CN103059152B - Isolates of hedysari polybotrys polysaccharide 3 and application thereof - Google Patents

Abstract

The invention provides four isolates of hedysari polybotrys polysaccharide 3, and researches on physical and chemical properties (including molecular weight and distribution, conformation, composite, polysaccharide content and protein content), chemical structure and anti-aging effect of the four isolates. Moreover, a pharmacology experiment discovers that the four uniform hedysari polybotrys polysaccharide components have remarkable anti-aging effect on D-gal aging mice, and have good effects of regulating blood sugar level, reducing blood fat, resisting radiation and improving immunity on experimental type 2 diabetic rats. Simultaneously, the invention further provides corresponding preparation methods and pharmaceutical preparations of the isolates of hedysari polybotrys polysaccharide 3.

Description

Isolate of hedysarum polybotys saccharide 3 and uses thereof

Technical field

The present invention relates to isolate of hedysarum polybotys saccharide 3 and uses thereof, belong to Natural Medicine Chemistry technical field.

Background technology

Red stilbene (Radix Hedysari) is the root of pulse family Hedysarum spp plant Hedysarum polybotrys Hand.-Mazz. (Hedysarum polybotrys Hand.-Mazz). be Gansu special product rare medicinal herbs, the traditional Chinese medical science it can be used as traditional Chinese medicine, it has cardiac stimulant clinically, hypoglycemic, diuresis, antiviral, anti-ageing effect of waiting for a long time.Hedysarum polybotys saccharide is as one of its main active ingredient, and wherein HPS-3 part has the significantly effect such as hypoglycemic and antioxidation in vitro.

The domestic and international chemical composition to red stilbene there has been more detailed report at present, and for hedysarum polybotys saccharide research, also just rests on the research of primary structure and some pharmacological actions.Liu Fangming extraction and isolation from red stilbene of Lanzhou University in 1997 goes out a kind of homopolysaccharide-dextran, and studies its structure; Lee's generation has just waited and has obtained 3 HPS components by dextrane gel column chromatography for separation purifying, only uses the monose composition of gc analysis 3 HPS components, and studies HPS anti tumor activity in vitro and structure activity relationship; 2009 Nian Ma garrison troops etc. have carried out pre-test to the purifying of the red stilbene acidic polysaccharose in Gangu and structure.Ma Dan etc. obtain HPS-3 sterling by substep alcohol deposition method and gel column chromatography, its composition of gas chromatography determination.Polysaccharide as biological polymeric compound, pharmacologically active, closely related with its physico-chemical property, therefore, understand its physico-chemical property and seem particularly important.

Recent year scholar in succession reports the herbal polysaccharides such as Liuwei Dihuang polysaccharides, Cactus Pedicel Crude polysaccharides, Schisandra chinensis polysaccharide, wheat-based diet, Root of Upright Ladybell polysaccharide, pholiota nameko polysaccharide, astragalus polysaccharides and Semen Nelumbinis polysaccharide and has anti-aging effects.But research that is anti-ageing about hedysarum polybotys saccharide, radioprotective effect, there is not been reported in home and abroad.And report also very few for polysaccharide for reducing blood sugar, raising immunizing power, this experiment inquired into first hedysarum polybotys saccharide anti-ageing, hypoglycemic and and the possible mechanism of action of reducing blood-fat, for Chinese medicine delay senility and hypoglycemic research open up new field, and to hedysarum polybotys saccharide radioprotective and improve immunizing power be studied.

Summary of the invention

The invention reside in four separated portions providing hedysarum polybotys saccharide 3, and its drug value is analyzed.

In order to solve the problems of the technologies described above, the invention provides following technical scheme:

The isolate of hedysarum polybotys saccharide 3, obtains through following steps:

Hedysarum polybotys saccharide 3 is after deproteinated, depigmentation, cross Sephadex gel column, first use distilled water wash-out, then with the NaAc-HAc buffering salt wash-out containing 0 ~ 2M different concns NaCl, obtain hedysarum polybotys saccharide 3-A, hedysarum polybotys saccharide 3-B, hedysarum polybotys saccharide 3-C, hedysarum polybotys saccharide 3-D 4 components.

Particularly, obtain through following steps:

(1) hedysarum polybotys saccharide 3 is through Sevag method deproteinated, hydrogen peroxide for decoloration, and hedysarum polybotys saccharide 3 solution after decolouring is concentrated into certain volume, is 50% ~ 90% alcohol settling 1 ~ 3 time with final concentration, lyophilize; Hedysarum polybotys saccharide 3 after decolouring, with after water dissolution, crosses DEAE-cellulose 52 column chromatography, and respectively with the NaAc-HAc buffering salt wash-out containing 0 ~ 2M different concns NaCl, the pH value of elute soln keeps 4 ~ 7, and phend-sulphuric acid spike, obtains 4 components;

(2) more than, 4 components cross Sephadex G-100, Sephadex G-75 gel column respectively repeatedly, use distilled water wash-out, obtain hedysarum polybotys saccharide 3-A, hedysarum polybotys saccharide 3-B, hedysarum polybotys saccharide 3-C, hedysarum polybotys saccharide 3-D 4 components.

