CN104277133A - Extraction and purification method and application of fermented astragalus polysaccharide - Google Patents
Extraction and purification method and application of fermented astragalus polysaccharide Download PDFInfo
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Abstract
The invention provides an extraction and purification method and application of a fermented astragalus polysaccharide. The extraction and purification method of the fermented astragalus polysaccharide comprises the following steps of (1) carrying out warm immersion extraction on fermented astragalus, concentrating an extracting solution by using a membrane intercepting ultrafilter, removing protein through a Sevag method, and carrying out dialysis, alcohol precipitation and washing to obtain a crude fermented astragalus polysaccharide; (2) purifying the fermented astragalus polysaccharide by adopting DEAE Sepharose FastFlow, eluting by using ultrapure water, concentrating and freeze-drying an eluant to obtain a neutral polysaccharide DF-1, eluting by using gradient NaCl, and concentrating, dialyzing, concentrating and freeze-drying the eluant to obtain a weakly acidic polysaccharide; (3) purifying the neutral polysaccharide DF-1 obtained from the step (2) by adopting a Sephacryl S-400 High Reslution preparation chromatographic column, eluting by using ultrapure water, concentrating and freeze-drying the eluant to obtain neutral polysaccharides SF-1 and SF-2. Compared with a crude drug APS, the FAPS (Fermented Astragalus Polysaccharide) extracted by applying the extraction and purification method provided by the invention is outstandingly increased in yield and content.
Description
Technical field
The present invention relates to extracting and purifying method and the application thereof of fermentation astragalus polysaccharides.
Background technology
Along with the development of modern science and technology and the day by day deep of modernization of cmm, research and development fermentation engineering being applied to Chinese medicine preparation appear in the newspapers repeatly.Existing scholar utilizes different strain to adopt solid and liquid fermentation process to carry out bio-transformation to Chinese medicine astragalus, and result of study shows, yield and the drug effect of the Radix Astragali polysaccharide and polyphenols after bio-transformation are all significantly increased.
About the separation purification method of biological polyoses, various report differs, traditional technology generally adopts decocting method, along with the development studied Polyose extraction purification process, according to the physics-chem characteristic of different biological polyoses, warm leaching method, saturated lime water formulation, Enzymatic extraction are developed.But the tcm product after bio-transformation, its physico-chemical property there occurs certain change, selects suitable pH to improving the output of polysaccharide and keeping its biological activity extremely important.Leavened prod pH value is about about 4, can saturated lime water extraction method (pH value is about 9 ~ 11) affect the active substance effect in leavened prod? H
2o
2decoloring method commonly uses ammoniacal liquor adjust ph to 9, needs 80 ~ 90 DEG C to heat in the reaction 1 hour, by increasing concentration of hydrogen peroxide to accelerate decolorization rate.Can the change of pH change the content of fermentation astragalus polysaccharides? a lot of enzymes that non-solution lactose suis LZMYFGM9 secretes can participate in biotransformation, the yield increasing fermentation astragalus polysaccharides can be assisted by Mierocrystalline cellulose Enzymatic Extraction? have not yet to see the comparative studies about bio-transformation type herbal polysaccharide extracting method.
Summary of the invention
The invention provides a kind of extracting and purifying method and application thereof of the astragalus polysaccharides that ferments.
The applicant utilizes a strain separation from the non-solution lactose suis LZMYFGM9 of chicken intestinal, and deposit number: be CGMCC No.4227, carry out deep liquid ferment altogether with Chinese medicine astragalus develops fermentation Radix Astragali product.Patent of the present invention is in the existing shake flask fermentation technical foundation in laboratory, and application 10L fermentor tank, by orthogonal experiment optimization for fermentation technology, sets up the scale up test technique of leavened prod.Research shows, this leavened prod can strengthen broiler growth performance as fodder additives, improves immunity of organisms.
The applicant is by comparing several method in prior art, optimize warm leaching method to extract, then be four empirical factors with extraction temperature, amount of water, extracting times, alcohol precipitation ethanol concn respectively, take polysaccharide content as Testing index, adopt orthogonal experiment to optimize extraction process, obtain optimum extracting method.Adopt film to retain ultra-fine filter and concentrate polysaccharide extraction liquid, then by Sevag method removing protein, dialysis method removes small molecules and two step column chromatographies are further purified Crude polysaccharides.Finally obtain the separation purification method of fermentation astragalus polysaccharides of high yield, low consumption.Meanwhile, also for the selection of the extracting and purifying method of other liquid fermenting herbal polysaccharide provides reference.
For solving the problems of the technologies described above, the technical scheme that the applicant adopts is as follows:
First object of the present invention is to provide a kind of extracting and purifying method of the astragalus polysaccharides that ferments, and step is as follows:
(1) the fermentation Radix Astragali is carried out warm lixiviate to get, concentrated extracting solution, by Sevag method removing protein, dialysis, concentrate dialysate, alcohol precipitation, washing obtains fermentation Radix Astragali Crude polysaccharides;
(2) carry out purifying with DEAE Sepharose FastFlow to fermentation Radix Astragali Crude polysaccharides, use ultrapure water wash-out, concentrated, freeze-drying elutriant, obtains neutral polysaccharide DF-1; With gradient NaCl wash-out, concentrated, dialysis, concentrated, freeze-drying elutriant, obtain weakly acidic polysaccharide;
(3) neutral polysaccharide DF-1 Sephacryl S-400 High Reslution preparative chromatography post step (2) obtained carries out purifying, uses ultrapure water wash-out, and concentrated, freeze-drying elutriant, obtains neutral polysaccharide SF-1 and SF-2.
Further, it is first get with the lixiviate of ethanol temperature that described temperature lixiviate is got, and obtains the dregs of a decoction, then the lixiviate of dregs of a decoction pure water temperature is got.
Further, it is by 20 times amount (volume: quality, mL:g) 85%(volume percent that the lixiviate of described ethanol temperature is got) airtight immersion 6 ~ 9h under ethanol room temperature, 85 DEG C of temperature leaching 1 ~ 1.5h, filter, filter residue repeats extraction 1 time again; It is in the dregs of a decoction, add 10 ~ 15 times amount (volume: quality, mL:g) pure water, soaking at room temperature 6 ~ 9 h that the lixiviate of described pure water temperature is got, then in 85 DEG C of water-bath temperature leaching 1 ~ 1.5h, inclines and supernatant liquor, then repeat temperature leaching 2 times; Preferably, it is by 20 times amount (volume: quality, mL:g) 85%(volume percent that the lixiviate of described ethanol temperature is got) airtight immersion 8h under ethanol room temperature, 85 DEG C of temperature leaching 1h, filter, filter residue repeats extraction 1 time again; It is the pure water adding 10 times amount in the dregs of a decoction that the lixiviate of described pure water temperature is got, soaking at room temperature 8h, then in 85 DEG C of water-bath temperature leaching 1h, inclines and supernatant liquor, then repeat temperature leaching 2 times.
Further, concentrated described in step (1)-(3) is all adopt film to retain the molecule that ultra-fine filter molecular weight cut-off is 5000 Dal, and ultra-fine filter operating pressure is 0.2 ~ 0.4 MPa, and temperature is 25 DEG C.
