CN102898538B - Abalone viscera acidic polysaccharose and health-care product containing abalone viscera acidic polysaccharose and application of abalone viscera acidic polysaccharose - Google Patents
Abalone viscera acidic polysaccharose and health-care product containing abalone viscera acidic polysaccharose and application of abalone viscera acidic polysaccharose Download PDFInfo
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Abstract
The invention provides abalone viscera acidic polysaccharose. According to the abalone viscera acidic polysaccharose, a main chain comprises rhamnose and glucuronic acid, wherein a C-2 position or C-3 position of the rhamnose in the main chain is subjected to sulphating; and the rhamnose in the main chain is connected with the rhamnose or galactose to form a branched chain structure. The abalone viscera acidic polysaccharose which is extracted from abalone viscera can promote the proliferation of hepatoma carcinoma cells (HepG2) in vitro obviously, and has a certain effect of dose dependency; and experiments of studying a repair effect of the abalone viscera acidic polysaccharose on the HepG2 cells which are subjected to oxidative damage and on rat hepatic tissues which are subjected to acute hepatic damage caused by carbon tetrachloride in vivo indicate that the abalone viscera acidic polysaccharose can be used as a functional factor for protecting liver.
Description
Technical field
The present invention relates to a kind of polysaccharide, relate in particular to a kind of from abalone internal organ extraction novel acid polysaccharide.
Background technology
Bao is subordinate to Mollusca (Mollusca), Gastropoda (Gastropoda), Probranchia (Prosobranchia), Archaeogastropoda (Archaeogastropoda), Bao Ke (Haliotidae), Bao genus (Haliotis), named abalone has 216 kinds to each sea area, the whole world, approximately 30 kinds of frequent specieses, distribute and mainly contain 7 kinds at coastal area of china.Haliotis discus hannai Ino and Haliotis diversicolor are respectively northern China and southern Representative Cultivars, and haliotis discus hannai Ino economic worth is higher compared with Haliotis diversicolor, and the abalone being produced with Shandong coastal waters is the most famous, are called " stepping on Lay abalone " in history.Abalone internal organ account for 15 ~ 25% of abalone gross weight, as the waste in the abalone course of processing, are dropped more or make feed, not only to environment, have also reduced the value of abalone simultaneously,
Carbohydrate is a member the most colourful in composition biopolymer family, 300 unnecessary kinds of polysaccharide compounds now from natural product, are isolated, they are present in each kind of plant, animal and microorganism cultures widely, have many important functions.Polysaccharide is the macromole active compound being polymerized by multiple monose or derivatives thereofs, is the requisite compositions of all lived organisms, closely related with maintaining biological function.Polysaccharide is nonspecific immunomodulator, has the biologic activity such as antitumor, anti-inflammatory, anticoagulation, antiviral, radioprotective, hypoglycemic, reducing blood-fat, and polysaccharide becomes one of important directions of current functional foodstuff exploitation just gradually.Polysaccharide starts from 1934 as medicine, and after the sixties, polysaccharide has caused the great interest of people as wide spectrum immunopotentiating agent.The eighties finds that again the sugar chain of polysaccharide has conclusive effect in molecular biology, can control cell fission and differentiation, regulates growth and the aging of cell.Recent two decades comes, and due to the development of molecular biology and cytobiology, finds that polysaccharide and conjugate participate in the various biological phenomenas of cell, and it is broken as the traditional concept of sustentacular tissue and energy derive.
Abalone internal organ acidic polysaccharose is abalone internal organ chief component composition, its complex structure, and have difference because of the difference of abalone species and growing environment.In recent years, Zhu Beiwei etc. have extracted abalone internal organ acidic polysaccharose ACPI and ACPII from abalone internal organ, and both,, all by being made up of rhamnosyl, Fucose, wood sugar, semi-lactosi, seminose, prove that it has antitumor, improve immunity of organisms, the multiple physiologically active such as anti-oxidant.