Described in step (1), hedysarum polybotys saccharide 3-A is spherical conformation, weight-average molecular weight Mw is 3 ~ 20KDa, rootmean-square rotation radius Rg is 20 ~ 70nm, polydispersity coefficient Mw/Mn is 1.000 ~ 1.300, the molar ratio of each monose composition of hedysarum polybotys saccharide 3-A is: glucose (Glc): semi-lactosi (Gal)=70 ~ 95: 1 ~ 10, and polysaccharide content is 80 ~ 100%; Hedysarum polybotys saccharide 3-A (is abbreviated as p) glucose by α-D-(1 → 4) pyrans and forms main chain, 1 branch is had in O-6 position every 3 ~ 7 saccharide residues, wherein branch contains → 1) Glc, → 1) Gal (4 →, every 8 ~ 12 saccharide residues have 1 sulfate, and sulfate is positioned at the O-6 position of Gal.

Structure is such as formula shown in I:

Wherein, n=1 ~ 14.

Described hedysarum polybotys saccharide 3-B is highly-branched degree structure, Mw is 10 ~ 200KDa, Rg is 11 ~ 25nm, Mw/Mn is 1.005 ~ 1.410, and the molar ratio of monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl (Rha)=10 ~ 55: 5 ~ 50; 1 ~ 15: 1 ~ 20, polysaccharide content is 70 ~ 99%; Hedysarum polybotys saccharide 3-B is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 7 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 13 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-B is mainly containing α-amino-isovaleric acid (Val) (37.5%) and glycine (Gly) (22.3%).

Structure is such as formula shown in II:

Wherein, n=1 ~ 85.

Described hedysarum polybotys saccharide 3-C conformation is a random ball of string, Mw is 15 ~ 200KDa, Rg is 10 ~ 23.5nm, Mw/Mn is 1.100 ~ 1.350, the molar ratio of monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=12 ~ 40: 15 ~ 65: 1 ~ 13: 0.15 ~ 3, and polysaccharide content is 80 ~ 98%; Hedysarum polybotys saccharide 3-C is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has comparatively higly branched chain, every 4 ~ 9 saccharide residues have 1 shorter or long-chain branch in O-6 position, containing → 1 on side chain) Ara (5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 11 ~ 16 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-C mainly contains aspartic acid (Asp) (19.8%), L-glutamic acid (Glu) (17.6%), Gly (15.7%) three seed amino acid.

Structure is as shown in formula III:

Wherein, n=1 ~ 93.

Described hedysarum polybotys saccharide 3-D is highly-branched degree structure, Mw is 10 ~ 150KDa, Rg is 10 ~ 25nm, Mw/Mn is 1.800 ~ 3.000, and the molar ratio of monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=15 ~ 60: 13 ~ 55: 0.5 ~ 10: 3 ~ 25.5.Polysaccharide content is 82 ~ 98.5%; Hedysarum polybotys saccharide 3-D is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 5 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →, → 1) Ara (3,5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 15 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-D mainly contains Gly (24.6%), Glu (17.3%), Asp (13.1%) three seed amino acid.

Structure is such as formula shown in IV:

Wherein, n=1 ~ 120.

What any one or any two of above-mentioned hedysarum polybotys saccharide 3-A, hedysarum polybotys saccharide 3-B, hedysarum polybotys saccharide 3-C and hedysarum polybotys saccharide 3-D were above is combined in the application prepared in antiaging agent, hypoglycemic drug, blood lipid-lowering medicine, raising immunity of organisms medicine, antiradiation drug.

Hedysarum polybotys saccharide 3 is preparing the application in antiaging agent, raising immunity of organisms medicine, antiradiation drug.

A kind of pharmaceutical preparation, comprises any one or any two above combinations of above-mentioned hedysarum polybotys saccharide 3-A, hedysarum polybotys saccharide 3-B, hedysarum polybotys saccharide 3-C or hedysarum polybotys saccharide 3-D, or hedysarum polybotys saccharide 3, and pharmaceutically acceptable auxiliary material.

The formulation of described preparation is solution, syrup, granule, capsule, powder, pill, tablet, aqueous injection, lyophilisate, patch, gelifying agent, film, pill, vina, extractum or sustained-release preparation.。

One, the conformation research of 4 components in hedysarum polybotys saccharide 3

1, HPS-3-B, HPS-3-C, the HPS-3-D aqueous solution is all positive to biuret and triketohydrindene hydrate, and the HPS-3-A aqueous solution is then negative; 4 component Pyrogentisinic Acid sulphate reagents all produce color reaction; DAD detector color atlas shows, and HPS-3-B, HPS-3-C, the HPS-3-D aqueous solution has more weak uv-absorbing at 280nm, and the HPS-3-A aqueous solution does not then absorb; And 4 components all have polysaccharide charateristic avsorption band at about 190nm.Above result shows that component HPS-3-A is polysaccharide; Component HPS-3-B, HPS-3-C, HPS-3-D is the polyose-protein composition containing a small amount of protein or polypeptide, through elemental analyser analysis, HPS-3-B, HPS-3-C, HPS-3-D have 0.3 ~ 0.5%N element (Kjeldahl determination is for containing protein 1.875 ~ 3.125%) existence also to demonstrate this point.

2, HPGPC method qualification polysaccharide fraction purity

Adopt HPGPC-DAD-RI coupling technique, chromatographic column: Ultrahydrogel 1000,500 Coupled columns, moving phase: ultrapure water, flow velocity: 0.8ml/min, column temperature: 40 DEG C, differential refraction detector temperature: 40 DEG C, sample size: 30 μ l.Sample solution concentration is 3mg/ml, 0.22 μm of membrane filtration.

