocyte; and it is indicated that the blood clam polysaccharide disclosed by the invention has immunological enhancement.
Description
Technical field
The present invention is specifically related to a kind of α of having-1, blood clam polysaccharide of 3-dextran main chain and its production and use; Belong to the natural polymer field.
Background technology
Blood clam (Arca subcrenata Lischke) is Mollusca (Mollusca) Bivalvia (Bivalvia) Taxodonta (Taxodonta) blood clam section (Arcidae), belongs to the marine products economic shellfish.Blood clam is often perched at the shallow sea silt that has fresh water to inject slightly, because special growing environment, so tender, delicious flavour of meat fertilizer, nutritive value is also very high, can improve the immunological competence of body, therefore caused the concern of Chinese scholars, domestic scholars has been extracted from blood clam and has been obtained some activeconstituentss at present.
Exist the natural product that has various peculiar properties and physiologically active in a large number in the ocean, its special chemical structure is that the Lu Sheng natural active matter is incomparable, and many new chemical structures are that terrestrial organism is unexistent, have significant pharmacological action.Polysaccharide is a big class marine bioactivity material wherein, by the polysaccharide that separates in the various marine organisms, can be divided into marine animal polysaccharide, Sargassum polysaccharides, marine microorganism polysaccharide from the source, proved to have various biological activitys and medicinal function, as antitumor, antiviral, anti-cardiovascular disease, anti-oxidant, immunomodulatory etc.
That the animal polysaccharide has is antitumor, improve immunity.Studies show that hojothuria leucospilota polysaccharide all has significant growth inhibition ratio to experiment mice mammary cancer, lung cancer, melanoma.The anticancer reason of polysaccharide is to have activated vivo immuning system mostly, has improved immunity of organisms.The abalone polysaccharide that extracts from haliotis discus hannai Ino can obviously increase the macrophage phagocytic ability, strengthens delayed hypersensitive reaction.This immuno-potentiation may be the mechanism of its antitumor action.Find that in addition hyriopsis cumingii polysaccharide, clam polysaccharide, pilose antler polysaccharides, silkworm chrysalis polysaccharide, loach polysaccharide, squid ink, shark cartilage, sea cucumber etc. have the activity of antitumor and strengthening immunity.It will be a new charming field that polysaccharide is applied to oncotherapy.The structure of polysaccharide and its biological function are closely related, and the parsing that therefore had the biologic activity polysaccharide structures in recent years is the focus in carbohydrate chemistry field, also is a difficult point simultaneously.
Summary of the invention
The objective of the invention is from blood clam, to extract a kind of polysaccharide, identify its chemical structure and determine its concrete active purposes.
Blood clam polysaccharide repeat unit structure is as follows:
The extraction flow process that the present invention relates to the blood clam polysaccharide is seen Fig. 1, with and structure identify, namely from blood clam, separate the blood clam polysaccharide that obtains, show through acid hydrolysis and GC result, the blood clam polysaccharide is for separating the dextran of a kind of novel structure obtain first from blood clam, and infers by chemical analysis and spectroscopic analysis and the repeat unit structure of blood clam polysaccharide among the present invention to contain 1 → 3,1 → 4 and 1 → 6 three kind of glycosidic link, main chain is made up of 1 → 3 glycosidic link, and side chain is made up of 1 → 4 and 1 → 6 glycosidic link.
Above-mentioned blood clam polysaccharide has tangible proliferation function to splenic lymphocyte when 50-200 μ g/mL, and is certain dose-dependently.The blood clam polysaccharide can stimulate Turnover of Mouse Peritoneal Macrophages RAW264.7 propagation significantly in addition, in 25-400 μ g/mL concentration range, compare significant difference (P＜0.05) with control group, and can significantly promote Turnover of Mouse Peritoneal Macrophages to engulf the ability of toluylene red.
Beneficial effect:
The blood clam polysaccharide is for separating a kind of α of having-1 that obtains, the dextran of the novel structure of 3-dextran main chain first from blood clam; The blood clam polysaccharide has significant immunoproliferation effect.