Further, described dialysis is the dialysis tubing supernatant liquor obtained after removing protein being placed in MWCO:7000, and flowing water is dialysed 48 h, and distill water dialysis 24 h, until without white precipitate.
Further, described alcohol precipitation is that in dialyzate, add ethanol contend percentage composition in dehydrated alcohol to solution be 80% carry out alcohol precipitation.
Further, described gradient NaCl wash-out is with 0.1 mol/L NaCl, 0.2 mol/L NaCl, 0.3 mol/L NaCl and 0.4mol/L NaCl wash-out.
Second object of the present invention is to provide the fermentation Radix Astragali Crude polysaccharides that application said extracted purification process obtains.
3rd object of the present invention is to provide fermentation Radix Astragali Crude polysaccharides and is preparing the application in liver protecting, suppression hepatic fibrosis medicines.
4th object of the present invention is to provide a kind of liver protecting, suppresses hepatic fibrosis medicines, and the effective constituent of described medicine is above-mentioned fermentation Radix Astragali Crude polysaccharides.
The present invention first removes non-saccharide portion with high concentration ethanol, greatly reduce the interference of impurity in Polyose extraction purge process, the ultra-fine filter of concentrated employing molecular weight cut-off 5000 Dal of polysaccharide liquid, decreases heating concentrated to the destruction of polysaccharide structures and the complex operations step of concentrating under reduced pressure process.Effectively slough polysaccharide color by DEAE Sepharose Fast Flow ion column chromatography, eliminate the decolorization process of polysaccharide.Invention significantly improves extraction efficiency and the purity of fermentation astragalus polysaccharides.
The inventive method is from the significantly different of other method:
1, first the molten part of alcohol is extracted with 85% ethanol in 85 DEG C of temperature leachings, fermentation astragalus polysaccharides is extracted again with the dregs of a decoction of drying, so first the non-polysaccharide material of alcohol dissolubility is extracted, not only eliminate the extensive work of removal of impurities in follow-up polysaccharide purification, also be convenient to the separation and Extraction of the effective constituent to the molten part of alcohol, be beneficial to abundant exploitation and the efficiency utilization of leavened prod effective constituent.
2, in the purifying of polysaccharide, do not have independent decolorization process, experiment shows, adopts conventional H
2o
2decoloring method and be not suitable for fermentation astragalus polysaccharides decolouring, adjustment polysaccharide soln pH to 9 is needed in this method decolorization, but fermentation Radix Astragali pH in biotransformation is 6.4, fermentation astragalus polysaccharides liquid is adjusted to alkalescence by acidity may make the physico-chemical property of polysaccharide change, and therefore should not use H
2o
2decolouring.Gac has stronger adsorption to polysaccharide, and gac is used for the loss that polysaccharide decolouring can cause polysaccharide.And directly go up DEAE Sepharose Fast Flow ion exchange column, effectively can remove pigment while purified polysaccharide, the fermentation astragalus polysaccharides that purifying obtains is mainly the neutral polysaccharide of white, eliminates decolorization process.Concentrate polysaccharide liquid with ultra-fine filter, decrease in the complex operations and long-time heating process heating concentrated and concentrating under reduced pressure and polysaccharide structures is destroyed.Present method in reduction energy consumption, time savingly obtain desirable decolouring and concentrated effect simultaneously.
3, the present invention follows the example of with cellulase assisted Extraction, compared with lime water extraction method, traditional decoction method, have Polyose extraction efficiency high, purity of polysaccharide is high, and the damage of polysaccharide biological activity is little, energy-conservation, easy and simple to handle, the advantages such as experiment condition controllability is strong.
4, apply extracting and purifying method of the present invention and extract gained fermentation Radix Astragali Crude polysaccharides (FAPS) compared with crude drug astragalus polysaccharides (APS), yield and content all have remarkable rising.The enzyme system result of study secreted during the fermentation to non-solution lactose suis LZMYFGM9 shows, participate in the key enzyme (UDPG-4-isomerase) of extracellular polysaccharide of bacteria synthesis in the process and participate in the enzyme (alpha-galactosidase of cellulose decomposition, Portugal poly-1,6-alpha-glucosidase, glucokinase) expression amount all comparatively ferment before significantly raise, illustrates that microorganism can cause the change of polysaccharide kind and quantity in the fermentation Radix Astragali to the biotransformation of the Radix Astragali.Pharmaceutical research shows that fermentation Radix Astragali Crude polysaccharides also can protect liver cell, suppresses hepatic fibrosis to a certain extent.Therefore, the astragalus polysaccharides that ferments demonstrates good development prospect.
Accompanying drawing explanation
Fig. 1 is DEAE Sepharose Fast Flow wash-out chromatograms (190 nm and 280 nm) of fermentation Radix Astragali Crude polysaccharides;
Fig. 2 is the polysaccharide curve (490nm) that the DEAE Sepharose Fast Flow elutriant phend-sulphuric acid of fermentation Radix Astragali Crude polysaccharides is described;
Fig. 3 is Sephacryl S-400 High Reslution wash-out chromatograms (190nm and 280nm) of fermentation Radix Astragali neutral polysaccharide component S F-1 and SF-2;
Fig. 4 is that the Sephacryl S-400 High Reslution elutriant phend-sulphuric acid of fermentation Radix Astragali neutral polysaccharide component S F-1 and SF-2 describes polysaccharide curve (490nm);
Fig. 5 is that HPGPC-DAD-RI, GPC-MALLS coupling measures fermentation Radix Astragali neutral polysaccharide component S F-1 absolute molecular weight and relative molecular weight collection of illustrative plates (690nm);
Fig. 6 is that HPGPC-DAD-RI, GPC-MALLS coupling measures fermentation Radix Astragali neutral polysaccharide component S F-2 absolute molecular weight and relative molecular weight collection of illustrative plates (690nm);
Fig. 7 is DEAE Sepharose Fast Flow wash-out chromatograms (190nm and 280nm) of astragalus polysaccharides;
Fig. 8 is the polysaccharide curve (490nm) that the DEAE Sepharose Fast Flow elutriant phend-sulphuric acid of astragalus polysaccharides is described;
Fig. 9 is Sephacryl S-400 High Reslution wash-out chromatograms (190nm and 280nm) of Radix Astragali neutral polysaccharide component S S-1;
Figure 10 is that the Sephacryl S-400 High Reslution elutriant phend-sulphuric acid of Radix Astragali neutral polysaccharide component S S-1 describes polysaccharide curve (490nm);
Figure 11 is that HPGPC-DAD-RI, GPC-MALLS coupling measures Radix Astragali neutral polysaccharide component S S-1 absolute molecular weight and relative molecular weight collection of illustrative plates (690nm);
Figure 12 (A-F) is hepatic tissue section H.E. dyeing (H.E. × 100); Wherein, A is Normal group; B is model group; C is fermentation Radix Astragali extract low dosage; D is dosage group in fermentation Radix Astragali extract; E is fermentation Radix Astragali extract high dose group; F is colchicine control group.
Figure 13 (A-F) is hepatic tissue section Masson dyeing (Masson × 100); Wherein, A is Normal group, and B is model group, and C is fermentation Radix Astragali extract low dose group, and D is dosage group in fermentation Radix Astragali extract, and E is fermentation Radix Astragali extract high dose group, and F is colchicine control group.