The visible HPS-3-A of Fig. 1 is single symmetrical peak, though and other 3 component peaks are unimodal, peak protracts, this is because HPS-3-B, HPS-3-C, HPS-3-D are polyose-protein composition, there is charged group in its molecular structure, with gel column generation adsorption, thus occurs leading peak.

3, GPC-MALLS chromatographiccondition

Adopt Ultrahydrogel 1000,500 Coupled columns gel chromatography, moving phase is for containing 0.02%NaN ₃0.1MNaNO ₃solution, flow velocity is 0.8ml/min, column temperature: 40 DEG C; The light source gas of MALLS is helium and neon, wavelength: 690nm.The refractive index of moving phase gets 1.330, and polysaccharide refractive index increment in the solution (dn/dc) is 0.135, and the instrument(al)constant of calibration laser is 8.1104 × 10 ^-61/ (V cm), calibration differential refraction detector instrument(al)constant is 2.2697 × 10 ^-41/ (V cm).

Precision takes sample HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, is mixed with 5mg/ml solution by moving phase, through 0.22 μm of membrane filtration, carries out GPC-MALLS analysis.

Before carrying out GPC-MALLS analysis, sample must be in complete dissolved state in the solution, and sample concentration can not lower than 2mg/ml.

The present invention adopts gel permeation chromatography-multiple angle laser light scattering (GPC-MALLS) coupling technique, to record in hedysarum polybotys saccharide 3 conformation of 4 components at solution state, can be hedysarum polybotys saccharide 3 research and development becomes medicine (as injection etc.) and provides basis.

By containing 0.02%NaN to moving phase ₃naNO ₃the investigation that the concentration (0 ~ 0.1mol/L) of solution, sample concentration (2 ~ 5mg/mL), column temperature (30 ~ 50 DEG C) and flow velocity (0 ~ 1mL/min) affect sample laser signal, result shows, having the greatest impact of flowing relative sample laser signal, secondly be sample concentration, the impact of column temperature and flow velocity is then less.For avoiding too high post pressure, shorten analysis time, flow velocity is set to 0.8mL/min simultaneously as far as possible.The increase of moving phase concentration and the increase (particularly the sample of molecular weight) of sample concentration, reduce noise largely, improve laser signal.Therefore, select containing 0.02%NaN ₃0.1MNaNO ₃solution is moving phase, and sample concentration is 5mg/mL, and column temperature is 40 DEG C, chooses an angle of 90 degrees laser signal (highly sensitive), and the sample chromatogram figure under this condition is shown in Fig. 2 ~ 5.

Compare with Fig. 1, the appearance time of HPS-3-A on MAALLS differential figure is substantially constant, and obvious delay all appears in other three's appearance times, and the kind of this and moving phase has much relations.Fig. 1 adopts pure water as moving phase, and Fig. 2, Fig. 3, Fig. 4 then all adopt containing 0.02%NaN ₃0.1MNaNO ₃solution is as moving phase, and HPS-3-A is as polysaccharide fraction, and the configuration of polysaccharide chain and shape of molecule size can not change with the concentration of ionogen (salt) within the scope of moving phase finite concentration; And HPS-3-B, HPS-3-C, HPS-3-D are as polyose-protein composition, wherein containing a small amount of polypeptide or protein, its configuration and shape of molecule size can be subject to the impact of ionogen (salt).Generally, when ionogen (salt) concentration is less, the polysaccharide protein composition of certain molecular weight can have comparatively polymer shape, along with the increase of ionogen (salt), shape of molecule can diminish gradually, because the molecular sieve effect of gel column goes out peak by molecular size, instead of press molecular size range by wash-out out.Direct relation is had with sample concentration and molecular weight because laser signal is strong and weak, and the concentration of HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D preparation is equal, therefore, from an angle of 90 degrees laser peak color atlas of 4 components, HPS-3-A molecular mass is minimum, and the molecular vibrational temperature of HPS-3-D is the widest.

Fig. 6 shows the molecular vibrational temperature of HPS-3 4 components, can find out that HPS-3-D molecular vibrational temperature scope is maximum.Result shows, and HPS-3-A weight-average molecular weight is 3 ~ 20KDa, and rootmean-square rotation radius Rg is 20 ~ 70nm, and polydispersity coefficient Mw/Mn is 1.000 ~ 1.300; HPS-3-B weight-average molecular weight is 10 ~ 200KDa, Rg be 10 ~ 25nm, Mw/Mn is 1.005 ~ 1.410; HPS-3-C weight-average molecular weight is 15 ~ 200KDa, Rg be 10 ~ 23.5nm, Mw/Mn is 1.100 ~ 1.350; HPS-3-D weight-average molecular weight is 11 ~ 150KDa, Rg be 11 ~ 25nm, Mw/Mn is 1.800 ~ 3.000.

As seen from Figure 7 in HPS-34 component, the rootmean-square rotation radius (Rg) of four, along with the increase of elution volume, all has the trend diminished gradually, illustrates that shape of molecule reduces gradually.From software statistics result, the Rg of four is less, wherein, the Rg of HPS-3-A is maximum, HPS-3-B, HPS-3-C, HPS-3-D three is more or less the same, this and weight-average molecular weight size order are inconsistent, and this causes because HPS-3-B, HPS-3-C, HPS-3-D cause molecular size to reduce in the shape generation shrinkage of ionogen (salt) molecules in solution.In general, the scope of MALLS Accurate Determining sample rootmean-square rotation radius (Rg) is 10nm ~ 500nm, and the Rg of HPS-3-B, HPS-3-C, HPS-3-D is all close to its measurement range lower limit, thus causes larger measuring error.