Summary of the invention
Technical problem to be solved by this invention is to be to provide a kind of novel abalone internal organ acidic polysaccharose, described polysaccharide is the polysaccharide extracting from abalone internal organ, it can obviously promote HepG2 cell proliferation outside human body or animal body, and can repair the HepG2 cell of oxidative damage, in human body or animal body, can improve the liver function situation that the medicines such as tetracol phenixin cause acute liver damage, thus can be for the reparation after injuries of tissues and organs.
The invention provides a kind of abalone internal organ acidic polysaccharose, described polysaccharide is mainly made up of rhamnosyl, glucuronic acid, semi-lactosi.
Wherein, the main chain of described abalone internal organ acidic polysaccharose is mainly made up of rhamnosyl and glucuronic acid, and the C-2 position of the rhamnosyl in main chain or C-3 position are by sulphating, thereby the rhamnosyl in described main chain is connected with rhamnosyl or semi-lactosi forms branched structure.
Wherein, the main chain of described abalone internal organ acidic polysaccharose mainly contain → 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(3SO
4)-(1 → (I) and → 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(2SO
4)-(1 → (II) two kinds of structural units, the ratio of described two kinds of structural units is 2:1, in main chain I, on sulfation rhamnosyl C-2, be connected with side chain α-L-Rhap, the C-3 position of the sulfation rhamnosyl of main chain II is connecting side chain α-L-Rhap-(4SO4)-(1 → 3)-β-D-Galp, the main structure of described polysaccharide is shown in formula 1
Formula 1.
Wherein, the molecular-weight average of described polysaccharide is 32.5Kda.
The present invention also provides the extracting method of above-mentioned polysaccharide, separation purification method and Structural Identification method.
It is a kind of for promoting medicine or the healthcare products of hepatocellular propagation that the present invention also provides, and it comprises above-mentioned abalone internal organ acidic polysaccharose.
The invention provides a kind of medicine for acute liver damage in dummy or healthcare products, it comprises above-mentioned abalone internal organ acidic polysaccharose.
The present invention also provides the application of above-mentioned abalone internal organ acidic polysaccharose in medicine or healthcare products for the preparation of the hepatocellular propagation of promotion.
The present invention also provides the application in medicine or the healthcare products of above-mentioned abalone internal organ acidic polysaccharose acute liver damage in for the preparation of dummy.
The technique effect that the present invention is useful is:
The abalone internal organ acidic polysaccharose that the present invention extracts from abalone internal organ can significantly promote the propagation of HepG2 cell in vitro, and its effect is certain dose-dependently, in the repair of the HepG2 cell by research abalone internal organ acidic polysaccharose to oxidative damage and body, tetracol phenixin is caused to Liver of Rats with Acute Liver Injury repair experiment and show, abalone internal organ acidic polysaccharose can be used as a kind of functional factor of liver protecting.
Brief description of the drawings
The primary structure unit of accompanying drawing 1 abalone internal organ acidic polysaccharose;
The high performance liquid phase separating spectrum of accompanying drawing 2 abalone internal organ acidic polysaccharose acid hydrolysis things;
The infrared spectra of accompanying drawing 3 abalone internal organ acidic polysaccharoses;
Accompanying drawing 4 abalone internal organ acidic polysaccharoses
1h NMR;
The DEPT spectrogram of accompanying drawing 5 abalone internal organ acidic polysaccharoses;
The part HMQC collection of illustrative plates of accompanying drawing 6 abalone internal organ acidic polysaccharoses;
Accompanying drawing 7 abalone internal organ acidic polysaccharoses
1h-
1h COSY spectrogram;
The proliferation function of the external HepG2 cell of accompanying drawing 8 abalone internal organ acidic polysaccharose;
Accompanying drawing 9 abalone internal organ acidic polysaccharoses on carbon tetrachloride-injured after the impact of rat blood serum ALT and AST;
Accompanying drawing 10 abalone internal organ acidic polysaccharoses on carbon tetrachloride-injured after the impact of rat liver content of triglyceride;
Accompanying drawing 11 abalone internal organ acidic polysaccharoses to carbon tetrachloride-injured after rat liver HE coloration result (400 ×).
