CN107056962A - A kind of mussel polysaccharide and its preparation method and application - Google Patents
A kind of mussel polysaccharide and its preparation method and application Download PDFInfo
- Publication number
- CN107056962A CN107056962A CN201710351379.4A CN201710351379A CN107056962A CN 107056962 A CN107056962 A CN 107056962A CN 201710351379 A CN201710351379 A CN 201710351379A CN 107056962 A CN107056962 A CN 107056962A
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- Prior art keywords
- mussel
- polysaccharide
- mussel polysaccharide
- precipitation
- glc
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Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 153
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 152
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
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Abstract
The application in reducing blood lipid, the medicine and health food of antiatherosclerosis is prepared the invention provides a kind of mussel polysaccharide extracted in mussel and its industrial extraction method and the mussel polysaccharide.The mussel polysaccharide, with（1 → 4) α D Glc be main chain, contain（1 → 2) α D Glc branches, are configured as α pyranoid form glucans, have 1 in average every 12 grape sugar backbones（1 → 2) glucose branch, relative molecular weight is 800 8000 KDa.The mussel polysaccharide is by slightly carrying, digesting, purify, drying and other steps extract from mussel category（Mytilus sp.）Boil shellfish to do, extraction conditions are gentle, product purity is high.Mussel polysaccharide energy substantial reduction in triglycerides, T-CHOL and the LDL-C of the present invention, with effect for reducing fat, the medicine for the treatment of or prevention hyperlipemia, atherosclerosis is being prepared, preparing in health products or special medicine purposes formula food with blood fat reducing function has extensive use.
Description
Technical field
The invention belongs to natural product extraction and application field, it is related to a kind of mussel polysaccharide and its preparation method and application.
Background technology
There is the number one killer cardiovascular and cerebrovascular disease of hyperlipidemia and human health caused by disorders of lipid metabolism
Close relationship, is to threaten one of most important pathogenic factor of human life and health.Popular hypolipidemic in the market
Thing is so that statins is most widely used, curative effect preferably, account for more than the 80% of lipid lowering agent gross sales amount.But statins
In the presence of the adverse reaction related to liver, kidney and internal dysbolism, in addition, also common have gastrointestinal reaction, rhinitis, nasosinusitis, head
Bitterly, the toxic side effect such as pharyngalgia, influenza syndrome, arthritis, pectoralgia, insomnia, musclar toxicity.This to substitute statins
The demand of product is extremely urgent.
Polysaccharide is the important component of all biologic artifacts, with unique biological activity, can improve body
Immunologic function, and all important vital movement processes are participated in, with the effect such as extensive Immune-enhancing effect, reducing blood lipid, antitumor,
Adverse reaction simultaneously and very little.Recently as the rise of exploitation marine resources, people generate greatly to marine polysaccharide
Interest, it was found that many have bioactivity polysaccharose substances, including the sulfated polysaccharide with antiviral activity, promote
Marine bacteria polysaccharide of knitting etc..
Mussel polysaccharide derives from marine animal mussel（Mytilus sp.）.Mussel is the important kind in seashells cultivation
Class, also known as marine rainbow, are commonly called as " marine egg ", the effects such as with obvious filling liver kidney, reducing blood lipid, there is very high medicine price cheating value.Grind
Study carefully and find that the function of mussel reducing blood lipid is derived mainly from its internal polysaccharide.At present, the chemical constitution of mussel polysaccharide is still not clear,
Its fat-reducing effect and toxicity assessment also contribute to it and further apply medicine and health food.Therefore, making a gift of for high-purity is prepared
Pattra sugar simultaneously carries out structure elucidation, efficacy study and toxicity assessment to it, to further being developed into natural origin reducing blood lipid
Medicine and health food are significant.
The content of the invention
The invention provides a kind of mussel polysaccharide and its industrial extraction method extracted in mussel.
What another object of the present invention was to provide mussel polysaccharide is preparing reducing blood lipid, the medicine of antiatherosclerosis and health care
Application in food.
To achieve the above object, the present invention is adopted the following technical scheme that.
A kind of mussel polysaccharide, with（1 → 4)-α-D-Glc be main chain, contain（1 → 2)-α-D-Glc branches, are configured as α-pyrrole
There is 1 in type of muttering glucan, average every 12 grape sugar backbones（1 → 2) glucose branch, its structural formula is：
；
The mussel polysaccharide relative molecular weight is 800-8000 KDa.
The mussel polysaccharide extracts from mussel category（Mytilus sp.）Animal, including Trachyostracous mussel（Mytilus coruscus）And Mytilus galloprovincialis（Mytilus edulis）.