When slope is 1, represent that polymer is bar-shaped arrangement in the solution, then represent a polymer linearly random ball of string in the solution when slope is 0.4 ~ 0.6, when slope is about 0.33, be then expressed as spherical-like morphology.The slope being calculated HPS-3-A by Fig. 8 is 0.35 ± 0.01, and known HPS-3-A is globosity in moving phase solution, and the slope being calculated HPS-3-C by Figure 10 is 0.66 ± 0.15, and known HPS-3-C is the linear polymer of random ball of string conformation.And poor to the sexual intercourse of Mw construction line as can be seen from the Rg of Fig. 9 and Figure 11, HPS-3-B and HPS-3-D, both similar U-shaped curves show that HPS-3-B and HPS-3-D is typical highly-branched degree structure, and the cladodification degree of HPS-3-D is higher than HPS-3-B.

Two, in hedysarum polybotys saccharide 3, the monose composition of 4 components is studied with polysaccharide content

1, thin-layer chromatography condition

1.1 developers: phthalic acid 1.6g is dissolved in water saturated propyl carbinol 100mL, add aniline 0.93g (being equivalent to 0.9mL).Developping agent: V (ethyl acetate): V (Glacial acetic acid): V (methyl alcohol): V (water)=7: 2: 2: 1.Join before use.Thin layer plate: silica-gel plate, uses 0.3molL ^-1naH ₂pO ₄cMC-Na solution bed board, 105 DEG C of activation 0.5h.

The preparation of 1.2 monose standard substance mixing solutionss

Take glucose, wood sugar, rhamnosyl, pectinose, each 3 ~ 5mg of semi-lactosi, add 100 μ L water dissolution respectively, obtain standard monosaccharide solutions.

1.3 sample preparations and thin-layer chromatography

Be dissolved in 4mL trifluoracetic acid (TFA) respectively by HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D 4 components, tube sealing, is hydrolyzed 6h at 110 DEG C.In vitro solution decompression evaporate to dryness, eliminates TFA.The 100 μ L that add water in sample again make sample dissolution, and point sample monose reference substance and sample, carry out thin-layer chromatography respectively, after launching colour developing, and 85 DEG C of heating 10min.

The TLC of HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D 4 fraction hydrolyzes liquid is shown in Figure 12,5 kinds of mixing monose standard substance separate substantially, monose standard substance show different colours at TLC, and pectinose, wood sugar are aobvious red, and glucose, rhamnosyl, semi-lactosi are then aobvious brown.Relatively 4 fraction hydrolyzes liquid and the Rf value mixing monose standard substance, and the color of TLC, can infer that HPS-3-A contains glucose, HPS-3-B, HPS-3-C, HPS-3-D three is all containing pectinose and semi-lactosi.

2. GC conditions

2..1 temperature programming: keep 4min at 120 DEG C, then with 5 DEG C of min ^-1rise to 190 DEG C, keep 4min, then with 3 DEG C of min ^-1rise to 210 DEG C, keep 10min.

Injector temperature: 250 DEG C; Detector temperature: 240 DEG C; Nebulizer gas pressure: 0.5Mpa.

2..2 the preparation of standard monose sugar nitrile acetic ester derivative

Take glucose, wood sugar, semi-lactosi, seminose, rhamnosyl, each 8mg of pectinose respectively; add 10mg oxammonium hydrochloride respectively, then add 0.5mL pyridine, 90 DEG C of reaction 30min; be chilled to room temperature; add 0.5mL acetic anhydride, 90 DEG C are continued heating acetylizes, add 1mL water and 1mL chloroform extraction 3 times after cooling; get chloroform layer; volatilize, residue adds the dissolving of 1mL chloroform and shakes up, and obtains standard monosaccharide derivatives.

2..3 polysaccharide hydrolysis and aldoononitrile acetate

Take each 10mg of HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D respectively, add 2moLL-1 trifluoroacetic acid 4mL, 90 DEG C of airtight hydrolysis 10h, evaporated under reduced pressure, adds MeOH evaporate to dryness again, repeats 3 times, thoroughly to remove excessive TFA.Identical with standard monosaccharide derivatives preparation process with aftertreatment, sample introduction standard monosaccharide derivatives and polysaccharide derivates respectively, GC analyzes.

See Figure 13 ~ 17, adopt the molar ratio of monose in chromatographic peak area normalization method working sample, determine the mixed polysaccharide that HPS-3-A is made up of pectinose, semi-lactosi, the molar ratio of each monose composition of HPS-3-A is: glucose: semi-lactosi=70 ~ 95: 1 ~ 10, HPS-3-B, HPS-3-C, HPS-3-D are all then the mixed polysaccharide by pectinose, glucose, semi-lactosi, rhamnosyl, and the molar ratio of HPS-3-B monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=10 ~ 55: 5 ~ 50: 1 ~ 15: 1 ~ 18; The molar ratio of HPS-3-C monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=12 ~ 40: 15 ~ 65: 1 ~ 13: 0.15 ~ 3; The molar ratio of HPS-3-D monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=15 ~ 60: 13 ~ 55: 0.5 ~ 10: 3 ~ 25.5.This is consistent with TLC analytical results.