Description of drawings
Fig. 1 is that the blood clam polysaccharide extracts schema
Fig. 2 is blood clam polysaccharide hydrolysate thin-layer chromatogram
Fig. 3 is the gas chromatogram of blood clam polysaccharide alditol acetate
Fig. 4 is NaIO
4The concentration of solution-light absorption value typical curve
Fig. 5 is the gas chromatogram of blood clam polysaccharide Smith degraded
Fig. 6 is methylate infrared spectrum after 3 times of blood clam polysaccharide
Fig. 7 is blood clam polysaccharide ultraviolet spectrometry collection of illustrative plates
Fig. 8 is blood clam polysaccharide infrared spectroscopy collection of illustrative plates
Fig. 9 is the blood clam polysaccharide
1H NMR collection of illustrative plates
Figure 10 is the blood clam polysaccharide
13C NMR collection of illustrative plates
Figure 11 is the H-H COSY collection of illustrative plates of blood clam polysaccharide
Figure 12 is the HMBC collection of illustrative plates of blood clam polysaccharide
Figure 13 is the HSQC collection of illustrative plates of blood clam polysaccharide
Figure 14 is the blood clam polysaccharide influences figure to the propagation of mouse spleen lymphocyte
Figure 15 is that the blood clam polysaccharide influences figure to mouse macrophage RAW264.7 propagation
Figure 16 is the blood clam polysaccharide is engulfed toluylene red to mouse macrophage RAW264.7 the figure that influences
Embodiment
The fresh blood clam of 20kg is shelled, collect human body part 5kg, clean with water rinse, filtered through gauze.The human body of collecting partly carries out homogenate with the tissue homogenate stamp mill.Add isopyknic acetone decolouring in the homogenate.Sample hot water extraction after the processing, centrifugal collection supernatant liquor, after rotary evaporation is concentrated into original solution 1/3 volume, add 1/4 volume Sevag reagent, thermal agitation 15min, the centrifugal 15min of 4000rpm gets supernatant, repeating aforesaid operations does not have milky white precipitate up to chloroform layer 8 times, and concentrating under reduced pressure is flung to organic solvent.Polysaccharide extraction liquid behind the Deproteinization is evaporated to 1/5 of original volume, the dehydrated alcohol that slowly adds 4 times of volumes, centrifugal collecting precipitation is spent the night in 4 ℃ of placements, to precipitate and use washing with acetone 3 times, get blood clam Crude polysaccharides (called after CASLP-1) 105g finally by lyophilize.Get the aqueous solution that the 500mg Crude polysaccharides is made into 100mg/mL, separate through DEAE-52 cellulose ion exchange column after the filtration.Use the distilled water wash-out, flow velocity 1.0mL/min, every pipe 5mL fraction collection, the phenolsulfuric acid method detects sugared content by pipe, draws elution curve, collects 10～26 pipes, merges postlyophilization and obtains 120mg polysaccharide P-1.Get polysaccharide fraction P-130mg that above-mentioned collection obtains through Sephacryl S-400 column chromatography (1.6cm * 80cm) be further purified, 0.15mol/L the NaCl eluant solution, flow velocity 0.5mL/min, every pipe 1.5mL fraction collection, detection method is the same, merge 10～15 pipe same composition, the dialysis desalting postlyophilization namely gets blood clam polysaccharide products ASLP-110mg.Schema as shown in Figure 1.
Physico-chemical property is measured: the molecular weight through efficient gel column chromatography (HPGPC) analysis revealed ASLP-1 is 8.5 * 10
5Da, specific optical rotation
Determination of chemical structure:
1, gets blood clam Polysaccharide A SLP-110mg and place an ampere pipe, add 2mL2.0mol/L trifluoroacetic acid (TFA), fill N
2Tube sealing is placed on 110 ℃ of hydrolysis 2h, and concentrating under reduced pressure is removed TFA (＜40 ℃), adds the methyl alcohol evaporate to dryness then, and repetitive operation 3 times is to remove TFA fully.The water that adds about 0.5mL in the sample makes sample dissolution again.Whether make tlc analysis with the Microcrystalline Cellulose plate, it is complete tentatively to judge whether to contain uronic acid and hydrolysis reaction, and the contained monose kind of test sample.The developping agent that chromatography is used is: ethyl acetate: pyridine: water: acetic acid=8: 5: 3: 1.Developer is aniline-phthalic acid, and colour temp is 110 ℃, 10min.Sample after hydrolysis chromatography in developping agent, colour developing back are that reference substance is formed to determine its monose with glucose, rhamnosyl, glucuronic acid, semi-lactosi.Result's ASLP-1 hydrolysate as can be seen from Figure 2 only has a brown spot, and its Rf value is consistent with the glucose standard substance, shows that ASLP-1 may be a kind of polysaccharide of being made up of glucose.