A kind of extracting method of above-mentioned mussel polysaccharide, comprises the following steps：
（1）Feedstock treating：Mussel heating, which is shelled, to be stayed meat and dry, pulverize machine crushing, is crossed 20-40 mesh sieves, is obtained mussel dry powder；
（2）Polyose extraction：Step（1）The water of 6 times of volumes, 100 DEG C of 2 h of extraction are added in obtained mussel dry powder；Filtering is removed
Mussel meat slag, obtains polysaccharide extraction liquid；
（3）Enzymolysis：By step（2）Obtained polysaccharide extraction liquid adjusts pH to 8.0, adds 0.2 %w.t alkali protease, 50 DEG C
Digest and inactivate 15-30 min after the completion of 2 h, enzymolysis under the conditions of 80 DEG C, obtain enzymolysis liquid；Using diatomite as filter aid, suction filtration mistake
Filter, the enzymolysis liquid clarified；
（4）Ethanol precipitation：In step（3）1.5 times of 90-95% are added in obtained polysaccharide solution（v/v）Ethanol precipitated,
6 h are stood, incline supernatant, obtains polysaccharide precipitation；
（5）Dehydration：In step（4）1 times of volume 90-95% is added in obtained polysaccharide precipitation（v/v）Ethanol be dehydrated；
（6）Filtration drying：The polysaccharide precipitation after dehydration is filtered with 400 eye mesh screens, precipitation is collected, 60 DEG C are dried in vacuo,
Obtain Thick many candies；
（7）Weight is molten：By step（6）Gained Thick many candies are dissolved in water, prepare 10% mussel Thick many candies solution, and 12000 rpm centrifuge 1h,
By supernatant with 0.45 μm of membrane filtration, polysaccharide filtrate is obtained；
（8）Ion-exchange chromatography：By step（7）Polysaccharide filtrate use DEAE- agaroses（FF）Column chromatography, with 0.05
Mol/L NaCl solutions are eluent, collect elution fraction；
（9）Ultrafiltration：With the filter membrane that molecular cut off is 5000 to step（8）Gained elution fraction carries out ultrafiltration desalination and concentration,
Obtain concentrate；
（10）Freeze-drying：By step（9）Concentrate is freeze-dried, and obtains mussel polysaccharide finished product.
The purity of above-mentioned mussel polysaccharide finished product is more than 99%.
Described mussel polysaccharide is preparing the application in being used to treat or prevent cardiovascular and cerebrovascular diseases medicament, described heart and brain
Vascular diseases include hyperlipemia, atherosclerosis.
The mussel polysaccharide answering in the health products or special medicine purposes formula food with blood fat reducing function are prepared
With.
The present invention has advantages below：
The mussel polysaccharide of the present invention derives from seashells animal mussel, is the important species in seashells cultivation, is also most
A kind of common edible shellfish, can be by propagating a large amount of acquisitions artificially, so that mussel polysaccharide carrys out source problem and is readily available
Solve, sea-farming industry development can be pulled again.The method that the present invention extracts mussel polysaccharide, mild condition, purity are high.This hair
The bright nontoxic level of mussel polysaccharide acute toxicity classification category, it is safe；And mussel polysaccharide energy substantial reduction in triglycerides, total courage are solid
Alcohol and LDL-C, with effect for reducing fat.The present invention mussel polysaccharide prepare treat or prevent hyperlipemia,
The medicine of atherosclerosis, prepare has extensively in health products or special medicine purposes formula food with blood fat reducing function
Using.
Brief description of the drawings
Fig. 1 is the HPGPC elution curves of mussel polysaccharide；
Fig. 2 is mussel polysaccharide PMP column front derivation liquid-phase chromatographic analysis spectrograms：A is the PMP column front derivation liquid phase colors of monose standard items
Spectrogram（Wherein, 1 is Man；2 be GlcN；3 be Rha；4 be GlcA；5 be GalA；6 be GalN；7 be Glc；8 be Gal；9 be Xyl；
10 be Ara；11 be Fuc）；B is the PMP column front derivation liquid chromatograms of mussel polysaccharide.