3. the determination of polysaccharide of 4 components in hedysarum polybotys saccharide 3

The preparation of standardized solution: each 20mg of various monose accurately taking drying, is dissolved in 100mL distilled water respectively.It is 40,60 that reference liquid is diluted to concentration respectively, 80,100,120,150 μ gm L ^-1solution, get each 0.2mL of this solution (sugary 8 ~ 30 μ g, as follows) be placed in 10mL tool plug test tube, after adding 5% phenol solution 0.5mL mixing, add rapidly the 2.5mL vitriol oil, mixing, 15min is incubated in 60 DEG C of water-baths, cool immediately, survey absorbancy at 490nm place, using distilled water as blank.

Various monose typical curve is within the scope of sugared content 0 ~ 30 μ g, and absorbancy and sugared content are good linear relationship.Corresponding linear equation is respectively:

Glc：A＝1.09×10-2c+0.004；

Man：A＝1.40×10-2c-0.0011；Gal：A＝0.87×10-2c-0.005；

Xyl：A＝1.03×10-2c-0.002；Ara：A＝0.98×10-2+0.003；

Rha：A＝0.92×10-2c-0.0013。

A: in the absorbancy at 490nm place, c: sugared content (μ g).

From the typical curve of various monose, its slope k known, and the correction coefficient k ' that can calculate various monose thus, that is they are to the Relative slope (table 1) of glucose.For the polysaccharide that known monose forms, available following formula calculates its correction coefficient, and k '=∑ ka, a is sugar composed weight percentage, and k ' is for it is relative to the correction coefficient of glucose.Applying this result, can glucose be just the content that standard test forms known polysaccharide.

The correction coefficient k ' of the various monose standard substance of table 1

Polysaccharide content is calculated for HPS-3-A, learnt by GC analytical results, the molar ratio of each monose composition of HPS-3-A is: glucose: semi-lactosi=83.83: 9.44, its k '=(1.00 × 92.56+7.44 × 0.80)/100=0.98, it is 0.304 that experiment records absorbance, because polysaccharide is that the monose being hydrolyzed generation participates in reaction, so what directly calculated by typical curve is contents of monosaccharides, corresponding polysaccharide content need be further converted to, transformation ratio f=polysaccharide molecular weight/effective molecular weight, for hexasaccharide f=[180n-18 (n-1)]/180n, for poly pentose f=[150n-18 (n-1)]/150n, for polysaccharide n > 10, if both also contain pentose, so 0.91 > f > 0.88 containing hexose.

Main containing hexose in HPS-3-A, therefore get f=0.90.So polysaccharide content is [(0.304-0.004)/1.10 × 10-2/0.98] × 0.9=24.5 (μ g) in HPS-3-A.

The determination of polysaccharide result of 4 components in table 2 hedysarum polybotys saccharide 3

As shown in Table 2, the RSD value of HPS-3-A is less than 5%, and other three's errors are comparatively large, and this is mainly due to the associated proteins containing 3% ~ 5% in these components, and Bradford method is deducted, and RSD value is all reduced within 5%.

For the mixed polysaccharide be made up of different monose, because the typical curve of different monose is different, form identical mixing monose production standard curve so should adopt with mixed polysaccharide.Analyzed by TLC and GC, obtain monose composition and the molar ratio thereof of polysaccharide, this can be selects suitable monose standard substance to make different monose typical curve and provide foundation, thus avoids because blindly choosing waste that monose standard substance cause and comparatively big experiment error.

Three, the chemical structure research of 4 components in hedysarum polybotys saccharide 3

1. the saccharide portion chemical structure research of 4 components in hedysarum polybotys saccharide 3

Adopt Partial acid hydrolysis, methylating is studied the structure of 4 fraction polysaccharide parts in hedysarum polybotys saccharide 3 in conjunction with technology such as compounds GC-MS.

4 components in 100mg hedysarum polybotys saccharide 3 put respectively in round-bottomed flask, add the TFA 50ml of 0.1mol/L, 100 DEG C of water-bath hydrolysis 1h, evaporated under reduced pressure, adds methyl alcohol evaporate to dryness, repeats 3 times, thoroughly to remove excessive TFA.Hydrolysate is dissolved in H ₂flowing water dialysis 2d (Mw=3500) in O, distill water dialysis 1d.Polysaccharide in dialysis tubing concentrates, lyophilize, gel column chromatography (Sephadex G-100,2.6 × 50cm) purifying, and flow velocity is 0.4ml/min, and the every 4ml of Fraction Collector collects a pipe, and concentrated, lyophilize obtains the secondary polysaccharide of 4 part.

Methylating of polysaccharide: first with boron deuterate sodium, 4 component sample and Partial acid hydrolysis sample are reduced before methylating, Hakomori method is adopted to carry out four times again, sample after reduction is placed in the flask that 5ml is with rubber cap respectively, add DMSO, ultrasonicly it is made to dissolve, slowly add 1.5ml SMSM, add within 10min.Mixture puts (20 ~ 25 DEG C) ultrasonic 30min in ultrasonic wave, leads to nitrogen in ultrasonic procedure by injection needles.Ultrasonic complete after reaction flask be placed in room temperature lucifuge and spend the night, under ice bath, slowly drip 2ml methyl iodide, add in 5min, seal after inflated with nitrogen, the ultrasonic 1h of normal temperature, solution is faint yellow clear liquor.