Embodiment
The invention provides a kind of acidic polysaccharose extracting from abalone internal organ, described abalone internal organ acidic polysaccharose is mainly made up of rhamnosyl, glucuronic acid, semi-lactosi.
The main chain of described polysaccharide comprises rhamnosyl and glucuronic acid, and the C-2 position of the rhamnosyl in main chain or C-3 position are by sulphating.
Further, the main chain of described abalone internal organ acidic polysaccharose is only made up of rhamnosyl and glucuronic acid.
The rhamnosyl in described main chain is connected with rhamnosyl or thereby semi-lactosi forms branched structure.
The main chain of described polysaccharide mainly contains → and 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(3SO
4)-(1 → (I) and → 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(2SO
4)-(1 → (II) two kinds of structural units, the ratio of described two kinds of structural units is 2:1, in main chain I, on sulfation rhamnosyl C-2, be connected with side chain α-L-Rhap, the C-3 position of the sulfation rhamnosyl of main chain II is connecting side chain α-L-Rhap-(4SO4)-(1 → 3)-β-D-Galp, the main structure of described polysaccharide is shown in formula 1
Formula 1.
Above-mentioned abalone internal organ acidic polysaccharose provided by the invention is the polysaccharide extracting from abalone internal organ.
The molecular-weight average of described abalone internal organ acidic polysaccharose is 32.5Kda.
The present invention also provides extracting method, separation purification method and the authentication method of above-mentioned abalone internal organ acidic polysaccharose.
It is a kind of for promoting medicine or the healthcare products of hepatocellular propagation that the present invention also provides, and it comprises above-mentioned abalone internal organ acidic polysaccharose.
The invention provides a kind of medicine for acute hepatocellular injury in dummy or healthcare products, it comprises above-mentioned abalone internal organ acidic polysaccharose.
The present invention also provides above-mentioned abalone internal organ acidic polysaccharose in the application for the preparation of promoting in medicine or the healthcare products of hepatocellular propagation.
The present invention also provides the application in medicine or the healthcare products of above-mentioned abalone internal organ acidic polysaccharose acute liver damage in for the preparation of dummy.
Below adopt embodiment and accompanying drawing to describe embodiments of the present invention in detail, to the present invention, how utilisation technology means solve technical problem whereby, and the implementation procedure of reaching technique effect can fully understand and implement according to this.
Embodiment 1: extraction and the separation and purification of abalone internal organ acidic polysaccharose, carry out successively following steps:
1), by dry 100g abalone internal organ pulverize thing, add the mixing solutions of the mixing chloroform-methanol (V:V, 2:1) of 2000mL, soak 12h, filter, filter residue dry.
2), get step 1) and dry gained abalone internal organ powder, add 3000mL cocktail buffer, and add 10g papoid, stirring reaction 24h under 60 DEG C of water-baths, the preparation method of this mixing buffered soln is as follows: in every liter of 0.1M acetic acid-sodium acetate buffer solution (pH6.0), add the EDTA of 5mmol and the halfcystine of 5mmol.
3), by step 2) centrifugal (2000g of enzymolysis after product of gained, 15min, 20 DEG C), be 10% cetylpyridinium chloride (CPC) aqueous solution to adding 80mL mass concentration in gained supernatant liquor, under room temperature, place after 24 hours, centrifugal (2000g, 15min), abandoning supernatant.
4), by step 3) (concentration of NaCl is 3mol/L for the aqueous ethanolic solution of the NaCl that is precipitated and dissolved in 1500mL of gained, ethanol: water=100:15, v/v) in, add again 3000mL 95% (volumetric concentration) ethanolic soln, place 24 hours for 4 DEG C, centrifugal (2000g, 15min), precipitation uses respectively 100mL80% and 95% (being volumetric concentration) ethanolic soln to wash 2 times, finally will be deposited in 60 DEG C and be dried 2 hours, dissolve with distilled water, it is the dialysis tubing desalination of 3500Da with the molecular weight that dams, concentrated, freeze-drying (in-62 DEG C), obtain abalone internal organ Crude polysaccharides 7.6g, extraction yield is about 15.2%.