Figure 14 (A-F) is transmission electron microscope observing hepatic tissue ultrastructure; Wherein, A: control rats liver cell golgi body form is normal, collagen-free fibroplasia (transmission electron microscope × 12000) in space of Disse; B: fatty apparition in fermentation Radix Astragali extract high dose group tenuigenin, nucleus, plastosome are normal, have no Collagen Proliferation in space of Disse, liver cell gap broadening (transmission electron microscope × 12000); C: have collagen fiber hyperplasia in middle dosage fermentation Radix Astragali extract group hepatic tissue space of Disse, fat vacuoleization obviously (transmission electron microscope × 12000); D: have a large amount of collagen fiber hyperplasia in low dosage fermentation Radix Astragali extract group hepatic tissue space of Disse, fat vacuoleization obviously (transmission electron microscope × 12000); E: have collagen fiber hyperplasia in colchicine group space of Disse, visible red cell (transmission electron microscope × 15000) in liver sinusoid; F: model group rats liver finestructure is fuzzy, and boundary is unclear.Collagenous fiber bundle transverse section (transmission electron microscope × 12000) a large amount of as seen in space of Disse.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
In the present invention, measurement of the polysaccharide content adopts phend-sulphuric acid, and step is as follows:
The making of a, typical curve: accurately take dextrose standard sample 10 mg of drying under reduced pressure to constant weight, be placed in 500 mL volumetric flasks, add water to scale, be made into the glucose solution of 0.2 mg/mL.Draw reference substance solution 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL and 1.3 mL respectively, 2.5 mL are settled to respectively with pure water, each precision draws 0.2 mL in tool plug test tube, add 5% phenol solution 0.1 mL, shake up, more vertically add rapidly the vitriol oil 0.5 mL, jolting, room temperature leaves standstill 10 min, and boiling water bath heats 15 min, cools in cold water.Retinue blank.490 nm wavelength places survey light absorption values, with glucose concn be X-coordinate (x), light absorption value (A) is ordinate zou (y), carries out linear regression, drawing standard curve.Typical curve equation is: y=0.0335x (R
2=0.9994) wherein: y is light absorption value, x is glucose concn (mg/L), R
2for relation conefficient;
B, determination of polysaccharide: obtained fermentation astragalus polysaccharides is made into the solution that concentration is 1.0 mg/mL with distilled water, get 0.2 mL measures 490 nm light absorption value by the method for step a, calculates the concentration of polysaccharide according to typical curve;
C, fermentation astragalus polysaccharides content (%)=(typical curve calculates gained polysaccharide quality/fermentation astragalus polysaccharides quality) × 100%;
D, fermentation astragalus polysaccharides yield (%)=(quality of the quality/fermentation Radix Astragali product of polysaccharide) × 100%.
embodiment 1
The bacterial classification fermented altogether with the Radix Astragali in the present embodiment be a strain be separated non-solution lactose suis in chicken intestinal (
streptococcus alactolyticus) LZMYFGM9, its deposit number is CGMCC No.4227.Preservation date: on October 19th, 2010; Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Depositary institution is called for short: CGMCC; Depositary institution address: No. 3, No. 1, BeiChen West Road, Chaoyang District, BeiJing City institute; The cultural characters of this bacterial classification is shown in Chinese patent literature ZL 201210141827.5(denomination of invention: a kind of non-solution lactose suis and application thereof).
In the present embodiment fermentation astragalus polysaccharides, the mensuration of protein content adopts Suo Laibao company BCA determination of protein concentration test kit to detect.
In the present embodiment fermentation Radix Astragali, the massfraction of the crude drug Radix Astragali is 78 %, and the quality namely containing the crude drug Radix Astragali in every 400g fermentation Radix Astragali lyophilized powder is 312g.
The preparation method of Radix Astragali lyophilized powder of fermenting in the present embodiment is: after being pulverized by the Radix Astragali crude drug medicine materical crude slice of cleaning, cross 20 mesh sieves, with the 10 L ferment tank Radixs Astragali, the bacterial concentration of fermentation strain non-solution lactose suis CGMCC No.4227 is 2 × 10
8individual/mL, connecing bacterium amount is that 5 %(account for fermentation cumulative volume), Radix Astragali consumption is 8 %(Radix Astragali quality: fermentating liquid volume, g:mL), temperature 39 DEG C, rotating speed 200 r/min, pH value 6.4, dissolved oxygen amount 10 %, neutralizing agent is the NaOH of 6 mol/L, fermentation time 48 h.After fermentation ends, freeze-drying fermentation astragalus liquid must ferment Radix Astragali product.
Wherein, the liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 25.79 parts; Peptone: 2.28 parts; Glucose: 0.60 part; Yeast powder: 1.56 parts; Magnesium sulfate: 0.04 part; Triammonium citrate: 0.40 part; Hydrogen sulfate dipotassium: 0.40 part; Sodium acetate: 1.00 parts; Astragalus membranaceus powder: 80 parts; Water: 1000 parts.
The extracting and purifying method of a kind of astragalus polysaccharides that ferments of the present invention, comprises the following steps:
(1) take fermentation Radix Astragali lyophilized powder 400g(and cross 20 mesh sieves), with 20 times amount (8000mL), airtight immersion 8h under volume percent 85% ethanol room temperature, 85 DEG C of temperature leaching 1h, filter, filter residue repeats extraction 1 time again.The dregs of a decoction in air dry oven 50 DEG C dry to without alcohol taste for extracting fermentation astragalus polysaccharides.
(2) in step (1) the gained dregs of a decoction, add 10 times amount (mL/g) pure water, under room temperature, soak 8h, then in 85 DEG C of water-bath temperature leaching 1h, incline and supernatant liquor.Again filter residue is repeated warm lixiviate to get and carry out twice: namely in the dregs of a decoction, add 10 times amount (mL/g) pure water, under room temperature, soak 8h, then in 85 DEG C of water-bath temperature leaching 1h, incline and supernatant liquor.Merge three supernatant liquors, after centrifugal, adopt Sartorius Vivaflow 200 molecular weight cut-off be the ultra-fine filter concentrated supernatant of 5000 Dal to 1:5, ultra-fine filter operating pressure is 0.2 ~ 0.4 MPa, and temperature is 25 DEG C.Auxiliary heating in water bath 90 DEG C is concentrated in 1:1(every milliliter extracting solution containing fermenting the Radix Astragali 1 g) again.
(3) by (the Sevag method: add 1/3 volume Sevag reagent (chloroform: propyl carbinol=4:1) of albumen in Sevag method removing step (2) gained concentrated solution, under low temperature, magneton stirs 30min, centrifugal 3600 r/min, 1min, Aspirate supernatant repetitive operation 5 times, until BCA test kit detection protein content is more stable, be about 2 μ g/mL).
(4) step (3) gained supernatant liquor is placed in the dialysis tubing of MWCO:7000, flowing water dialysis 48h, distill water dialysis 24h.Use 1%AgNO
3solution and 1%BaCl
2detect dialysis-effect, until without white precipitate.Dialyzate ultra-fine filter is concentrated into 1:1(crude drug Radix Astragali quality again: concentrated solution volume, g:mL), the operating pressure of ultra-fine filter is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.