Methylate the separation of polysaccharide: drive remaining methyl iodide with nitrogen, and completely, namely lyophilize obtains the polysaccharide that methylates to transfer in dialysis tubing dialysis in distilled water after adding distilled water.Above methylation procedure is repeatedly until methylate completely.Whether completely methylating of polysaccharide adopt infrared spectra to confirm, at 3400cm ^-1the OH stretching vibration at place disappears for methylation reaction is complete.

The preparation of partial methylation alditol acetate: be divided into hydrolysis, reduction, acetylize three step.The polysaccharide that methylates is put in sealing Teflon test tube and is added 100 DEG C, 88% formic acid (2ml) hydrolysis 6h.Rotary evaporation is except after formic acid removal, and residuum adds 2mol/L trifluoracetic acid 3ml 100 DEG C and is hydrolyzed 6h again, steams altogether repeat to eliminate to trifluoracetic acid for several times by adding the decompression of 3ml methyl alcohol.Final hydrolyzate is dissolved in 2ml distilled water, adds sodium borohydride 25mg, shakes up, sealing, ambient temperatare puts 2 hours, makes it be converted into the sugar alcohol that methylates accordingly, reduction vaporization, to dry, adds 3ml methyl alcohol and 1 acetic acid makes unnecessary reforming sodium borohydride be borate, evaporated under reduced pressure solvent.In triplicate, finally add 5ml methyl alcohol and dry up, repeat twice.

The sugar alcohol that methylates is placed in test tube, adds 2ml acetic anhydride, sealing, 100 DEG C are heated 1 hour, take out, be cooled to room temperature, evaporated under reduced pressure, adding toluene 40 DEG C steams to dry altogether, add 3ml chloroform again to dissolve, wash 3 times with distilled water 3ml, chloroform layer anhydrous sodium sulfate drying, be concentrated into 300 μ l and namely obtain part esterification alditol acetate, Gc-ms.

Hedysarum polybotys saccharide 3-A (is abbreviated as p) glucose by α-D-(1 → 4) pyrans and forms main chain, every 3 ~ 7 saccharide residues have 1 branch in O-6 position, wherein branch contains → 1) Glc, → 1) Gal (4 →, sulfate, every 8 ~ 12 saccharide residues have 1 sulfate, and sulfate is in the O-6 position of Gal; Hedysarum polybotys saccharide 3-B is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 7 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 13 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-C is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has comparatively higly branched chain, every 4 ~ 9 saccharide residues have 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →; With → 1) Rha (2 →, also have non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 11 ~ 16 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-D is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 5 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →, → 1) Ara (3,5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 15 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi.

2. the amino acid analysis of 4 components in hedysarum polybotys saccharide 3

In order to study kind amino acid contained in glycoconjugate and quantity accurately, automatic analyzer for amino acids is adopted to detect so general.First the glycoconjugate sample prepared taking certain mass is dissolved in the hydrochloric acid soln of 0.5ml6mol/L, after moving into hydrolysis pipe tube sealing, at 110 DEG C of hydrolysis 20 ~ 24h, cuts tube sealing, after draining, carries out amino acid analysis with amino acidanalyser.

Owing to not containing protein in HPS-3-A, aminoacids content is 0; HPS-3-B is mainly containing 2 seed amino acids such as Val (37.5%) and Gly (22.3%); HPS-3-C is mainly containing Asp (19.8%), 3 seed amino acids such as Glu (17.6%), Gly (15.7%), HPS-3-D is then mainly containing Gly (24.6%), 3 seed amino acids such as Glu (17.3%), Asp (13.1%).

3. the analysis of polysaccharide and albumen mode of connection

Getting 4 each 10mg of component in hedysarum polybotys saccharide 3 is dissolved in 5ml 0.1mol/L NaOH solution, and 30 DEG C of reaction 20h, measure the change of 240nm place aminoacids content before and after alkaline purification.

In hedysarum polybotys saccharide 3,4 components are after β-eliminative reaction, and HPS-3-B, HPS-3-C, HPS-3-D three has obvious absorption peak at 240nm place, therefore can illustrate that it is O-glycoprotein compositions.

Four, the research of the anti-aging effects of 4 components in hedysarum polybotys saccharide 3 and 3

1. material: HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3,70 kunming mices, weight (20.0 ± 2.0g), male and female half and half.Free diet water under room temperature ordinary ray, adaptability is divided into 7 groups after feeding 1 week at random, i.e. Normal group, aging model group, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group 5 treatment groups, often organizes 10, male and female half and half.D-semi-lactosi, superoxide-dismutase, Malondialdehyde Kit, Selenoperoxidase (GSH-Px) testing cassete.

2. administration: kunming mice is divided into 7 groups, except Normal group, 4 components and hedysarum polybotys saccharide 3 groups every neck dorsal sc injection 50g/l D-semi-lactosi 0.5ml in aging model group and hedysarum polybotys saccharide 3, carry out modeling.Normal group neck dorsal sc injection Isodose physiological saline.After modeling completes, Normal group and aging model group every intraperitoneal injection of saline 0.5ml; Give the hedysarum polybotys saccharide for the treatment of group abdominal injection corresponding dosage weight 0.5ml respectively, continuous 6 weeks.