Fig. 3 is mussel polysaccharide spectrum analysis spectrogram：A is the ultraviolet absorption curve of mussel polysaccharide；B is infrared for mussel polysaccharide
Absorption curve；
Fig. 4 is mussel polysaccharide1H-NMR collection of illustrative plates；
Fig. 5 is mussel polysaccharide13C-NMR collection of illustrative plates
Fig. 6 is the DEPT collection of illustrative plates of mussel polysaccharide；
Fig. 7 is the HH-COSY collection of illustrative plates of mussel polysaccharide；
Fig. 8 is the HSQC collection of illustrative plates of mussel polysaccharide；
Fig. 9 is the TOCSY collection of illustrative plates of mussel polysaccharide；
Figure 10 is the HMBC collection of illustrative plates of mussel polysaccharide；
Figure 11 is mussel polysaccharide methylated derivative GC-MS total ion current figures；
Figure 12 is the-O- methyl-D-glucose alcohol mass spectrograms of bis--O- acetyl group -1- deoxidations -2,3,4,6- of 1,5- four：A is mussel
Mass spectrogram obtained by polysaccharide；B is CCRC standard mass spectrograms；
Figure 13 is the-O- methyl-D-glucose alcohol mass spectrograms of tri--O- acetyl group -1- deoxidations -3,4,6- of 1,2,5- three：A is mussel
Mass spectrogram obtained by polysaccharide；B is CCRC standard mass spectrograms；
Figure 14 is the-O- methyl-D-glucose alcohol mass spectrograms of tri--O- acetyl group -1- deoxidations -2,3,6- of 1,4,5- three：A is that mussel is more
Sugared gained mass spectrogram；B is CCRC standard mass spectrograms；
Figure 15 is the-O- methyl-D-glucose alcohol mass spectrograms of tetra--O- acetyl group -1- deoxidations -3,6- of 1,2,4,5- two：A is that mussel is more
Sugared gained mass spectrogram；B is CCRC standard mass spectrograms.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but the present invention is not limited by following embodiments
System.
It is prepared by the extraction of the mussel polysaccharide of embodiment 1.
（1）Feedstock treating：Production Trachyostracous mussel heating in Zhejiang Province Shengsi County, which is shelled, stays jerky dry, is crushed with pulverizer, mistake
20 mesh sieves, obtain mussel dry powder；
（2）Polyose extraction：Step（1）The water of 6 times of volumes, 100 DEG C of 2 h of extraction are added in obtained mussel dry powder；
（3）Slagging-off：Mussel meat slag and polysaccharide extraction liquid are separated with 50 eye mesh screens；
（4）Enzymolysis：By step（3）Obtained polysaccharide extraction liquid adjusts pH to 8.0 with NaOH, adds the alkaline egg that mass ratio is 0.2%
20 min are inactivated under the conditions of 80 DEG C after the completion of white enzyme, 50 DEG C of 2 h of enzymolysis, enzymolysis, enzymolysis liquid is obtained；
（5）Filtering：Using diatomite as filter aid, suction filtration filtration, the polysaccharide solution clarified；
（6）Ethanol precipitation：In step（5）The edible alcohol that 1.5 times of volume mass fractions are 93% is added in obtained polysaccharide solution
Polysaccharide precipitation is carried out, 6 h are stood, incline supernatant, obtains polysaccharide precipitation；
（7）Dehydration：In step（6）The edible alcohol that 1 times of volume mass fraction of addition is 93% in obtained polysaccharide precipitation is to polysaccharide
Precipitation is dehydrated；
（8）Filtration drying：The polysaccharide precipitation after dehydration is filtered with 400 eye mesh screens, precipitation is collected, 60 DEG C are dried in vacuo,
Obtain Thick many candies.
（9）Weight is molten：By step（8）Gained Thick many candies are dissolved in water, prepare 10% mussel Thick many candies solution, 12000 rpm centrifugations
1 h is to remove insoluble matter；
（10）Filtering：Step（9）Centrifugation gained supernatant obtains polysaccharide filtrate with 0.45 μm of membrane filtration；
（11）Ion-exchange chromatography：Use DEAE- agaroses（FF）With 0.05 after column chromatography polysaccharide, polysaccharide filtrate loading
Mol/L NaCl solutions are eluted, and collect elution fraction；
（12）Ultrafiltration：With the filter membrane that molecular cut off is 5000 to step（11）Gained elution fraction carries out ultrafiltration desalination and dense
Contracting；
（13）Freeze-drying：Concentrate is freeze-dried, and obtains mussel polysaccharide sample.
The Structural Identification of the mussel polysaccharide of embodiment 2.
2.1 chemical analysis
Total sugar content is determined using sulfuric acid/phynol method：The mussel polysaccharide solution that 1 mL concentration is 100 μ g/mL is drawn, 1 is added
The % phenol solutions of mL 6, are rapidly added the 5 mL concentrated sulfuric acids, and vibration is allowed to mix, and after the min of boiling water bath 20, is cooled to room temperature 490
Its light absorption value is determined at nm wavelength.Standard curve is done with concentration of glucose-absorbance, equation of linear regression is：Y=3.8108X+
0.0415（R2=0.9996）, calculate sample total sugar content.