(5) in step (4) gained concentrated solution, adding ethanol content in dehydrated alcohol to solution is 80%(volume ratio), alcohol precipitation 8h under room temperature, centrifugal 3000r/min, 5min, abandon supernatant.With 4 times amount (mL/g) pure water dissolution precipitation, 80% alcohol alcohol precipitation, centrifugal, abandon supernatant, repeatable operation 2 times.Use dehydrated alcohol 1:1(mL/g) wash 2 times, centrifugal 3000r/min, 5min; Abandon supernatant, use acetone, ether 1:1(mL/g respectively) respectively wash 1 time, centrifugal 3000r/min, 5 min; Abandon supernatant, precipitation is placed in 50 DEG C of air dry ovens and dries to (24 h), and must ferment Radix Astragali Crude polysaccharides (FAPS) 18 g, and polysaccharide yield reaches 4.5%, and it is 66.3% that phend-sulphuric acid measures its mass fraction of polysaccharide without ether taste substantially.
(6) Biologic DuoFlow chromatographic system, with DEAE Sepharose FastFlow preparative chromatography post (Φ 1 cm × 17.5 cm), accurately take step (5) gained fermentation Radix Astragali Crude polysaccharides 100 mg, be dissolved in 1mL pure water (swelling 12h in advance, auxiliary 60 DEG C of heating in water bath if desired), centrifugal 10000g/min, 5min.Get supernatant liquor, cross 0.45 μm of filter membrane, filtrate is slowly splined on DEAE Sepharose Fast Flow chromatography column.Then use respectively ultrapure water, 0.1 mol/L NaCl, 0.2 mol/L NaCl, 0.3 mol/L NaCl and 0.4 mol each 41 mL(3 times column volume of L NaCl) wash-out successively.Liquid chromatogram (see figure 1) under flow velocity 1 mL/min, on-line checkingi 280 nm and 190 nm, auto partial sampler collects elutriant, often pipe 2 mL.Tracing detection is carried out with phend-sulphuric acid, with light absorption value (A) under 490 nm, wash-out pipe number is mapped, describe elution curve, collect each elution peak respectively, concentrate and (be concentrated into 1:1(crude drug Radix Astragali quality with ultra-fine filter: concentrated solution volume, g:mL), the operating pressure of ultra-fine filter is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.), dialysis (supernatant liquor is placed in the dialysis tubing of MWCO:7000, flowing water dialysis 48h, distill water dialysis 24h.Use 1%AgNO
3solution and 1%BaCl
2detect dialysis-effect, until without white precipitate), concentrated (dialyzate ultra-fine filter being concentrated into 1:1(crude drug Radix Astragali quality again: concentrated solution volume, g:mL)), freeze-drying.Wherein, neutral polysaccharide DF-1 is ultrapure water wash-out gained, and it is that 92.1 %(are shown in Fig. 2 that phend-sulphuric acid measures its mass fraction of polysaccharide).Gradient NaCl wash-out obtains weakly acidic polysaccharide.
(7) Biologic DuoFlow chromatographic system, with Sephacryl S-400 High Reslution preparative chromatography post (Φ 1 cm × 95 cm), accurately take fermentation astragalus polysaccharides DF-1 40 mg that step (6) ultrapure water wash-out obtains, (swelling 12 h) to be in advance dissolved in 1 mL ultrapure water, centrifugal 10000 g/min, 5 min.Get supernatant liquor, cross 0.45 μm of filter membrane, filtrate is slowly splined on Sephacryl S-400 High Reslution chromatography column.With ultrapure water 220 mL(3 times of column volume) wash-out, the liquid chromatogram (see figure 3) under flow velocity 0.4 mL/min, on-line checkingi 190 nm and 280 nm, auto partial sampler collects elutriant, often pipe 2 mL.Tracing detection is carried out with phend-sulphuric acid, with light absorption value (A) under 490 nm, wash-out pipe number is mapped, describe elution curve, collect each elution peak respectively, concentrate and (be concentrated into 1:1(crude drug Radix Astragali quality with ultra-fine filter: concentrated solution volume, g:mL), the operating pressure of ultra-fine filter is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.), freeze-drying.The operating pressure of the ultra-fine filter used in concentration process is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.Ultrapure water wash-out obtains SF-1 and SF-2 two neutral polysaccharide components, phend-sulphuric acid measure its mass fraction of polysaccharide be respectively 97.5% and 97.0%(see Fig. 4).
(8) HPGPC-DAD-RI coupling technique measures the purity of polysaccharide, and GPC-MALLS coupling technique analyzes the absolute molecular weight of polysaccharide fraction, adopts Ultrahydrogel
tM1000,500 Coupled columns gel chromatographies, moving phase is for containing 0.2 % NaN
30.9 mol/L NaCl solution, flow velocity 1 mL/min, column temperature 30 DEG C; MALLS light source gas is helium and neon, determined wavelength 690 nm.By moving phase, SF-1 and SF-2 is mixed with 50mg/mL, through 0.22 μm of membrane filtration, loading analyzes relative molecular weight and absolute molecular weight (see Fig. 5, Fig. 6).Analytical results shows that the weight-average molecular weight of SF-1 is 1.008 × 10
5dal, number-average molecular weight is 9.39 × 10
4dal; The weight-average molecular weight of SF-2 is 6.386 × 10
4dal, number-average molecular weight is 6.340 × 10
4dal(is in table 1).
embodiment 2
The extraction purification of crude drug astragalus polysaccharides comprises the following steps:
(1) take crude drug astragalus membranaceus powder 312 g(and cross 20 mesh sieves), with airtight immersion 8 h under 20 times amount (6240 mL) 85% ethanol room temperature, 85 DEG C of temperature leaching 1 h, filter, filter residue repeats extraction 1 time again.The dregs of a decoction in air dry oven 50 DEG C dry to without alcohol taste for extracting crude drug astragalus polysaccharides.
(2) in step (1) the gained dregs of a decoction, add 10 times amount (mL/g) pure water, under room temperature, soak 8 h, then in 85 DEG C of water-bath temperature leaching 1 h, incline and supernatant liquor.Again this step is repeated twice.Merge three supernatant liquors, after centrifugal, adopt Sartorius Vivaflow 200 molecular weight cut-off be the ultra-fine filter concentrated supernatant of 5000 Dal to 1:5, more auxiliary heating in water bath 90 DEG C to be concentrated in 1:1(every milliliter extracting solution containing the crude drug Radix Astragali 1 g).
(3) (1/3 volume Sevag reagent (chloroform: propyl carbinol=4:1) is added with albumen in Sevag method removing step (2) gained concentrated solution, under low temperature, magneton stirs 30min, centrifugal 3600r/min, 1min, Aspirate supernatant repetitive operation 5 times, it is more stable that BCA test kit detects protein content, is about 2 μ g/mL).
(4) step (3) gained supernatant liquor is placed in the dialysis tubing of MWCO:7000, flowing water dialysis 48h, distill water dialysis 24h.Use 1% AgNO
3solution and 1% BaCl
2detect dialysis-effect, until without white precipitate.Again dialyzate is concentrated into 1:1.