3. experimental index measures: in Mouse Blood, experimental index measures: superoxide dismutase activity, mda content in serum, in Mouse whole blood, and the vigor of Selenoperoxidase.After 6 weeks, under anesthesia, mouse is extractd eyeball and get blood, a part of conventional separation of serum, at 4 DEG C, the centrifugal 5min of 3000r/min, gets supernatant to be measured; Another part whole blood is to be measured; Superoxide dismutase activity, mda content, Selenoperoxidase (GSH-Px) measures and is strictly undertaken by test kit schedule of operation.

4. Immune Organs Index measures: after 6 weeks, claim quality and take mouse thymus and spleen, claim quality with electronic balance by mouse, represents mouse spleen index and thymus index with the internal organs quality (mg/g) that wets.

5. evaluation criteria: mouse 1 drug administration by injection every day, after 6 weeks, to detect in mice serum superoxide dismutase activity and mda content with spectrophotometer and measures Mouse whole blood Glutathione Peroxidase vigor.

6. MAIN OUTCOME MEASURES: measure superoxide dismutase activity, mda content, whole blood Glutathione Peroxidase Activity and mouse spleen index and thymus index in serum.

7. statistical analysis: statistical treatment data acquisition SPSS10.0 statistics software processes, many groups of test of significance user difference analysis of asking difference and two groups compare to be checked with t.

In table 3 hedysarum polybotys saccharide 3 and 3,4 components are on the impact (n=10 .x ± s) of D-gal Aging mice Thymus and Spleen index

Group	Dosage mgkg -1	Number of animals n	Thymus index mg (10g) -1	Index and spleen index mg (10g) -1
					Control group	-	10	22.17±2.64	45.63±6.91
Model group	-	10	19.50±2.54**	40.98±5.75**
					HPS-3-A	100	10	27.96±5.32△△	50.97±8.94△△
HPS-3-B	100	10	28.98±4.32△△	51.27±7.34△△
					HPS-3-C	100	10	30.12±5.31△△	52.03±5.23△△
HPS-3-D	100	10	32.95±4.13△△	54.15±6.16△△
					HPS-3	100	10	28.96±5.82△△	50.17±9.74△△

Note: t checks, and compares with control group, * * P < 0.01: compare with model group, △ △ P < 0.01

As shown in table 3, mouse continuous subcutaneous injection D-gal is after 6 weeks, and Thymus and Spleen index declines more to some extent with control group, and all has statistical significance, gavages 100mgkg simultaneously ^-1d ^-1hPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 all can make mouse aging thymus index and index and spleen index rebound significantly (P < 0.01), and HPS-3-D action effect is best.

In table 4 hedysarum polybotys saccharide 3 and 3,4 components are on the impact (GSH-Px) (n=10 .x ± s) of exhausted mining areas serum superoxide dismutases, mda and whole blood Glutathione Peroxidase

Compare with control group, P ^*< 0.05; With aging model group P ^△< 0.05

As shown in table 4, aging model group compares with Normal group, and in mice serum, superoxide dismutase activity significantly reduces, and mda content significantly raises (P < 0.05), proves that aging model is successfully set up.HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 treatment group compares with model group, in mice serum, superoxide dismutase activity all significantly raises, mda content all significantly reduces, and whole blood Glutathione Peroxidase Activity all obviously raises (P < 0.05 or P < 0.01).

The D-gal semi-lactosi Aging mice that this experiment copies is slow in action, the loose tarnish of fur, and weight increasing amount obviously declines, and in serum, superoxide dismutase activity significantly reduces simultaneously, and mda level obviously raises, and conforms to the conclusion of above-mentioned report.This experimental result shows, in hedysarum polybotys saccharide 3 and 3,4 component groups compare with aging model group, and in mice serum, superoxide dismutase activity all obviously raises, and the equal content of mda obviously reduces, and has statistical significance.What in hedysarum polybotys saccharide 3 and 3,4 components all obviously can resist D-gal semi-lactosi causes solarization, superoxide dismutase activity in body is significantly improved, mda level then obviously declines, thus infer that in hedysarum polybotys saccharide 3 and 3,4 components can by improving superoxide dismutase activity, strengthen its Scavenging activity to free radical, anti-lipid peroxidation, reduces mda content, thus alleviates the damage of body tissue to delay senility.In addition, in hedysarum polybotys saccharide 3 and 3,4 components also have regulating effect to immunologic function, and it significantly can increase spleen index and the thymus index of mouse.

Five, the research of 4 component hypoglycemic activities in hedysarum polybotys saccharide 3 and 3

The hypoglycemic activity of 4 components on Experimental Mice diabetes and the impact of glucose tolerance in mice in 5.1 hedysarum polybotys saccharides 3 and 3

5.1.1. laboratory animal cleaning grade Kun Ming mice, weight (20g ± 2) g, Lanzhou University's animal center provides.

5.1.2. medicine and reagent tetraoxypyrimidine (ALX) (Sigma-A7413), Glucose Oxidase kit (Sichuan Mai Ke Science and Technology Ltd., lot number: 0606101).

5.1.3. healthy Male Kunming strain mice 80 is got in animal model and administration, and adaptability raises 1w, after fasting (can't help water) 16h, and the fresh ALX solution (220mg/kg) of abdominal injection 2%.72h after last administration, fasting (can't help water) 5h, blood is got in docking, surveys fasting blood sugar (FPG) by glucose oxidase (GOD-PAP) method.FPG > 11.1mmol/L, and there is many drinks, many foods, diuresis person, be judged to modeling success.Get the successful mouse of modeling 60, be divided into model group, HPS-3-A group, HPS-3-B group, HPS-3-C group, HPS-3-D group, HPS-3 group (each group dosage is 100mg/kg) at random, between group, blood sugar difference is not more than 1.1mmol/L, often organizes 10.Every day gastric infusion 1 time, continuous 2w.Model control group gavage isometric(al) physiological saline.