Glucuronic acid content is determined using sulfuric acid/carbazole method：Draw the mussel polysaccharide solution that 0.5 mL concentration is 50 μ g/mL
And blank reagent.3 mL sodium tetraborates-sulfuric acid solution is added in ice-water bath, the min of boiling water bath 15 is cooled to immediately after taking-up
Room temperature, plus the mL of 0.1% carbazole solution 0.1, its light absorption value is determined after shaking up at 530 nm wavelength.With glucuronic acid concentration-
Absorbance does standard curve, and equation of linear regression is：Y=4.1536X+0.0252（R2=0.9990）, calculate sample uronic acid and contain
Amount.
Sulfate radical content uses BaCl2- gelatin turbidimetry for Determination：Draw the mussel polysaccharide that 0.5 mL concentration is 10 mg/mL
Solution and blank reagent, sequentially add the mL of 3% trifluoroacetic acid solution 3.8, the mL of barium chloride-gelatin solution 1.0, shake up, room temperature is put
15 min are put, using 1 mol/L HCl as blank control, its light absorption value is determined at 500 nm.Done with sulfate concentration-absorbance
Standard curve, equation of linear regression is：：Y=0.4733X-0.0035（R2=0.9977）, calculate sample sulfated polysaccharide content.
Measurement result result is as follows：The total sugar content of mussel polysaccharide sample is 99.2%, and each batch is without obvious uronic acid group
Divide and exist, do not contain sulfate radical.Chemical analysis results show that mussel polysaccharide is a kind of neutral polysaccharide with higher degree, no
Contain obvious uronic acid and sulfated polysaccharide composition.
2.2 relative molecular weights are determined
Mussel polysaccharide molecular weight is measured using high performance liquid chromatography-molecular exclusion chromatography.Chromatographic condition is as follows：Shimadzu
LC-20AT high performance liquid chromatographs；Chromatographic column is TSK-gel GMPWXL（Exclusion limit 5,000,000）；Column temperature：35 ℃；Mobile phase：
0.05 mol/mL sodium nitrate solutions；Flow velocity：0.6 mL/min；Detector：RID-20AT differential refraction detectors；Applied sample amount 20
μL。
It is respectively 180,55500,102000,291600,534000,1185000,2990000Dal by weight average molecular weight
Glucan control series product draw using the logarithm value of molecular weight as ordinate, using the retention time of corresponding chromatographic peak as abscissa
Three standard curves, obtaining regression equation is：y = -0.00315x3 +0.16469x2- 3.47013x+29.24623, R2
=0.9988.Precision weighs the mg of mussel polysaccharide sample 50, plus flows phased soln and be settled to 10 ml, HPLC detections, gpc analysis.
The HPGPC elution curves of mussel polysaccharide are as shown in figure 1, in addition to main polysaccharide component is contained in 11.9 min, also contain
There is a small amount of low molecule impurity.Calculated according to standard curve, the average molecular mass of mussel polysaccharide main polysaccharide component is
1212.7 KDa。
2.3 monosaccharide composition analysis
Using column front derivation high performance liquid chromatography（PC-HPLC）Determine the monose composition of mussel polysaccharide.Chromatographic condition is as follows：
The high performance liquid chromatographs of UltiMate 3000；Chromatographic column is Boston Geen ODS C18 chromatographic columns（The mm of 250 mm × 4.6,
5 μm）；Column temperature：30 ℃；Mobile phase：Phosphate buffer（pH 6.7）/CH3CN（82:18, V/V）；Flow velocity：1.0 mL/
min；Detector：PDAD（DAD, 245 nm）.
Precision weighs mussel polysaccharide in right amount, with 2 mol/L trifluoroacetic acids（TFA）Complete hydrolysis.Take mussel polysaccharide degradation solution
100 μ L, carry out derivatization reaction with PMP in the basic conditions, after chloroform extraction processing, HPLC analyses.As a result as shown in Fig. 2
As a result show, glucose component is comprised only in mussel polysaccharide.
2.4 ultraviolet and infrared spectrum analysis
Mussel polysaccharide solution is prepared by solvent of pure water and uses ultraviolet-visible spectrophotometer in the range of wavelength 190-500 nm
Scanning, as a result as shown in Figure 3A.From collection of illustrative plates, mussel polysaccharide is in the range of 190-500 nm without obvious UV absorption, symbol
Close the architectural feature of polysaccharide.