(5) in step (4) gained concentrated solution, dehydrated alcohol is added to 80%(volume ratio), alcohol precipitation 8h under room temperature, centrifugal 3000r/min, 5min, abandon supernatant.With 2 times amount (mL/g) pure water dissolution precipitation, 80% alcohol alcohol precipitation, centrifugal, abandon supernatant, repeatable operation 2 times.Use dehydrated alcohol 1:1(mL/g) wash 2 times, centrifugal 3000 r/min, 5min; Abandon supernatant, use acetone, ether 1:1(mL/g respectively) respectively wash 1 time, centrifugal 3000 r/min, 5min; Abandon supernatant, precipitation is placed in 50 DEG C of air dry ovens and dries to (24 h), and obtain crude drug astragalus polysaccharides (APS) 8.7g, polysaccharide yield reaches 2.8%, and it is 39.8 % that phend-sulphuric acid measures its mass fraction of polysaccharide without ether taste substantially.
(6) Biologic DuoFlow chromatographic system, with DEAE Sepharose Fast Flow preparative chromatography post (Φ 1cm × 17.5cm), accurately take step (5) gained crude drug astragalus polysaccharides (APS) 100mg, be dissolved in 1 mL pure water (swelling 12 h in advance, auxiliary 60 DEG C of heating in water bath if desired), centrifugal 10000 g/min, 5 min.Get supernatant liquor, cross 0.45 μm of filter membrane, be splined on DEAE Sepharose Fast Flow chromatography column.Then use ultrapure water 34 mL(2.5 times of column volume respectively), 0.1 mol/L NaCl 48 mL(3.5 times column volume), 0.2 mol/L NaCl 48 mL(3.5 times of column volume), 0.3 mol/L NaCl 41 mL(3 times of column volume), 0.4 mol L NaCl 41 mL(3 times of column volume) wash-out successively.Flow velocity 1 mL/min, detects the liquid chromatogram (see figure 7) under 280 nm and 190 nm, and auto partial sampler collects elutriant, often pipe 2 mL.Tracing detection is carried out with phend-sulphuric acid, with light absorption value (A) under 490 nm, wash-out pipe number is mapped, describe elution curve, collect each elution peak respectively, concentrate and (be concentrated into 1:1(crude drug Radix Astragali quality with ultra-fine filter: concentrated solution volume, g:mL), the operating pressure of ultra-fine filter is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.), dialysis (supernatant liquor is placed in the dialysis tubing of MWCO:7000, flowing water dialysis 48h, distill water dialysis 24h.Use 1%AgNO
3solution and 1%BaCl
2detect dialysis-effect, until without white precipitate), concentrated (dialyzate ultra-fine filter being concentrated into 1:1(crude drug Radix Astragali quality again: concentrated solution volume, g:mL)), freeze-drying.Wherein, neutral polysaccharide DS-1 is ultrapure water wash-out gained, and it is that 89.8%(is shown in Fig. 8 that phend-sulphuric acid measures its mass fraction of polysaccharide).Gradient NaCl wash-out obtains weakly acidic polysaccharide.
(7) Biologic DuoFlow chromatographic system, with Sephacryl S-400 High Reslution preparative chromatography post (Φ 1 cm × 95 cm), accurately take crude drug Radix Astragali neutral polysaccharide DS-1 40 mg that step (6) ultrapure water wash-out obtains, (swelling 12 h) to be in advance dissolved in 1 mL ultrapure water, centrifugal 10000 g/min, 5 min.Get supernatant liquor, cross 0.45 μm of filter membrane, be splined on Sephacryl S-400 High Reslution chromatography column.With ultrapure water 220 mL(3 times of column volume) wash-out, flow velocity 0.4 mL/min, detects the liquid chromatogram (see figure 9) under 190 nm and 280 nm, and auto partial sampler collects elutriant, often pipe 2 mL.Tracing detection is carried out with phend-sulphuric acid, with light absorption value (A) under 490 nm, wash-out pipe number is mapped, describe elution curve, collect each elution peak respectively, concentrate and (be concentrated into 1:1(crude drug Radix Astragali quality with ultra-fine filter: concentrated solution volume, g:mL), the operating pressure of ultra-fine filter is 0.2 ~ 0.4MPa, and temperature is 25 DEG C.), freeze-drying.Ultrapure water wash-out obtains SS-1 neutral polysaccharide component, and it is that 96.2 %(are shown in Figure 10 that phend-sulphuric acid measures its mass fraction of polysaccharide).
(8) HPGPC-DAD-RI coupling technique measures the purity of polysaccharide, and GPC-MALLS coupling technique analyzes the absolute molecular weight of polysaccharide fraction, adopts Ultrahydrogel
tM1000,500 Coupled columns gel chromatographies, moving phase is for containing 0.2 % NaN
30.9 mol/L NaCl solution, flow velocity 1 mL/min, column temperature 30 DEG C; MALLS light source gas is helium and neon, determined wavelength 690 nm.By moving phase, SS-1 is mixed with 50 mg/mL, through 0.22 μm of membrane filtration, loading analyzes relative molecular weight and absolute molecular weight (see Figure 11).Analytical results shows that the weight-average molecular weight of SS-1 is 1.836 × 10
5dal, number-average molecular weight is 1.649 × 10
5dal(is in table 1).
Table 1 ferments the characterization of molecules parameter of neutral polysaccharide 3 components of the Radix Astragali and the crude drug Radix Astragali
Be 180,000 dalton by the absolute molecular weight of the inventive method extraction purification crude drug Radix Astragali neutral polysaccharide component, the absolute molecular weight of fermentation Radix Astragali neutral polysaccharide component (SF-1 and SF-2) is 6 ~ 100,000 dalton, the absolute molecular weight of Radix Astragali neutral polysaccharide component is reduced through fermentation, illustrate that the structure of neutral polysaccharide in probiotic bacterium biotransformation there occurs noticeable change, infer that the suppression rat liver fibrosis that fermentation astragalus polysaccharides has and fermentation Radix Astragali neutral polysaccharide can more effectively activate the outer dendritic cell vaccination of Mice Body compared with Radix Astragali neutral polysaccharide active different from polysaccharide structures closely related.
Fermentation astragalus polysaccharides and crude drug astragalus polysaccharides are through Sepharose Fast Flow ion-exchange chromatography purifying, neutral polysaccharide component and acidic polysaccharose component can be obtained respectively with the NaCl solution wash-out of ultrapure water and different ionic strength, experimental result display fermentation astragalus polysaccharides and crude drug astragalus polysaccharides detect wherein not containing polysaccharide through phend-sulphuric acid in 0.4 mol/L NaCl wash-out gained position, therefore in actually operating, use ultrapure water, 0.1 mol/L NaCl, 0.2 mol/L NaCl and 0.3 mol/L NaCl wash-out respectively.
embodiment 3
The difference of the present embodiment and embodiment 1 is:
(1) the fermentation Radix Astragali lyophilized powder 400g(taking preparation in embodiment 1 crosses 20 mesh sieves), with 20 times amount (8000mL), airtight immersion 9h under volume percent 85% ethanol room temperature, 85 DEG C of temperature leaching 1h, filter, filter residue repeats extraction 1 time again.The dregs of a decoction in air dry oven 50 DEG C dry to without alcohol taste for extracting fermentation astragalus polysaccharides.