5), by abalone internal organ Crude polysaccharides through DEAE-52 anion-exchange column and S-200 gel-filtration column separating purification, concrete separating step is as follows:
About 1.0g abalone internal organ Crude polysaccharides is dissolved in to about 30mL 0.1M NaAc-HAc(pH 6.0) buffered soln, cross DEAE-52 anion-exchange column (2.6 × 20cm), adopt 0-1.2mol/L NaCl buffer salt solution (0.1M NaAc-HAc, pH6.0) to carry out gradient elution, collect cut 5mL/ pipe, with phenol sulfuric acid and Folin-phenol mensuration polysaccharide and protein content, draw elution curve, collect sample according to elution curve, flowing water dialysis 24h, distill water dialysis 24h, freeze-drying.
The about 100mg of polysaccharide that gets above-mentioned separation is dissolved in 1mL ultrapure water, cross Sephacryl S-200 gel column (1.6 × 100cm), adopt 0.2mol/L NaCl to carry out wash-out, collect cut 2min/ pipe, according to the polysaccharide content in phenolsulfuric acid method detector tube, collect peak point sample, flowing water dialysis 24h, distill water dialysis 24h, freeze-drying, obtains abalone internal organ acidic polysaccharose sterling.Gained polysaccharide detects purity and molecular weight by liquid phase TSK4000 pillar, and result shows that the molecular-weight average of abalone internal organ acidic polysaccharose is about 32.5KDa, and purity is 99.1%.
Embodiment 2 carries out structure elucidation to the abalone internal organ acidic polysaccharose of embodiment 1 gained
1), get above-described embodiment 1 gained abalone internal organ acidic polysaccharose 2.0mg in ampere bottle, add 1mL 2molL-1TFA, inflated with nitrogen tube sealing, is hydrolyzed 8h at 110 DEG C.Be cooled to subsequently room temperature, at the temperature of 50 DEG C, volatilize TFA, be successively adjusted to neutrality with 2molL-1,0.3molL-1NaOH solution respectively, ultrapure water is settled to 1mL, gets 450 μ L 50 μ L internal standard substance lactose and carries out PMP derivatize.Chromatographic condition: chromatographic instrument: Agilent 1100 high performance liquid chromatographs, chromatographic column: ZORBAX EclipseXDB-C18 separator column (4.6 × 150mm, 5 μ m), detector: UV-detector, 245nm, flow velocity: 1.0mLmin-1, column temperature: 25 DEG C, moving phase: solvent orange 2 A: 10% (V/V) acetonitrile+0.1molL-1 ammonium acetate-acetate buffer (pH 5.5), solvent B:25% (V/V) acetonitrile+0.1molL-1 ammonium acetate-acetate buffer (pH 5.5), gradient mode: time gradient: 0 → 40min, concentration gradient: 45 → 100% solvent B, sampling volume: 10 μ L.See Fig. 2, result shows that the monose of abalone internal organ acidic polysaccharose consists of rhamnosyl (Rha), glucuronic acid (GlcA) and semi-lactosi (Gal), and its ratio is 6.40:3.69:1.0.
2), get above-described embodiment 1 gained abalone internal organ acidic polysaccharose 2mg, add 1mL concentration 2molL
-1at the temperature that TFA is 110 DEG C, be hydrolyzed 10h, 80 DEG C of left and right volatilize TFA, with ultrapure water ultrasonic dissolution, constant volume is to 25mL, get 2.5mL and adopt ion-chromatographic determination sulfate content, make standard substance Criterion curve with potassium sulfate, result shows that the sulfate of the abalone internal organ acidic polysaccharose obtaining is 15.2%.