2, the blood clam polysaccharide among the present invention is reduced into corresponding sugar alcohol under the effect of sodium borohydride after being hydrolyzed into monose, through the acetyl reaction, obtains corresponding alditol acetate derivative, makes non-volatile polysaccharide become volatile, heat stable derivative.By gas chromatographic analysis, the ASLP-1 hydrolysate only produces a peak, and its retention time is 19.743min (Fig. 3), shows that ASLP-1 is made up of a kind of monose.The glucose standard substance carry out parallel running, and glucose standard substance appearance time is 19.800min, and is in full accord with the ASLP-1GC collection of illustrative plates.This result is consistent with above-mentioned thin layer chromatography result, therefore shows that the blood clam Polysaccharide A SLP-1 among the present invention is a kind of dextran.
3, NaIO
4The drafting of solution typical curve: the NaIO that accurately prepares 15mM, 10mM, 8mM, 6mM, 4mM, 2mM with redistilled water
4Solution all dilutes after 250 times and to compare zeroing with redistilled water and measure its light absorption value at 223nm place, drafting light absorption value A
223-concentration C typical curve is seen Fig. 4.
Take by weighing 10mg ASLP-1, place brown reaction flask, add the NaIO of 30mL15mM
4, vibration makes its dissolving, jumps a queue to place the dark place's oxidation of 4 ℃ of refrigerators, and interrupted oscillation, extract reaction solution 200 μ L at interval respectively at 24h, 48h, 72h, 96h, 120h, dilute 250 times and measure its light absorption value at the 223nm place (distilled water zeroing), calculate NaIO according to typical curve
4Consumption, treat NaIO
4Light absorption value reach when stablizing, add ethylene glycol and also stir 30min to reduce unreacted NaIO
4Get the solution after 2.0mL reaction finishes, make indicator with phenolphthalein, with 0.5mM NaOH titration and calculate the burst size of formic acid.According to NaIO
4Consumption and the burst size of the formic acid mol ratio of calculating the different sugar glycosidic bond.The ASLP-1 polysaccharide is through the NaIO of 15mM
4Oxidation 48h reaches balance, IO in the reaction
4 -Consumption be 0.042mmol, the formic acid burst size is about 0.0135mmol.Can know by inference according to principle, 1 → 4 or (with) 1 → 2 glycosidic link and 1 → 6 glycosidic link or (with) ratio of non-reduced end is 1: 1.1.