(2) in step (1) the gained dregs of a decoction, add 15 times amount (mL/g) pure water, under room temperature, soak 9h, then in 85 DEG C of water-bath temperature leaching 1h, incline and supernatant liquor.Again filter residue is repeated warm lixiviate to get and carry out twice: namely in the dregs of a decoction, add 15 times amount (mL/g) pure water, under room temperature, soak 9h, then in 85 DEG C of water-bath temperature leaching 1h, incline and supernatant liquor.
All the other steps are all identical with embodiment 1.Extract to obtain fermentation Radix Astragali Crude polysaccharides (FAPS) 17.75 g, polysaccharide yield reaches 4.42%, and it is 67.4% that phend-sulphuric acid measures its mass fraction of polysaccharide.
embodiment 4
The difference of the present embodiment and embodiment 1 is:
(1) the fermentation Radix Astragali lyophilized powder 400g(taking preparation in embodiment 1 crosses 20 mesh sieves), with 20 times amount (8000mL), airtight immersion 6h under volume percent 85% ethanol room temperature, 85 DEG C of temperature leaching 1.5h, filter, filter residue repeats extraction 1 time again.The dregs of a decoction in air dry oven 50 DEG C dry to without alcohol taste for extracting fermentation astragalus polysaccharides.
(2) in step (1) the gained dregs of a decoction, add 12 times amount (mL/g) pure water, under room temperature, soak 6h, then in 85 DEG C of water-bath temperature leaching 1.5h, incline and supernatant liquor.Again filter residue is repeated warm lixiviate to get and carry out twice: namely in the dregs of a decoction, add 12 times amount (mL/g) pure water, under room temperature, soak 6h, then in 85 DEG C of water-bath temperature leaching 1.5h, incline and supernatant liquor.
All the other steps are all identical with embodiment 1.Extract to obtain fermentation Radix Astragali Crude polysaccharides (FAPS) 16.9 g, polysaccharide yield reaches 4.59%, and it is 66.7% that phend-sulphuric acid measures its mass fraction of polysaccharide.
embodiment 5
The difference of the present embodiment and embodiment 1 is:
The liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 26.35 parts; Peptone: 2.20 parts; Glucose: 0.67 part; Yeast powder: 1.53 parts; 0.05 part, magnesium sulfate; Triammonium citrate 0.30 part; Hydrogen sulfate dipotassium 0.50 part; Sodium acetate 0.92 part; Astragalus membranaceus powder: 90 parts; Water: 900 parts.
All the other steps are all identical with embodiment 1.
embodiment 6
The difference of the present embodiment and embodiment 1 is:
The liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 24.00 parts; Peptone: 2.39 parts; Glucose: 0.50 part; Yeast powder: 1.68 parts; 0.03 part, magnesium sulfate; Triammonium citrate 0.50 part; Hydrogen sulfate dipotassium 0.30 part; Sodium acetate 1.10 parts; Astragalus membranaceus powder: 100 parts; Water: 1100 parts.
All the other steps are all identical with embodiment 1.
embodiment 7
Adopt the pharmaceutical research of the inventive method purifying gained fermentation Radix Astragali Crude polysaccharides (FAPS):
One, FAPS is to the restraining effect research of Liver Fibrosis
(1) test method
SD male mouse counterpoise 215 ± 20 g, is divided into 6 groups at random, often organizes 10.Be divided into control group, model group, fermentation Radix Astragali Crude polysaccharides low dose group (FAPS
l) (50mgkg
-1d
-1), dosage group (FAPS in fermentation Radix Astragali Crude polysaccharides
m) (100mgkg
-1d
-1), fermentation Radix Astragali Crude polysaccharides high dose group (FAPS
h) (200mgkg
-1d
-1), colchicine group (Col.) (0.2mgkg
-1d
-1).After modeling starts, except control group dorsal sc injection respective amount peanut oil, all the other each group is all given dorsal sc injection 50 % CCl
4sterilizing peanut oil solution, 2mL/kg, weekly twice, totally 8 weeks.After injection first, administration group every day, gavage gave fermentation Radix Astragali Crude polysaccharides, colchicine solution respectively, and model group and control group gavage give the normal saline solution of corresponding dosage simultaneously, totally 8 weeks.Weigh once weekly, and according to body weight change adjustment dosage.
Take the method for modeling drug intervention simultaneously to inquire into the effect (table 2) of medicine to rat liver fibrosis.Put to death fasted for one day prior and can't help water, femoral artery sacrificed by exsanguination, collection blood and hepatic tissue do every detection experiment.Every detection data all carry out variance analysis with SPSS17.0 statistical software.
Table 2 tests grouping and administration process
(2) Radix Astragali Crude polysaccharides (FAPS) is fermented on the impact of rat body weight
During off-test, control rats body weight is significantly higher than model group (P<0.01), is also significantly higher than FAPS
lgroup, FAPS
mgroup and Col. group (P<0.05).Control group and FAPS
hgroup comparing difference is not significantly (P>0.05).Model group mean body weight only has 306 g, by giving FAPS(50 to rat model, and 100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after, administration group rat body weight mean is all higher than model group (table 3).
Table 3 is tested front and back rat body weight and is compared
(3) Radix Astragali Crude polysaccharides (FAPS) is fermented to the biochemical liver function test of rat blood
Compared with control group, ALT, AST, ALP in model group rats serum are active significantly raises (P<0.01); FAPS(50 is given, 100,200mgkg to rat model
-1d
-1), Col.(0.2mgkg
-1d
-1) after, obviously can reduce ALT, AST, ALP in rat blood serum active, especially FAPS
hgroup ALT, AST, ALP are active significantly reduces (P<0.01), shows that FAPS is to CCl
4the Liver Damage in Rats caused has provide protection (table 4).
Table 4 FAPS is on the impact of hepatic fibrosis rat blood serum ALT, AST and ALP activity that CCl4 induces
(4) Radix Astragali Crude polysaccharides (FAPS) is fermented on the impact of Hyp content in P III NP, C IV, LN, HA and hepatic tissue in hepatic fibrosis rats serum
Experimental result display (table 5), compared with control group, in model group rats serum, in P III NP, C IV, LN, HA and liver tissue homogenate, the content of Hyp significantly raises (P<0.01); By giving FAPS(50 to modeling rat, 100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after, in its serum, in P III NP, C IV, LN and hepatic tissue, HA level obviously reduces.FAPS
hc IV activity and control group comparing difference remarkable (P>0.05) of group.Col. the active remarkable reduction (P<0.05) compared with model group of the HA organized.For Hyp in hepatic tissue, by giving FAPS(50 to rat model, 100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after, can obviously reduce Hyp content in liver homogenate, especially FAPS
hgroup and Col. group more all significantly can reduce Hyp activity (P<0.05) with model group.Above 5 index determining results illustrate that FAPS and Col. is to CCl
4the rat liver fibrosis of induction has certain restraining effect, and can reduce tissue collagen fiber, alleviate extent of liver fibrosis, with the increase of dosage in the FAPS gradient concentration of this Setup Experiments, this restraining effect strengthens to some extent.