3), above-described embodiment 1 gained abalone internal organ acidic polysaccharose and KBr compacting in flakes, use Vector22 type Fourier infrared spectrograph, scanning 4000～400cm
-1the spectral absorption value of wave-number range.The results are shown in Figure 3, result shows that abalone internal organ acidic polysaccharose is at 3600-3200cm
-1all there is a wide O-H stretching vibration peak, at 2936cm
-1near absorption peak is the absorption peak of rhamnosyl methyl, at 1641cm
-1and 1389cm
-1neighbouring (carboxyl C=O is flexible, and C-O is flexible) located absorption, illustrates in abalone internal organ acidic polysaccharose and contains uronic acid, and this is consistent with monose compositional analysis result, the stretching vibration peak of 1231(S=O) and 843cm
-1(stretching vibration peak of C-O-S) located strong absorption, further shows that abalone internal organ acidic polysaccharose is rich in sulfate; 1051cm
-1locate the stretching vibration that strong absorption peak is C-O-C or C-O-H.
4), get the abalone internal organ acidic polysaccharose 50mg of gained in above-described embodiment 1, with 0.5mL D
2o (concentration is 99.96%) exchanges 2 times continuously, through 0.5mL D
2after O (concentration is 99.96%) dissolves, by Bruker AVANCE III 600 nuclear magnetic resonance spectrometers mensuration
1h NMR, DEPT,
1h-
1h COSY, TOCSY, NOESY,
1h-
13c HMQC, HMBC.Condition determination: 60 DEG C, 600MHz; Interior mark: DSS.For sugar compounds,
1proton (H-1) signal in H NMR spectrum (Fig. 4) on C-1, conventionally at δ 4.5～5.6ppm, is more easily resolved, and the proton signal of other C-2～C-6 all concentrates on δ 3.2～4.8ppm, and the intersection that overlaps each other is resolved difficulty.At should be-CH of δ 1.1ppm left and right
3, in abalone internal organ acidic polysaccharose, the fignal center at this place is higher, and in conjunction with monose composition, this place should be Rha-CH
3proton.Concentrate on the proton signal that δ 3.2～4.6ppm is C-2～C-6, its intersection that overlaps each other, resolves difficulty.Chemical shift should be β-D-GlcUA at the 1H of 4.60ppm left and right
1h, chemical shift is at the anomer hydrogen chemical shift data that should be α configuration rhamnosyl of the fignal center of 5.0ppm and 5.32ppm left and right.
In the DEPT spectrum (Fig. 5) of abalone internal organ acidic polysaccharose, there is 1 carbon signal (δ 16.85) in its high field region (δ 0～50), is Rha-CH
3carbon signal.Between δ 65～78, fignal center is comparatively complicated, is difficult to divide, and should be C-2, C-3, the chemical shift of C-4, should be C-2 at the carbon signal of δ 78.59, C-3, in C-4, on certain carbon, having substituting group, is secondary carbon at the fignal center at δ 63.2 places, should be attributed on Gal-C-2 explanation C-6 replacement has occurred; The fignal center occurring between δ 98～106 is anomeric carbon district, and this district's signal is more complicated, and more difficult definite anomeric carbon number needs further to identify by two-dimensional spectrum figure.
The HMQC spectrum anomeric carbon district signal (Fig. 6) of abalone internal organ acidic polysaccharose determines and in abalone internal organ acidic polysaccharose, mainly contains 7 kinds of sugar units, and determines 7 kinds of anomer hydrogen signals according to anomeric carbon signal is corresponding, in conjunction with
1h-
1h TOCSY composes (Fig. 7), from
1h-
1in H COSY, anomer hydrogen signal sets out, and determines in turn H-2, H-3, and H-4, H-5, H-6 signal, determines the chemical shift of each sugar unit hydrogen, then according to HMQC spectrum, the carbon signal of each sugar unit is belonged to, shown in Table.
It is methyl that the anomer hydrogen chemical shift of saccharide residue A-D is greater than 4.80, C-6 position, illustrates that A-D is α configuration rhamnosyl (α-L-Rha); It is lower that the anomer hydrogen chemical shift of saccharide residue F, G is less than 4.80, H-2 chemical shift, is illustrated as β-D-GlcA; Saccharide residue E should be β-D-Gal.For the position of substitution ownership of sulfate group, due to sulphating can cause carbon and hydrogen signal all can be to low mobile saccharide residue A-4 (C4/H4,80.1/4.64), B-2 (C2/H2,80.3/4.27), D-3 (C3/H3,4.53/80.7), A is described, B, D is respectively α-L-Rha (4SO
4), α-L-Rha (2SO
4), α-L-Rha (3SO
4).