The dry mg of mussel polysaccharide 2 is taken with drying tabletting after the mg of KBr 200 grindings, in Fourier transform infrared spectroscopy
Instrument is in 4000-500 cm-1Wave band is scanned, as a result as shown in Figure 3 B.From collection of illustrative plates, mussel polysaccharide is in 3391 cm-1In the range of
Strong absorption, the stretching vibration peak for being O-H in the presence of wide；In 2928 cm-1Be absorbed as on sugared ring in methine and methylene
C-H stretching vibrations, show to contain methylene and methine in sample molecule structure.In 1154 cm-1、1081 cm-1、1021 cm-1Absworption peak be sugared ring C-O-H, C-O-C feature skeletal vibration；In 1417 cm-1、1368 cm-1、1238 cm-1For pyrans
Sugared ring O-H in-plane deformation absorption of vibrations, 577 cm-1For O-H out-of-plane deformation absorption of vibrations.848 cm-1The absorption at place shows
Sugared ring is α-configuration；928 cm-1The absorption at place shows that sugared ring is D-form；1646 cm-1Place to be absorbed as polysaccharose substance normal
The micro-moisture associate hydrogen bond seen is caused.It is possible thereby to which initial guess, mussel polysaccharide is α-D- pyranoid form glucans.
2.5 nuclear magnetic resonance spectroscopy
2.5.1 one-dimensional nuclear magnetic resonance wave spectrum（1D-NMR）
Instrument and reagent：Agilent DD2-500 nuclear magnetic resonance chemical analysers（500MHz）；Heavy water（D2O, 99.96%）.
The mg of mussel polysaccharide 20 is taken, with 1 mL D2O is exchanged 3 times, and D is used again after freezing2O dissolves, using deuterated acetone as internal standard,
Recorded at 20 DEG C1H-NMR、13C-NMR and DEPT collection of illustrative plates.
In mussel polysaccharide1H-NMR collection of illustrative plates（Fig. 4）In, there is end group H1 signal at the ppm of δ 5.24, and positioned at compared with
Low field（Chemical shift>δ5.0 ppm）, it is wider unimodal, show mussel polysaccharide is configured as α-configuration.In δ 3.0 ~ 4.0
Signal at ppm is sugared ring H2 ~ H6 signals, need to be by H H-COSY, TOCSY and HSQC etc. because its chemical shift is overlapping serious
Collection of illustrative plates is identified.
In mussel polysaccharide13C-NMR collection of illustrative plates（Fig. 5）In, there is end group C1 signal at the ppm of δ 99.74, and positioned at compared with
High-Field（Chemical shift<δ102 ppm）, meet the feature of α-configuration polysaccharide, with it1Sentencing in H-NMR collection of illustrative plates and in infrared spectrum
It is disconnected consistent；The ppm signals of δ 60.48 occurred in high field region, may be the sugared ring C6 of mussel polysaccharide signal；In the ppm models of δ 69 ~ 78
Signal in enclosing is the sugared ring C2 ~ C5 signals of mussel polysaccharide；Do not occur C signal in the range of the ppm of δ 80 ~ 90；Show that mussel is more
Sugar is pyranoid form, and is not present（1→3）Glycosidic bond.
In the DEPT of mussel polysaccharide（Fig. 6）In collection of illustrative plates, the ppm of δ 60.4 signal is negative peak, and it is Asia to show corresponding C signal
Methyl（-CH2）, may be the C6 signals in the sugared ring structure of mussel polysaccharide, this with its13The supposition of C-NMR collection of illustrative plates is consistent.Cause exists
In the DEPT collection of illustrative plates of mussel polysaccharide, other positions show to be not present in mussel polysaccharide structure without negative spike（1→6）Glucosides
Key.
2.5.2 two dimensional NMR wave spectrum（2D-NMR）
Instrument and reagent：Agilent DD2-500 nuclear magnetic resonance chemical analysers（500MHz）；Heavy water（D2O, 99.96%）.
Test method：The mg of mussel polysaccharide sample 20 is taken, with 1 mL D2O is exchanged 3 times, and D is used again after freezing2O dissolves, with deuterium
It is internal standard for acetone, HSQC, HH-COSY, TOCSY and HMBC collection of illustrative plates is recorded at 20 DEG C.
According to the HH-COSY collection of illustrative plates of mussel polysaccharide（Fig. 7）, with reference to HSQC collection of illustrative plates（Fig. 8）, TOCSY collection of illustrative plates（Fig. 9）、HMBC
Collection of illustrative plates（Figure 10）, mussel polysaccharide C and H chemical shift ownership can be confirmed（Table 1）.
The mussel polysaccharide of table 11H-NMR and13C-NMR signals assignments
In the HMBC collection of illustrative plates of mussel polysaccharide, H1 can be respectively seen（δ5.24 ppm）With C4（δ76.74 ppm）Coupling phase
OFF signal, C1（δ99.74 ppm）With H4（δ3.51 ppm）Coupling coherent signal；H1（δ5.24 ppm）With C2（δ77.26
ppm）Coupling coherent signal, C1（δ99.74 ppm）With H2（δ3.47 ppm）Coupling coherent signal；Show in mussel polysaccharide
In the presence of（1→4）Glycosidic bond and（1→2）Glycosidic bond.