4, the Smith degraded is to carry out acid hydrolysis or part acid hydrolysis after the reduction of periodate oxidation product.Detect with GC through after the acid hydrolysis, can infer forming of glycosidic link by the product of degraded.Get 10mg ASLP-1, through NaIO
4Behind the complete oxidation, add 2mL ethylene glycol and and stir 30min and make reaction terminating, reaction solution is to flowing water dialysis 2 days, redistilled water dialysis 1 day.Liquid is evaporated to 10mL in the bag, adds 30mg NaBH
4, stirring reaction 24h in dark place under the room temperature transfers PH=4～5 with 0.1M acetic acid, again to remove excessive N aBH
4, use flowing water, distill water dialysis to thoroughly desalination successively, lyophilize gets polysaccharide polyol.Get polysaccharide polyol and place an ampere pipe, add the 2mL2.0mol/L trifluoroacetic acid, fill N
2Tube sealing is placed on 110 ℃ of hydrolysis 2h, and concentrating under reduced pressure is removed TFA (＜40 ℃), adds the methyl alcohol evaporate to dryness then, and repetitive operation three times is to remove TFA fully.Sample after the hydrolysis is dissolved in the distilled water about 3mL, adds the 25mg sodium borohydride, and reductase 12 h under room temperature (shake once every now and then) destroys excessive N aBH with Glacial acetic acid
4, between pH value to 4～5 of regulator solution, evaporated under reduced pressure adds 3mL methyl alcohol and a Glacial acetic acid, and evaporated under reduced pressure so repeats 3 times (not adding Glacial acetic acid for the last time) lyophilize again.Sample after the reduction adds the 2mL anhydrous pyridine, and ultrasonic dissolving fully back adds the 1mL acetic anhydride, 100 ℃ of reactions of close plug 2h.After reaction solution is cooled to room temperature, add 5mL distilled water, regulate pH to neutral with HCl again.Product after the acetylize is replaced solution transfer in separating funnel with chloroform and distilled water.After the jolting, water layer is removed in static, layering, with isopyknic distilled water wash 1 time, remove water layer again, chloroform layer is with anhydrous sodium sulfate drying, place 10min after the jolting, refilter and remove the sodium sulfate solid, chloroform is concentrated into the laggard promoting the circulation of qi phase of 0.1mL chromatogram (GC) analysis.GC analyzes as Fig. 5, the result shows in the Smith degraded product of ASLP-1 glycerine (retention time is 12.531min), tetrahydroxybutane (retention time is 15.453min) and glucose (retention time is 20.782min), and the ratio that draws the three after the peak area normalization method is 2: 2: 7.Therefore, 1 → 6 glycosidic link and non-reduced terminal 1 →, the mol ratio of 1 → 4 glycosidic link and 1 → 2 and 1 → 3 glycosidic link is 2: 2: 7.
5, methylating of polysaccharide: take by weighing ASLP-15mg in reaction flask, vacuum-drying 6h, dried sample adds the 4mL DMSO that handled with dry 4A molecular sieve, and the room temperature magnetic agitation is dissolved the back at N fully until the polysaccharide sample
2Protection adds the NaOH powder art 20mg of the drying of grinding down, and stirring at room 3h uses ice bath 5min then instead; after solution in the question response bottle is freezing fully; dropwise add 0.3mL methyl iodide (this process needs 15min), reactant can thaw at leisure simultaneously, and clarification gradually; until becoming bright yellow solution; return to room temperature, continue magnetic agitation reaction 3h, the room temperature underpressure distillation; remove excessive to the greatest extent methyl iodide; add the 2mL chloroform in the reaction solution, the vortex vibration mixes, leave standstill treated layering in several minutes after; sucking-off lower floor chloroform repeats this operant response liquid 4 combined chloroform phases of chloroform extraction mutually; add 2mL water, the vortex vibration mixes centrifugal 3000r/min; 5min; remove water, repeat this operation, chloroform is added to a small amount of anhydrous sodium sulphate with 2mL water treatment 5 times to chloroform mutually; fully mix, to remove residual moisture.A small amount of cotton is filled in glass drop pipe thickness intersection, with chloroform washing 1 time, chloroform phase liquid after will dewatering then adds for wide mouthful from dropper, filters through cotton, collects filtrate residue adding minimum of chloroform and washs, washing lotion is also filtered merging filtrate through cotton, reduction vaporization is to doing, again with carrying out lyophilize, vacuum-drying 5h then behind the 1mL dissolved in distilled water, after repeating aforesaid operations 3 times again, the sample that takes a morsel carries out infrared detection.The 3300cm of raw sample on the IR spectrum
-1Locate strong and wide hydroxyl peak and disappear, and 2900cm
-1When the methyl peak at place strengthens relatively, see Fig. 6, oneself is methylated interpret sample fully.If sample does not methylate fully, continue again to methylate 1～2 time.