Further by
1h-
1h NOESY and
1h-
13between C HMBC heteronuclear
1h-
1h and
1h-
13c coupling coherent signal (table 2) is determined mode of connection and the order of connection between saccharide residue.It is as shown in the table, can observe anomer hydrogen and the coherent signal that is connected saccharide residue position connection hydrogen by NOE signal, that is: A H-1/E H-3, B H-1/G H-3, C H-1/D H-2, D H-1/F H-3, E H-1/B H-3, F H-1/D H-4, G H-1/BH-4, has determined that the mode of connection between residue is, A (1 → 4) E, B (1 → 3) G, C (1 → 2) D, D (1 → 3) F, E (1 → 3) B, F (1 → 4) D, G (1 → 4) B.
1h-
13c HMBC has further determined that NOESY composes determined information, and comprises anomeric carbon simultaneously and be connected saccharide residue position and connect the coherent signal of hydrogen, and anomer hydrogen and the coherent signal that is connected saccharide residue position connection carbon are as shown in table 2.Combined sulfur acidic group replaces mode of connection and the order of connection between site and definite saccharide residue, finally determines that abalone internal organ acidic polysaccharose is mainly made up of following two structural unit I and II, and according to
1the integration of corresponding anomer hydrogen signal ratio in H NMR, the ratio of determining two structural units is 2:1.
Table 1 abalone internal organ acidic polysaccharose
1h and
13the chemical shift ownership of C
The ownership of table 2 abalone internal organ acidic polysaccharose NOESY spectrum and HMBC spectrum
The above analysis can draw the following conclusions:
Therefore, the primary structure unit of the abalone internal organ acidic polysaccharose in the present invention, as shown in Fig. 1 Chinese style 1, mainly comprises two kinds of structural units of I and II, and its ratio is 2:1, is to produce from Qingdao the acidic polysaccharose that separates a kind of novel structure obtaining abalone internal organ first.
Embodiment 3 abalone internal organ acidic polysaccharoses promote cell-proliferation activity
1), mtt assay detect polysaccharide to the proliferation function of HepG2 cell by the HepG2 cell in logarithmic phase, making density with RPMI – 1640 perfect mediums (contain 10% new-born calf serum) is 2 × 10
4individual mL
-1cell suspension, be inoculated in 96 well culture plates 100 μ L/ holes.After 24h is adherent, be replaced with concentration and be respectively 25 μ gmL
-1, 50 μ gmL
-1, 100 μ gmL
-1, 200 μ gmL
-1with 400 μ gmL
-1polysaccharide, each concentration is established 4 multiple holes, 200 μ L/ holes.Blank group adds the perfect medium of equivalent.37 DEG C, 5%CO
2under condition, cultivate respectively after 24h, 48h and 72h, discard substratum, (preparation of PBS liquid, final concentration is 0.5mgmL to add MTT liquid
-1).Continue to cultivate after 4h, suck supernatant liquor, add acidifying Virahol, piping and druming to blue crystallisate dissolves completely, and microplate reader is surveyed the each hole of 570nm absorbancy (A).MTT/%=A
570(sample sets)/A
570(blank group) × 100%.
2), EdU method detects the proliferation index of polysaccharide to HepG2 cell
Cell fabric swatch and culture condition are the same.Add after sample cultivation 48h, abandon substratum, every hole adds the EdU substratum of 100 μ L50 μ M to hatch 2h, abandons nutrient solution, and PBS cleans cell 2 times, each 5min.Then every hole adds the PBS cell stationary liquid incubated at room 30min of 100 μ L containing 4% paraformaldehyde, cell is fixed, dye through EdU afterwards, under fluorescent microscope, take pictures, specifically carry out programming and experimental implementation according to IX-51 type inverted fluorescence microscope system and the explanation of Edu fluorescent microscope detection kit.