Comprehensive 1D-NMR and 2D-NMR to mussel polysaccharide is analyzed, can primarily determine that mussel polysaccharide be with（1→4)-α-
D-Glc is main chain, is contained（α-pyranoid form glucan of 1 → 2)-α-D-Glc branches.
2.6 monose connected modes are analyzed
Mussel polysaccharide is subjected to methylation reaction combination GC-MS analyses and determines monose connected mode.
Methylation reaction is carried out to mussel polysaccharide using the Hakomori methods of improvement.Weigh and fully dried through phosphorus pentoxide
The mg of polysaccharide component 2 after 48 h, adds anhydrous DMSO, in N2The h of magnetic agitation 1 makes sample fully dissolve in atmosphere.Rapidly join
Dry NaH powder, continues the h of stirring reaction 1.The mL of iodomethane 1 is added dropwise, in N2The h of lucifuge stirring reaction 1.5 in atmosphere, so
Afterwards plus the mL terminating reactions of pure water 1.With the multiple extractive reaction liquid of chloroform, merge chloroform layer, be dried under reduced pressure and produce the polysaccharide that methylates.
Mussel polysaccharide is methylated into sample using the complete sour water solutions of 2 mol/L TFA, through boron deuterate sodium reduction, with pyridine and
GC-MS analyses are carried out after acetic anhydride generation acetyl derivatives.Mass spectral analysis condition is：Gas chromatographic column：Agilent
DB-225（0.25 mm×30 m×0.25 µm）；Carrier gas：Helium；Flow rate of carrier gas：1.0 mL/min；Injector temperature：250
℃；Detector：Mass detector（MSD）；Sample size：1 µL.
The total ion current figure that mussel polysaccharide methylated derivative carries out GC-MS analyses is as shown in figure 11.Can from figure
Go out, the main appearance at 21.48 min, 24.42 min, 25.13 min and 27.82 min of mussel polysaccharide.Controlled with reference to U.S.'s assistant
Sub- university's saccharide complex research center（Complex Carbohydrate Structure Database, CCSD）GC/MS
Database, it is as follows that the chromatographic peak in the sample GC-MS that methylated to mussel polysaccharide analyses carries out interpretation of mass spectra ownership.
Chromatographic peak at 21.48 min, is-O- acetyl group -1- the deoxidations -2,3 of 1,5- bis-, 4,6- tetra--O- methyl Ds-Portugal
Grape sugar alcohol（Figure 12）, by end Glc- (1 → generations.
Chromatographic peak at 24.42 min, is 1,2,5- tri--O- acetyl group -1- deoxidations -3, the 4,-O- methyl Ds of 6- tri--Portugal
Grape sugar alcohol（Figure 13）, produced by → 2)-Glcp- (1 →.
Chromatographic peak at 25.13 min, is the Isosorbide-5-Nitrae,-O- acetyl group -1- deoxidations -2,3 of the 5- tri-,-O- methyl Ds of 6- tri--Portugal
Grape sugar alcohol（Figure 14）, produced by → 4)-Glcp- (1 →.
Chromatographic peak at 27.82 min, is-O- the methyl Ds of 1,2,4,5- tetra--O- acetyl group -1- deoxidations -3,6- bis--Portugal
Grape sugar alcohol（Figure 15）, it is by → 2,4)-Glc- (1 → produce.
Attribution analysis is carried out to the GC-MS results of mussel polysaccharide, find to mainly contain in mussel polysaccharide end → (Glc, →
4)-Glc- (1 →, → 2)-Glc- (1 → and → 2,4)-Glc- (1 → etc. connected mode.Each signal is integrated, Ke Yiji
Calculate mole composition of different glycosidic bond connected modes in mussel polysaccharide（Table 2）.
The methylation analysis results of the mussel polysaccharide of table 2
According to mussel polysaccharide methylate sample derive through boron deuterate sodium reduction and acetylation after GC-MS analysis results, can be true
The connected mode for determining mussel polysaccharide is mainly → 4)-Glcp- (1 →, also containing → 2)-Glcp- (1 → and → 2,4)-Glcp- (1
→ etc. connected mode, this matches with its 2D-NMR analysis result.The mol ratio of three kinds of glycosidic bond connected modes is about successively
11:1:1。
The chemical constitution of 2.7 mussel polysaccharides
The chemical analysis and ultraviolet spectra of comprehensive mussel polysaccharide（UV）, infrared spectrum（IR）, NMR spectrum（NMR）And makings
Combination analysis（GC-MS）Deng modern instrumental analysis result, it may be determined that mussel polysaccharide is that a kind of purity is higher, with（1→4)-
α-D-Glc are main chain, are contained（Have 1 in α-pyranoid form glucan of 1 → 2)-α-D-Glc branches, and average every 12 glucose
The glucose branch of individual end.