Add 4mL2mol/L TFA in the sample behind the exhaustive methylation, seal back 110 ℃ of following hydrolysis 4h, the solution decompression evaporate to dryness in the reaction flask adds 3mL methyl alcohol again, and evaporate to dryness repeats 3 times to eliminate excessive TFA.Sample after the hydrolysis is dissolved in the distilled water about 3mL, add the 25mg sodium borohydride, reductase 12 h under room temperature (or jolting) destroys excessive sodium borohydride with Glacial acetic acid, between pH value to 4～5 of regulator solution again, evaporated under reduced pressure, add 3mL methyl alcohol and a Glacial acetic acid, evaporated under reduced pressure so repeats 3 times again, do not add Glacial acetic acid for the last time, then vacuum-drying 4h.Dried sample adds the 2mL anhydrous pyridine, and ultrasonic dissolving fully back adds the 1mL acetic anhydride, 100 ℃ of reactions of close plug 2h.After reaction solution is cooled to room temperature, add 5mL distilled water, regulate pH to neutral with HCl again.Product after the acetylize is replaced solution transfer in separating funnel with chloroform and distilled water.After the jolting, water layer is removed in static, layering, with isopyknic distilled water wash 1 time, removes water layer again, and chloroform layer is placed 10min with anhydrous sodium sulfate drying after the jolting, refilter to remove the sodium sulfate solid, chloroform is concentrated into the coupling of the laggard promoting the circulation of qi matter of 0.1mL analyzes.
45 derivative quasi-molecular ions that methylate have appearred in ASLP-1, and its retention time is respectively 11.382min, 12.579min, 14.735min, 15.985min and 18.246min.The base peak of m/z43 is arranged in the mass spectrum of all ASLP-1 part methyl alditol acetate derivatives, is to lose ethanoyl ion (CH
3CO
+) form.Each mass spectrum of online information retrieval is tentatively determined each mass spectral ownership; According to elementary fragment and secondary fragment in the cracking rule ownership mass spectrum of part methyl alditol acetate derivative, by resolving, five compositions should be 1 successively in the GC spectrogram; 5-two-oxy-acetyl-2,3,4; 6-four-oxygen-methyl-glucose (is abbreviated as 2,3,4; 6-Me4-Glc); 1,4,5-, three-oxy-acetyl-2; 3,6-, three-oxygen-methyl-glucose (is abbreviated as 2,3; 6-Me3-Glc); 1,3,4; 5-four-oxy-acetyl-2,6-two-oxygen-methyl-glucose (is abbreviated as 2,6-Me2-Glc) with 1; 3,5,6-, four-oxy-acetyl-2; 4-two-oxygen-methyl-glucose (is abbreviated as 2,4-Me2-Glc); 1,5; 6-three-oxy-acetyl-2,3,4-three-oxygen-methyl-glucose (is abbreviated as 2; 3; 4-Me3-Glc); 1,3,5-, three-oxy-acetyl-2; 4; (be abbreviated as 2,4,6-Me3-Glc) five component mol ratios are about 1: 3.3: 1 to 6-three-oxygen-methyl-glucose: 1: 8.2.Hence one can see that, ASLP-1 be a kind of contain non-reduced terminal 1 →, by the dextran of forming of 1 → 3,1 → 4 and 1 → 6 glycosidic link, four ratio is about 2: 16: 6: 2.
6, part acid hydrolysis: take by weighing the ASLP-1 of 10mg, place an ampere pipe, add the trifluoroacetic acid TFA5mL of 0.3mol/L, tube sealing, 100 ℃ of hydrolysis 18h use among the NaOH and unnecessary TFA, dialysis, lyophilize gets hydrolysate (called after mASLP-1).MASLP-1 is through NaIO
4Oxidation and NaBH
4Get corresponding sugar alcohol after the reduction, sugar alcohol generates the laggard promoting the circulation of qi analysis of hplc of derivative of sugared ethanol ester through hydrolysis and derivatize, the part acid hydrolysis products mASLP-1 of ASLP-1, carrying out GC after periodate oxidation, Smith degraded and TFA hydrolysis analyzes, only detect glucose, show that mASLP-1 only contains 1 → 3 glycosidic link, namely the main chain of ASLP-1 is made up of 1 → 3 glycosidic link.