See Fig. 8, in figure, a is that MTT measures the external impact on HepG2 cell proliferation, figure b is that Edu method is measured the proliferation index to HepG2 cell, MTT experiment and EdU experimental result show, compared with control group, the cell survival rate of abalone internal organ acidic polysaccharose obviously increases, and presents certain dose-effect relationship, illustrates that it has good cultivation effect.
Embodiment 4 repairs of abalone internal organ acidic polysaccharose to liver injury
1), the hepG2 cytothesis effect of external abalone internal organ acidic polysaccharose to oxidative damage
The HepG2 cell of taking the logarithm vegetative period, through 0.25% trysinization, making density with RPMI perfect medium (containing 10% new-born calf serum) is 1 × 10
5the cell suspension of individual/mL, adds 96 well culture plates, every hole 100 μ L.Cultivate after 24h, add 200 μ L tertbutyl peroxides (t-BHP), t-BHP activity is 200 μ mol/L, and control group adds the serum free medium of equivalent, cultivate 4h, discard nutrient solution, PBS cleans 2-3 time, abandons nutrient solution, add the abalone internal organ acidic polysaccharose of 200 μ L different concns, control group and model group add the substratum (containing 2% serum) of equivalent, hatch after 24h, and mtt assay detects cell survival rate.
Experimental data represents with x ± SE, adopts SPSS11.0 software to carry out ONE WAY ANOVA and analyzes, taking P<0.05 as there were significant differences.
The oxidative damage of the HepG2 cell that experimental result demonstration abalone internal organ acidic polysaccharose causes tertbutyl peroxide has significant repair.
The repairing effect of table 3 abalone internal organ acidic polysaccharose to oxidative damage HepG2 cell
| Normal group | Model group | 50ug/mL | 100ug/mL | |
| Repair | 100±3.0 | 57.5±2.4# | 85.4±2.0 * | 110±3.0 ** |
*p < 0.05,
*p < 0.01, with model group comparison;
#p < 0.05, with normal group comparison
2), abalone internal organ acidic polysaccharose causes the repair of acute liver injury of rats to tetracol phenixin
Laboratory animal is SD rat, male, body weight 180g-200g, and purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., credit number: SCXK (capital) 2009-0007.Rat basal feed adaptability was fed after 1 week, was divided at random 4 groups by body weight: normal group, model group, abalone internal organ acidic polysaccharose low dose group and abalone internal organ acidic polysaccharose high dose group, 9 every group.Each group respectively at the corresponding tested material of gavage 9 o'clock every days.Abalone internal organ acidic polysaccharose low dose group and high dose group are respectively according to the dosage gavage abalone internal organ acidic polysaccharose of (100mg/kg), (300mg/kg), and normal and model group is distinguished the physiological saline of gavage same volume, continuous 5 days.Test the CCl that before gavage, 1h is 50% to model group, abalone polysaccharide dosage group abdominal injection volume fraction respectively the 1st day and the 4th day
4peanut oil, 2ml/kg B.W., the peanut oil of normal group injection same volume.
Test the 7th day, after etherization rat, aorta abdominalis is got whole blood and is put to death, and measures gpt (ALT), glutamic-oxal(o)acetic transaminase (AST) activity in serum.; Get lobus sinister hepatic tissue, 10% neutral formalin is fixedly made pathological section, carries out HE dyeing, changes at the pathology of light Microscopic observation hepatic tissue.Liver fat is pressed the method extracting of Folch etc., and liver TC and TG are by kit measurement.
See Fig. 9,10, gavage abalone internal organ acidic polysaccharose has suppressed the activity of liver injury indicator enzyme ALT and AST in serum effectively, and can improve impaired the followed fat of liver function and excessively accumulate.