The impact of Hyp content in hepatic fibrosis rat blood serum P III NP that table 5 FAPS induces CCl4, C IV, LN, HA and liver tissue homogenate
(5) Radix Astragali Crude polysaccharides (FAPS) is fermented on the impact of Antioxidant Indexes in liver tissues of rats with hepatic fibrosis and serum
1. to ferment the impact of Radix Astragali Crude polysaccharides (FAPS) on liver tissues of rats with hepatic fibrosis T-AOC, GSH, MDA and protein content
Detected result display (table 6), compared with control group, in model group rats liver homogenate, protein content and T-AOC significantly decline (P<0.01), and MDA(P<0.05) and GSH(P<0.01) content significantly raise; Give rat model FAPS
h(200 mgkg
-1d
-1) after, obviously can reduce the MDA content of modeling rat, but difference is not significantly (P>0.05), significantly can raise T-AOC and protein content (P<0.01).Rat model gives FAPS(50,100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after, GSH content decreases but not statistically significant (P>0.05).
Table 6 FAPS is to CCl
4the impact of T-AOC, GSH, MDA and total protein content in the hepatic fibrosis liver tissues of rats of induction
T-AOC can reflect the integrality of body oxidant defense system, this defense system comprises enzymatic and non-enzymatic diplobiont system, enzymatic system mainly comprises SOD, GSH-Px, CAT, GST etc., and non-enzymatic reaction system then mainly comprises VITAMIN (vitamin-E, vitamins C, carotene etc.), amino acid (halfcystine, tryptophane etc.) and metalloprotein (Transferrins,iron complexes, lactoferrin etc.).Result of study shows, the T-AOC content in model group rats hepatic tissue significantly reduces, and compares there is significant difference (P<0.01) with normal group, shows that the resistance of oxidation of model group reduces; And FAPS
h, FAPS
mcCl can be significantly improved
4the content (P<0.01) of T-AOC in the liver tissues of rats with hepatic fibrosis of induction.GSH also plays an important role in the apoptosis also suppressing antioxidant radical peroxidation to cause thus, necrosis and homeostasis change etc.In model group liver tissue homogenate, GSH content is significantly higher than control group may be because after tissue damaged, the metabolic enzyme GSH-Px biosynthesis block of GSH, thus GSH content is raised, and does not represent its function of detoxification and strengthens.After giving drug intervention (FAPS and Col.), GSH-Px is increased, thus the GSH that dysregulation raises reduce gradually.
2. ferment astragalus polysaccharides to the impact of hepatic fibrosis rats GSH-Px in serum, GST activity
Detected result display (table 7), compared with control group, in model group rats serum, GST and GSH-Px is active significantly declines (P<0.01), and MDA content significantly raises (P<0.01); By giving rat model FAPS
h(200 mgkg
-1d
-1) after, the MDA content of rat obviously reduces, but difference is not significantly (P>0.05).And the active significantly rising (P<0.05) of GST and GSH-Px.
The impact of the active and MDA content of GST, GSH-Px in the hepatic fibrosis rat blood serum that table 7 FAPS induces CCl4
All to some extent with oxidative stress (Oxidative Stress, OS) in the pathogenic course of many liver injury factors, if these causes of disease are not dispelled, long term can cause the generation of hepatic fibrosis and even liver cirrhosis in liver.Active oxygen species in Liver Fibrosis Model animal serum increases, and in hepatic tissue, hydroxyl pancreatic desoxyribonuclease, MDA and associated products increase, and they are mainly distributed in around piecemeal necrosis district and portal vein, and its result causes oxidative stress index and MDA, SOD, GSH-P in blood
xdeng significantly change.This research have detected the content of MDA, GSH-Px in fibrosis model rat tissue.Found that, in model group rats hepatic tissue, oxide M DA is significantly higher than control group (P<0.05), and hints model group fibrosis rat vivo oxidation stress excessively produce; And the content of corresponding Antioxidative Defense System as GSH-Px significantly declines (P<0.01), prompting rat anti-oxidative defense miopragia.Give FAPS
hthe MDA value of rising can be reduced after treatment, and raise GSH-Px activity.Show lipid peroxidation and meta-bolites thereof important role in the process of mediation hepatic fibrosis generation, the treatment plan of current anti-hepatic fibrosis comprises Antioxidation Treatment, and prompting antioxygenation may be FAPS
hplay one of effect of anti-hepatic fibrosis.
(6) Radix Astragali Crude polysaccharides (FAPS) is fermented to hepatic fibrosis rats Histopathology Effect
1. pathology is analysed
Control rats hepatic tissue outward appearance is normal, and color is dark red, smooth surface, clear-cut margin, and quality is soft.The enlargement of model group hepatic tissue, in khaki color, there is coarse particles shape on surface.Col. organize outward appearance and model group similar.FAPS drug intervention group liver is brown color, slightly increases, the visible small-particle sample thing in surface.
2. histological examination
(1) H.E. dyeing
Control group liver cell plate centered by central vein radially arranges, and without obvious boundary between liver cell, liver cell nuclear is large and justify, be positioned at central authorities, liver lobule structural integrity, has a small amount of reticular tissue between leaflet, visible interlobular artery, interlobular veins and interlobular bile duct.Fat-free cavity in hepatic tissue, acellular sex change, necrosis and collagen fiber hyperplasia.Be assessed as 0 grade.
Model group rats hepatic cords arrangement disorder, mesenchymal cell showed increased in portal area and fibrous septum, the inflammatory cell (neutrophil leucocyte and lymphocyte) of central vein and portal area and being dispersed in property non-viable non-apoptotic cell (downright bad in leaflet, clasticity is downright bad or bridging necrosis) increase, and with Macrovesicular steatosis and ballooning degeneration, have a small amount of cellular swelling.Central vein has and departs from or extinction tests.Liver cell nuclear engrain, kytoplasm loosens light dye; Fibrillar connective tissue hyperplasia, collegen filament stretch and form fibrous septum in liver lobule, hold liver parenchyma centrilobular vein, form pseudolobuli.In pseudolobuli, liver cell has the pathological changes such as atrophy, the change of balloon sample, lipoid degeneration.The feature main manifestations of liver tissue lesions is: hepatic tissue periphery pathology is comparatively light, and center is heavier; With regard to a liver lobule, be usually expressed as pathology near central vein comparatively light, and periphery pathology is heavier.Be assessed as 3 grades.
By giving FAPS(50 to rat model, 100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after, hepatocellular degeneration and degree of necrosis reduce, and all have improvement in various degree to degree of hepatic fibrosis.Especially FAPS
hobviously alleviate with the hepatocellular balloon sample change of Col., fat-like sex change etc., but still visible a small amount of cell infiltration, accidental spotty necrosis.Be assessed as 1 grade.FAPS
land FAPS
mlesion degree difference compared with model group little, be assessed as 2-3 level.
By experimental result known fermentation astragalus polysaccharides, the repair of hepatic fibrosis pathology is strengthened with the increase of fermentation astragalus polysaccharides dosage.H.E. Figure 12 (A-F) is shown in dyeing.
(2) Masson dyeing
Collegen filament specificly can be dyed blueness by Masson staining, and nucleus is purple, and kytoplasm is red, sees Figure 13 (A-F).