7, ultraviolet spectral analysis: the blood clam Polysaccharide A SLP-11mg among the present invention is made into the 1.0mg/mL aqueous solution, scan in the scope of 200～600nm with ultraviolet spectrophotometer, see Fig. 7, it does not all have charateristic avsorption band at 260nm and 280nm place, shows that the ASLP-1 polysaccharide does not have protein and nucleic acid.
8, Infrared spectroscopy: the blood clam Polysaccharide A SLP-1 1.0mg of drying, with the KBr compressing tablet, at 4000～400cm
-1Scope in carry out Infrared spectrum scanning, see Fig. 8, as can be seen: at 3600～3200cm
-1(the 3384.54cm of place
-1) a stronger broad peak appears, and (stretching vibration OH) exists intermolecular and intramolecular hydrogen bond for hydroxyl; At 3000～2800cm
-12 absorption peaks that occur a little less than, be the stretching vibration of C-H, 1400～1200cm
-1(the 1364.06cm of place
-1) the peak be the angle vibration of C-H.These two groups of charateristic avsorption bands that the peak is polysaccharide.1649.40cm
-1The absorption peak that the place occurs is owing to solvent (water) causes.1154～1022cm
-1Scope is the stretching vibration of C-O, 1024.6cm
-1The main composition monose of the strong absorption explanation ASLP-1 at place is glucose; 901.5cm
-1Absorption peak is arranged and at 905～884cm
-1The place does not have the peak of suction and shows that ASLP-1 only contains the α glycosidic link; At 901.5cm
-1The absorption peak at place and 1024.6 and 954.4cm
-1Show that ASLP-1 is made up of α-D-Glucopyranose.
9, nucleus magnetic resonance (NMR) is analyzed: get the blood clam Polysaccharide A SLP-1 30mg after the lyophilize, oil pump vacuum-drying is dissolved in 0.5mL D under the room temperature after 3 hours
2Among the O, measure it in the AVANCE500 nuclear magnetic resonance analyser
1H NMR and
13C NMR spectrum, measuring temperature is 30 ℃.In addition, also measured ASLP-1's
1H-
12D NMR such as H COSY, HSQC and HMBC spectrum.See Fig. 9,
1H NMR has four anomeric proton signals in the low place of δ＞4.5ppm, and its chemical shift is respectively δ 5.45ppm, δ 5.27ppm and δ 4.70ppm, points out this polysaccharide to contain three kinds of α glycosidic links.Because three class anomeric proton H signal proportion differences illustrate that three kinds of glycosidic links are unbalanced in this polysaccharide.See Figure 10, from
13C NMR as seen, three fignal centers appear in the low place of δ 90～110ppm, are the fignal center of anomeric carbon, its chemical shift is respectively δ 97.02,98.91 and 100.97ppm, with
1Three anomer hydrogen fignal centers in the H NMR spectrum are corresponding, and the chemical shift that can draw corresponding C-H signal according to H-H COSY (seeing Figure 11), HMBC (seeing Figure 12) and the HSQC (seeing Figure 13) of blood clam polysaccharide is again according to the front
1H NMR and
13The CNMR analytical results carries out the part ownership with the C-H signal of correspondence and sees Table 1
Table 1 blood clam polysaccharide
1H-
1The concrete signal ownership of H COSY, HSQC and HMBC
Really the mode of connection that contains three kinds of glycosidic links in the basic repeating unit of its presentation of results composition ASLP-1, and saccharide residue is configured as the α type.Again according to methylating and the result of conclusive evidence polysaccharide structures common chemical methods commonly used such as periodate oxidation, Smith degraded, part acid hydrolysis proves that ASLP-1 contains 1 → 3,1 → 4 and 1 → 6 three kind of mode of connection, therefore, the fignal center of three anomeric carbons and anomeric proton belongs to 1 → 3,1 → 4 and 1 → 6 respectively.
10, comprehensive analysis described above can get following result:
(1) monose compositional analysis result shows: the blood clam polysaccharide among the present invention is dextran.