The mussel polysaccharide blood fat reducing function of embodiment 3 is studied.
3.1 experimental animal
Male SD rat is selected, experimental animal is pleased by Jinan friend Co., Ltd's offer, animal productiong licensing number is provided：SCXK (Shandong)
20140007.Animal quality certification number：No.37009200002065, the g of body weight 170 ± 10.
3.2 test method
It is randomly divided into 6 groups, respectively blank group, model group, L1 groups（100 mg/kg）, L2 groups（200 mg/kg）, L3 groups（400
mg/kg）, L4 groups（800 mg /kg）.Whole rats feed basal feed and observed 1 week, are weighed after the h of fasting 12, the intraocular corner of the eyes takes
Blood, 25 DEG C of 1 h of standing of room temperature, 3000 rpm centrifuge 10 min, take serum, with automatic biochemistry analyzer measure TC, TG, HDL-C
With LDL-C levels.Blank group all the time feed by basal feed, and remaining each group rat is fed with high lipid food（High lipid food is formulated
It is respectively 5% for cholesterol 1%, cholate 0.1%, lard 10%, yolk powder and whole milk powder, being purchased from the magnificent Fukang biotechnology in Beijing has
Limit company）.The intraocular corner of the eyes takes blood after 4 weeks（Take the h of fasting 12 before blood）, determine serum TC（T-CHOL）, TG（Triglycerides）,
HDL-C（HDL-C）And LDL-C（LDL-C）Level.After being fed 4 weeks with high lipid food
Each group rat blood serum total cholesterol level and triglyceride levels are compared with blank group significant difference（p<0.05）, show fat
Metabolic disorder model is successfully established.
After hyperlipoidemia is successfully established, blank group and model group daily gavage distilled water 1 mL, L1, L2, L3, L4
Group is respectively according to the daily gastric infusion of corresponding dosage 1 time, continuous 8 weeks.Being administered after 8 weeks Rat Septal curfew eats 12 h, take blood and
Liver, determines serum TC, TG, HDL-C and LDL-C levels.
3.3 statistical analysis
Measurement data is represented with x ± SD, is analyzed using the software statistics of SPSS 13.0.If the variance of measurement data is neat, using list
Analysis of variance LSD inspections are compared between being organized；If the heterogeneity of variance of measurement data, examined using two independent sample nonparametrics
Test.P<0.05 thinks statistically significant.
3.4 result of the test
In administration process after hyperlipidemia model foundation, continuous use 8 weeks is compared with model group, all dosage groups of mussel polysaccharide
Rat blood serum triglyceride levels can be significantly reduced（P<0.01）, wherein 400 mg/kgd, 800 mg/kgd dosage groups
It can obviously reduce serum total cholesterol and LDL-C content（P<0.05）（Table 3）.Prompting mussel polysaccharide has aobvious
Hypolipemic function is write, serum glycerine can be reduced under the conditions of a reduction serum triglyceride level, high dose under the conditions of low dosage
Three esters, T-CHOL and low-density lipoprotein cholesterol level.
Serum TC, TG, HDL-C, LDL-C that front and rear each group rat is administered in table 3 compare (mmol/L)
Note：N=10,# #RepresentP<0.01, compared with normal group；* representP<0.05, * * is representedP<0.01 is compared with model group
The mussel polysaccharide acute toxicity test of embodiment 4.
4.1 experimental animal
Kunming kind（KM）Mouse, SPF grades, 40, male and female half and half please experimental animal by Jinan friend and breed Co., Ltd's production, close
Lattice card number：37009200000705, experimental animal production licence number：SCXK（Shandong）2014-0007.Weight range is 18.4-
22.0 g。
4.2 test method
Mouse point sex, experimental group and control group, every group 20, male and female half and half are randomly divided into by body weight.0.6 g/mL is prepared to make a gift of
Gastric infusion 2 times in pattra sugar juice, experimental group one day, each mL/kg of administered volume 40 is spaced about 10 h, total dosage
For 48 g/kgd.Control group gives pure water with method gavage.
At once the response situation of animal is observed after administration, poisoning symptom, appearance sign and the poisoning symptom of animal is recorded
Time of occurrence, duration and recovery situation etc..The administration same day answers close observation, and then daily morning observation is once, continuous to see
Examine 14 days, the 3rd before medicine, after medicine, respectively claim a body weight within 7,10,14 days.Body weight value is represented with x ± SD, is carried out with DAS1.0 softwares
T is examined and compared between group, to observe the influence that medicine increases to the weight of animals.