See Figure 11, histological observation shows that normal rats liver is sorrel, quality softness; Under light microscopic, observe liver cell structural integrity, have no the obviously pathological change such as sex change, necrosis, liver lobule boundary is clear, and caryoplasm is evenly distributed.Model group rat liver volume increases, and model group central vein around obviously increases because of the gap that apoptosis produces, and dosage group is significantly improved.The apoptosis signs such as a large amount of karyopyknosises of model group, dissolving, dosage group is obviously improved, and in the section of high dose group, there is proliferative cell in particularly high dose group.So the acute liver damage tool that abalone internal organ acidic polysaccharose causes for tetracol phenixin has some improvement.
All above-mentioned these intellecture properties of primary enforcement, do not set restriction this product innovation of other forms of enforcement and/or novel method.Those skilled in the art will utilize this important information, and foregoing amendment, to realize similar implementation status.But all modifications or transformation belong to the right of reservation based on product innovation of the present invention.
The above, be only preferred embodiment of the present invention, is not the restriction of the present invention being made to other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (6)
1. an abalone internal organ acidic polysaccharose, is characterized in that: described polysaccharide is mainly made up of rhamnosyl, glucuronic acid, semi-lactosi;
The main chain of described polysaccharide is mainly made up of rhamnosyl and glucuronic acid, and the two or three-digit of the rhamnosyl in main chain is by sulphating, thereby the rhamnosyl in described main chain is connected with rhamnosyl or semi-lactosi forms branched structure.
2. abalone internal organ acidic polysaccharose as claimed in claim 1, is characterized in that: the main chain of described polysaccharide mainly contains → and 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(3SO
4)-(1 → (I) and → 3)-β-D-GlcpA-(1 → 4)-α-L-Rhap-(2SO
4)-(1 → (II) two kinds of structural units, the ratio of described two kinds of structural units is 2:1, in main chain I, on sulfation rhamnosyl C-2, be connected with side chain α-L-Rhap, the C-3 position of the sulfation rhamnosyl of main chain II is connecting side chain α-L-Rhap-(4SO4)-(1 → 3)-β-D-Galp, the main structure of described polysaccharide is shown in formula 1
3. abalone internal organ acidic polysaccharose as claimed in claim 1 or 2, is characterized in that: the molecular-weight average of described abalone internal organ acidic polysaccharose is 32500Da.
4. for promoting hepatocellular propagation or for repairing medicine or the healthcare products of acute liver damage in human body or animal body body, it is characterized in that, comprising: the abalone internal organ acidic polysaccharose described in claim 1 or 2.
5. the application of the abalone internal organ acidic polysaccharose described in claim 1 or 2 in medicine or healthcare products for the preparation of promotion human body or the hepatocellular propagation of animal body.
6. the application in medicine or the healthcare products of the acute liver damage in for the preparation of reparation human body or animal body body of the abalone internal organ acidic polysaccharose described in claim 1 or 2.
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| CN105087713A (en) * | 2015-09-01 | 2015-11-25 | 集美大学 | Preparation method of abalone viscera polysaccharide |
| CN107498067B (en) * | 2017-09-21 | 2019-10-18 | 厦门医学院 | The method that Bao internal organ degradation product green quickly prepares nano silver colloidal sol |
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| CN101724092A (en) * | 2009-12-23 | 2010-06-09 | 福州大学 | Method for extracting oxidation resistant water-soluble matter of abalone shell |
| CN102839149A (en) * | 2012-09-21 | 2012-12-26 | 中国海洋大学 | Application of abalone viscera polysaccharide-alternative serum in cell culture medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724092A (en) * | 2009-12-23 | 2010-06-09 | 福州大学 | Method for extracting oxidation resistant water-soluble matter of abalone shell |
| CN102839149A (en) * | 2012-09-21 | 2012-12-26 | 中国海洋大学 | Application of abalone viscera polysaccharide-alternative serum in cell culture medium |
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| Title |
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| 三种色谱法分析鲍鱼生殖腺多糖的单糖组成;宋叶涵等;《食品与机械》;20120331;第28卷(第2期);44－47页 * |
| 宋叶涵等.三种色谱法分析鲍鱼生殖腺多糖的单糖组成.《食品与机械》.2012,第28卷(第2期), |
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