(2) periodate oxidation, Smith degraded and the result that methylates show the ASLP-1 polysaccharide among the present invention be a kind of contain non-reduced terminal 1 →, by the dextran of forming of 1 → 3,1 → 4 and 1 → 6 glycosidic link, its ratio is about 2: 16: 6: 2.
(3) the acid-hydrolyzed result of part shows that the main chain of this polysaccharide is 1 → 3 glycosidic link.
(4) do not contain protein and nucleic acid in the UV analysis revealed polysaccharide of the present invention, the IR analysis revealed it by glucose that α-the D-Glucopyranose is formed.
(5) the NMR analytical results shows and contains 1 → 3,1 → 4 and 1 → 6 three kind of glycosidic link in the basic repeating unit of forming ASLP-1, and saccharide residue is configured as the α type.
Therefore, the structure of the basic repeating unit of the blood clam polysaccharide among the present invention is as shown below, and it is a kind of novel texture polysaccharide of finding from blood clam.
Material and reagent blood clam polysaccharide (self-control detects through HPLC and is homogeneous polysaccharide), LPS (Sigma), CCK-8 (Sigma), MTT (Sigma), RPMI Medium 1640 (Gibco), splenic lymphocyte parting liquid (Hangzhou folium ilicis chinensis biomaterial company limited)
Instrument Bechtop (Suzhou Decontamination Equipment Plant), CO
2Incubator (U.S. ThermoForma company), Model680 type enzyme-linked immunosorbent assay instrument (U.S. Bio-Rad company)
The blood clam polysaccharide is to the influence of mice spleen lymphocytes proliferation
Get 6-8 ICR mouse in age in week, the cervical vertebra dislocation is put to death, and sterile preparation splenic lymphocyte suspension does not have phenol red medium with the RPMI1640 that adds calf serum and adjusts mouse spleen lymphocyte concentration to 2 * 10
6/ mL adds 100 μ L cell suspensions in the every hole of 96 orifice plates, add different concns blood clam polysaccharide nutrient solution, and final concentration is respectively 6.25,12.5,25,50,100,200,400 μ g/mL, every Kongzui final volume is 200 μ L, sets blank hole and positive controls (LPS 50 μ g/mL) simultaneously.All establish 4 multiple holes, put 37 ℃, 5%CO for every group
2After cultivating 36h in the incubator, every hole adds CCK-8 20 μ L, continues to cultivate 1h, and the OD value is detected at microplate reader 450nm place.Result such as Figure 14 show that the blood clam polysaccharide has significantly proliferation function to splenic lymphocyte, and be dose-dependently when 50-200 μ g/mL.
The blood clam polysaccharide is to the influence of the peritoneal macrophage RAW264.7 of mouse
1 blood clam polysaccharide is to the influence of normal RAW264.7 cell-proliferation activity
Every hole adding density is 1.0 * 10 in 96 orifice plates
5The RAW264.7 cell 100 μ L of/mL, the nutrient solution of the ASLP-1 of adding different concns after 6h is adherent, final concentration is respectively 25,50,100,200,400 μ g/mL, every Kongzui final volume is 200 μ L, sets blank group, positive controls (LPS 50 μ g/mL) simultaneously.After hatching 24h, add the MTT20 μ L/ hole of concentration 5mg/mL, put 37 ℃, 5%CO
2Incubator is abandoned supernatant after cultivating 4h, adds DMSO 150 μ L/ holes, and shake well 10min measures absorbancy at microplate reader 450nm wavelength place.Result such as Figure 15, the blood clam polysaccharide can the remarkable propagation that must promote Turnover of Mouse Peritoneal Macrophages.
2 blood clam polysaccharide are to the influence of normal RAW264.7 cytophagy toluylene red function
Every hole adding density is 1.0 * 10 in 96 orifice plates
5The RAW264.7 cell 100 μ L of/mL, in the blood clam polysaccharide nutrient solution of the adherent back adding of 12h different concns, final concentration is respectively 25,50,100,200,400 μ g/mL, every Kongzui final volume is 200 μ L, sets blank and positive control (LPS 50 μ g/mL) simultaneously.Each concentration is established 4 multiple holes.Place 37 ℃, 5%CO
2Add 100 μ L/ holes, 0.1% toluylene red after cultivating 24h in the incubator, put 37 ℃, 5%CO
2Incubator is abandoned supernatant after cultivating 30min, and PBS liquid 200 μ L/ holes are washed 3 times, add lysis liquid (mixed solution of Glacial acetic acid and equal-volume dehydrated alcohol) 200 μ L/ hole 30min after, in microplate reader 490nm wavelength place mensuration absorbancy.Result such as Figure 16, the blood clam polysaccharide can significantly strengthen macrophage phagocytic toluylene red ability.