Control rats liver lobule structural integrity, liver cell arrangement architecture is normal, around portal area blood vessel and central vein, only have a small amount of collegen filament to dye.Model group liver lobule is destructurized, has pseudolobuli to be formed.Portal area is strongly painted, and the fibrous septum having thickness to differ is formed, and extends towards periphery hold pseudolobuli from portal area and central vein district.Administration group by giving FAPS(50 to rat model, 100,200 mgkg
-1d
-1), Col.(0.2 mgkg
-1d
-1) after intervention, visible Liver Collagen fibrous septum narrows, painted thin out, and the hyperplasia of collegen filament has minimizing in various degree, but still the thinner Fiber Distribution of visible part is around blood vessel and sinus hepaticus.
(3) transmission electron microscope observing
Control group: liver cell structural form is normal, and cellularstructure is complete, circular nucleus is positioned at cell central authorities, and kernel is placed in the middle, and Distribution of chromatin is even.Rough surfaced endoplasmic reticulum is abundanter, and Mitochondrial Shape complete (circular or oval), glycogenosome is obvious.Hepatic stellate cell is positioned at space of Disse, and core is irregular, and kytoplasm inner cell organ is relatively less, the fat granule that visible electron density is lower.Can see the Kupffer's cells that there is more projection on surface in sinus hepaticus, its kytoplasm has lysosome.
Model group: cellularstructure is fuzzy, boundary is unclear.Occur that cavity and a large amount of fat drip in most liver cell endochylema.Part of hepatocytes necrosis or apoptosis.Visible inflammatory cell infiltration around non-viable non-apoptotic cell.Mitochondrial swelling is out of shape, and ridge reduces, and structure is unintelligible.Between liver cell, gap is broadening, has a large amount of collagenous fiber bundle in interstitial.
Administration group liver cell structure is fuzzy compared with control group, the collagenous fiber bundle of visible a small amount of fat vacuole and hyperplasia.But relatively and model group collagen fiber hyperplasia have alleviating in various degree.HSCs with kupffer quantity in hepatic tissue is also not identical.
Integrated comparative respectively organizes pathology figure, FAPS under Electronic Speculum
hgroup (fermentation astragalus polysaccharides 200 mgkg
-1d
-1) and Col. group (colchicine 0.2 mgkg
-1d
-1) lesion degree is relatively light.Transmission electron microscope observing is shown in Figure 14 (A-F).
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. ferment the extracting and purifying method of astragalus polysaccharides, it is characterized in that: step is as follows:
(1) fermentation Radix Astragali lyophilized powder is carried out warm lixiviate to get, concentrated extracting solution, by Sevag method removing protein, dialysis, concentrate dialysate, alcohol precipitation, washing obtains fermentation Radix Astragali Crude polysaccharides;
(2) carry out purifying with DEAE Sepharose FastFlow to fermentation Radix Astragali Crude polysaccharides, use ultrapure water wash-out, concentrated, freeze-drying elutriant, obtains neutral polysaccharide DF-1; With gradient NaCl wash-out, concentrated, dialysis, concentrated, freeze-drying elutriant, obtain weakly acidic polysaccharide;
(3) neutral polysaccharide DF-1 Sephacryl S-400 High Reslution preparative chromatography post step (2) obtained carries out purifying, uses ultrapure water wash-out, and concentrated, freeze-drying elutriant, obtains neutral polysaccharide SF-1 and SF-2.
2. extracting and purifying method according to claim 1, is characterized in that: it is first get with the lixiviate of ethanol temperature that described temperature lixiviate is got, and obtains the dregs of a decoction, then the lixiviate of dregs of a decoction pure water temperature is got.
3. extracting and purifying method according to claim 2, it is characterized in that: it is by 20 times amount (volume: quality, mL:g) 85%(volume percent that the lixiviate of described ethanol temperature is got) airtight immersion 6 ~ 9h under ethanol room temperature, 85 DEG C of temperature leaching 1 ~ 1.5h, filter, filter residue repeats extraction 1 time again; It is in the dregs of a decoction, add 10 ~ 15 times amount (volume: quality, mL:g) pure water, soaking at room temperature 6 ~ 9 h that the lixiviate of described pure water temperature is got, then in 85 DEG C of water-bath temperature leaching 1 ~ 1.5h, inclines and supernatant liquor, then repeat temperature leaching 2 times; Preferably, it is by 20 times amount (volume: quality, mL:g) 85%(volume percent that the lixiviate of described ethanol temperature is got) airtight immersion 8h under ethanol room temperature, 85 DEG C of temperature leaching 1h, filter, filter residue repeats extraction 1 time again; It is the pure water adding 10 times amount in the dregs of a decoction that the lixiviate of described pure water temperature is got, soaking at room temperature 8h, then in 85 DEG C of water-bath temperature leaching 1h, inclines and supernatant liquor, then repeat temperature leaching 2 times.
4. extracting and purifying method according to claim 1, is characterized in that: concentrated described in step (1)-(3) is all adopt film to retain the molecule that ultra-fine filter molecular weight cut-off is 5000 Dal, and ultra-fine filter operating pressure is 0.2 ~ 0.4 MPa, and temperature is 25 DEG C.
5. extracting and purifying method according to claim 1, is characterized in that: described dialysis is the dialysis tubing supernatant liquor obtained after removing protein being placed in MWCO:7000, and flowing water dialysis 48h, distill water dialysis 24h, until without white precipitate.
6. extracting and purifying method according to claim 1, is characterized in that: described alcohol precipitation is that in dialyzate, add ethanol contend percentage composition in dehydrated alcohol to solution be 80% carry out alcohol precipitation.
7. extracting and purifying method according to claim 1, is characterized in that: described gradient NaCl wash-out is with 0.1 mol/L NaCl, 0.2 mol/L NaCl, 0.3 mol/L NaCl and 0.4mol/L NaCl wash-out.
8. application rights requires the fermentation Radix Astragali Crude polysaccharides that the extracting and purifying method described in 1 ~ 7 obtains.
9. the Radix Astragali Crude polysaccharides that ferments is preparing the application in liver protecting, suppression hepatic fibrosis medicines.
10. liver protecting, a suppression hepatic fibrosis medicines, is characterized in that: the effective constituent of described medicine is fermentation Radix Astragali Crude polysaccharides according to claim 8.
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| CN108359024A (en) * | 2018-04-09 | 2018-08-03 | 天津科技大学 | A kind of molten oligosaccharide of Astragenol and preparation method thereof and its antitumor application thereof |
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| CN109265576A (en) * | 2018-09-27 | 2019-01-25 | 广东药科大学 | A kind of macromolecule astragalus polyose and the preparation method and application thereof with anti-inflammatory activity |
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| CN111978429A (en) * | 2020-09-16 | 2020-11-24 | 上海交通大学 | Extraction method of flammulina velutipes root polysaccharide |
| CN113024679A (en) * | 2021-02-25 | 2021-06-25 | 广西壮族自治区农业科学院 | Method for extracting selenium polysaccharide and polyphenol from selenium-rich moringa seeds |
| CN113637091A (en) * | 2021-08-17 | 2021-11-12 | 浙江宜格企业管理集团有限公司 | Extraction method of high-purity low-molecular-weight astragalus polysaccharide, product and application thereof |
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