4.3 result of the test
The 3rd after medicine, 7,10,14 days experimental groups and control group body weight it is basically identical（Table 4）.Mouse cardinal symptom is shown as after medicine about
Occur substantially recovering in the reduction of mouse number of activities, about 20 min in 2 min, other observations are shown no obvious abnormalities.Observation terminates
Animal cut open inspection is put to death afterwards, and the main organs such as brain, the heart, liver, spleen, lung, kidney do not have the visible exception of naked eyes.
Mussel polysaccharide mouse maximum dosage-feeding is 48 g/kgd, is approximately equivalent to people plan dosage (16 mg/kg daily
d）3000 times, the nontoxic level of acute toxicity classification category.
Influence (x ± SD, n=20) of the mussel polysaccharide gastric infusion of table 4 to mouse weight
In summary, the mussel polysaccharide of doses can significantly reduce triglycerides and low-density lipoprotein courage in rat blood serum
The content of sterol；Rat blood serum triglyceride levels can be significantly reduced, serum total cholesterol and low-density lipoprotein is significantly reduced
Cholesterol level；The nontoxic level of mussel polysaccharide acute toxicity classification category.
Claims (5)
1. a kind of mussel polysaccharide, it is characterised in that with（1 → 4)-α-D-Glc be main chain, contain（1 → 2)-α-D-Glc branches,
It is configured as α-pyranoid form glucan, has 1 in average every 12 grape sugar backbones（1 → 2) glucose branch, its structural formula is：
；
The mussel polysaccharide relative molecular weight is 800-8000 KDa.
2. mussel polysaccharide according to claim 1, it is characterised in that extract from mussel category（Mytilus）Animal, including thickness
Shell mussel（Mytilus coruscus）And Mytilus galloprovincialis（Mytilus edulis）.
3. a kind of extracting method of mussel polysaccharide as claimed in claim 1, it is characterised in that comprise the following steps：
（1）Feedstock treating：Mussel heating, which is shelled, to be stayed meat and dry, pulverize machine crushing, is crossed 20-40 mesh sieves, is obtained mussel dry powder；
（2）Polyose extraction：Step（1）Added in obtained mussel dry powder at the water of 6 times of volumes, 100 DEG C and extract 2 h；Cross elimination
Except mussel meat slag, polysaccharide extraction liquid is obtained；
（3）Enzymolysis：By step（2）Obtained polysaccharide extraction liquid adjusts pH to 8.0, adds 0.2 %w.t alkali protease, 50 DEG C
Digest and inactivate 15-30 min after the completion of 2 h, enzymolysis under the conditions of 80 DEG C, obtain enzymolysis liquid；Using diatomite as filter aid, suction filtration mistake
Filter, the enzymolysis liquid clarified；
（4）Ethanol precipitation：In step（3）1.5 times of 90-95% are added in obtained polysaccharide solution（v/v）Ethanol precipitated,
6 h are stood, incline supernatant, obtains polysaccharide precipitation；
（5）Dehydration：In step（4）1 times of volume 90-95% is added in obtained polysaccharide precipitation（v/v）Ethanol be dehydrated；
（6）Filtration drying：The polysaccharide precipitation after dehydration is filtered with 400 eye mesh screens, precipitation is collected, 60 DEG C are dried in vacuo,
Obtain Thick many candies；
（7）Weight is molten：By step（6）Gained Thick many candies are dissolved in water, prepare 10% mussel Thick many candies solution, and 12000 rpm centrifuge 1 h,
By supernatant with 0.45 μm of membrane filtration, polysaccharide filtrate is obtained；
（8）Ion-exchange chromatography：By step（7）Polysaccharide filtrate use DEAE- agaroses（FF）Column chromatography, with 0.05
Mol/L NaCl solutions are eluent, collect elution fraction；
（9）Ultrafiltration：With the filter membrane that molecular cut off is 5000 to step（8）Gained elution fraction carries out ultrafiltration desalination and concentration,
Obtain concentrate；
（10）Freeze-drying：By step（9）Gained concentrate is freeze-dried, and obtains mussel polysaccharide finished product.
4. a kind of mussel polysaccharide as claimed in claim 1 is being prepared for treating or preventing answering in cardiovascular and cerebrovascular diseases medicament
With, it is characterised in that affiliated cardiovascular and cerebrovascular disease includes hyperlipemia, atherosclerosis.
5. a kind of mussel polysaccharide as claimed in claim 1 is preparing health products or special medicine purposes with blood fat reducing function
Application in formula food.
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| CN111139275A (en) * | 2019-12-31 | 2020-05-12 | 山东省药学科学院 | Improved method for preparing mussel oligosaccharide by using yeast |
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