Therefore, from embodiment 2 as can be seen the blood clam polysaccharide be a kind of immunostimulant, enhancing immunity activity significantly, and undertaken by different immunomodulatory modes is for follow-up anti-tumor experiment is laid a good foundation.
Claims (3)
1. one kind has α-1, the blood clam polysaccharide of 3-dextran main chain, it is characterized in that containing 1 → 3,1 → 4 and 1 → 6 three kind of glycosidic link in the repeated structural unit of described polysaccharide, wherein main chain is 1 → 3 glycosidic link, side chain is made up of 1 → 4 and 1 → 6 two kind of glycosidic link, saccharide residue is the α configuration, and its relative molecular weight is 8.5 * 10
5Da, specific optical rotation
2, a kind of α-1 that has according to claim 1, the blood clam polysaccharide of 3-dextran main chain is characterized in that:
Described repeated structural unit structure is as follows:
2. the preparation method of the polysaccharide that is extracted by blood clam according to claim 1 is characterized in that realizing as follows:
A, fresh blood clam 20kg is shelled, collect human body part 5kg, and clean with water rinse, filtered through gauze is to remove moisture content as far as possible, and the human body of collecting partly carries out homogenate with the tissue homogenate stamp mill, adds isopyknic acetone in the homogenate and decolours; Sample hot water extraction after the processing, determine to extract top condition with orthogonal experiment method, centrifugal collection supernatant liquor, after rotary evaporation is concentrated into original solution 1/3 volume, add 1/4 volume Sevag reagent, thermal agitation 15min, the centrifugal 15min of 4000rpm, get supernatant, repeat aforesaid operations and do not have milky white precipitate up to chloroform layer 8 times, concentrating under reduced pressure is flung to organic solvent; Polysaccharide extraction liquid behind the Deproteinization is evaporated to 1/5 of original volume, slowly adds the dehydrated alcohol of 4 times of volumes, 4 ℃ of placements are spent the night, and centrifugal collecting precipitation will precipitate and use washing with acetone 3 times, get blood clam Crude polysaccharides 105g finally by lyophilize;
B, blood clam Crude polysaccharides 500mg is made into the aqueous solution of 100mg/mL, separate through DEAE-52 cellulose ion exchange column after the filtration, use the distilled water wash-out, flow velocity 1.0mL/min, every pipe 5mL fraction collection, the phenolsulfuric acid method detects sugared content by pipe, draws elution curve, collect 10～26 pipes, merge same composition and lyophilize and obtain 120mg polysaccharide P-1;
The component P-130mg that c, above-mentioned collection obtain is through Sephacryl S-400 column chromatography (1.6cm * 80cm) be further purified, 0.15mol/L the NaCl eluant solution, flow velocity 0.5mL/min, every pipe 1.5mL fraction collection, detection method is the same, merge 10～15 pipes, the dialysis desalting postlyophilization namely gets blood clam polysaccharide 10mg.
3. by the described a kind of α-1 that has of claim 1, the application of the blood clam polysaccharide of 3-dextran main chain in preparation immunomodulator, antitumor drug or anti-oxidation medicine.
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| CN106349403A (en) * | 2016-08-19 | 2017-01-25 | 哈尔滨商业大学 | Yantai sweet potato polysaccharide with alpha-1,3-glucan chain and preparation method of Yantai sweet potato polysaccharide |
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| CN112390898A (en) * | 2019-08-18 | 2021-02-23 | 于荣敏 | Arca inflata reeve immunoregulation and anti-tumor polysaccharide and preparation method and application thereof |
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