5.1.4. Testing index: general status: observe the situations such as diet, drinking-water, urine volume, weight, the mental status, hair color; Blood sugar: measure by GOD-PAP method, the 1st, 2w fasting 3h, the blood glucose value after gavage 5h; Sugar tolerance: administration 1 time after 2w docking blood sampling, and 0.5h gives glucose 2.5g/kg gavage upon administration, after gavage glucose 0.5,2h docking blood sampling surveys blood glucose value, calculates Area under the curve of blood glucose.The change of each time point Area under the curve of blood glucose after more each treated animal blood glucose value and gavage glucose.Area under the curve of blood glucose=0.25 × (0h blood glucose value+4 × 0.5h blood glucose value+3 × 2h blood glucose value).

5.1.5.HPS-3 and the hypoglycemic activity of 4 components is tested in 3

Animal model and administration: get healthy Male Kunming strain mice 120, adaptability raises 1w, and mouse is divided into Normal group and experimental group at random, by " 3 " method establishment tetraoxypyrimidine tissue of experimental diabetic mice model.Get the successful mouse of modeling 84, to divide into groups administration by table 5, between group, blood sugar difference is not more than 1.1mmol/L.Normal group gavage isometric(al) physiological saline.Testing index: with content under " 4 " item.

5.1.6. statistical procedures is with SPSS 15.0 processing data, and adopt replicate measurement variance analysis (P < 0.05), all data represent with .x ± s.

In 5.2 hedysarum polybotys saccharides 3 and 3,4 components are to the hypoglycemic activity of experimental rat diabetes B

5.2.1. animal Wistar rat, SPF level, body weight 180 ~ 220g, ♀, laboratory animal occupancy permit number: SCXK (sweet) 2004-0006) provided by Gansu Chinese of Traditional Chinese Medicine's scientific experiment center.

5.2.2. (lot number: 0906066), is provided by Shanghai Shi Guibao pharmaceutical Co. Ltd of Sino-U.S. reagent glucophage metformin hydrochloride tablet; ACCU-CHEK (lot number: 22967331) is provided by German ROCHE company Active type blood sugar test paper.

5.2.3. method

The foundation of rat model of type 2 diabetes mellitus model, grouping and administration

Get female wistar rat 100 in 7 ~ 8 week age, by body weight random selecting 10 only as blank, feed with basal feed.Raise with high-sugar-fat-diet 4 weeks for all the other 90, the disposable 30mg/kg that presses injects streptozotocin (STZ), continues to raise with high-sugar-fat-diet 4 weeks making experimental type 2 diabetes mellitus models.Choose the rat 70 of fasting plasma glucose >=11.1mmoL/L after 8 weeks, be divided into diabetes B model group, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-35 treatment group (100mg/kgd) and N1,N1-Dimethylbiguanide group (100mg/kgd) at random by body weight.Blank group and diabetes B model group administered dose physiological saline.The equal gastric infusion of each group above, successive administration was drawn materials after 5 weeks, surveyed blood glucose target.Blood is got in docking the day before yesterday of drawing materials, and survey rat blood sugar with blood glucose monitoring system, result is with mmol/L.

5.2.4. statistical procedures

With SPSS 15.0 processing data, adopt replicate measurement variance analysis (P < 0.05), all data represent with .x ± s.

In table 5 hedysarum polybotys saccharide 3 and 3,4 components are on the impact of tissue of experimental diabetic mice blood sugar

Note: contrast with blank group and model group, * P < 0.05.

From table 5,5 treatment groups and blank group, model group compare, and in different time points, mouse blood sugar all reduces statistical significance (P < 0.05), and in display HPS-3 and 3, after 4 components treatments, mouse blood sugars all reduce.

In table 6 hedysarum polybotys saccharide 3 and 3,4 components are on the impact of tissue of experimental diabetic mice sugar tolerance

Note: contrast with blank group and model group, * P < 0.05 ^{☆ ☆}p < 0.01

All glucose mouse blood sugar can be reduced to some extent at 2h by 4 components in the visible HPS-3 and 3 of table 6, compare with model group with blank group, all there is statistical significance (P < 0.05), HPS-3-A (P < 0.01); The reduction of 5 group Area under the curve of blood glucoses is compared with model group with blank group, all has statistical significance (P < 0.05), HPS-3-A (P < 0.01); Illustrate that model group exists impaired glucose tolerance, in HPS-3 and 3,4 components all can improve diabetes B and insulin resistant.

In table 7HPS-3 and 3,4 components are on the impact of rat model of type 2 diabetes mellitus blood sugar

Note: contrast * P < 0.05 with blank group and contrast with model group ^△p < 0.05 and MET group contrast, ^☆p < 0.05

From table 7, rat model of type 2 diabetes mellitus model group blood glucose value is very high, and the blood glucose value that HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group and N1,N1-Dimethylbiguanide (MET) are organized is all lower, compares with model group and there is statistical significance (P < 0.05).Illustrate that comparatively MET compares, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 have hypoglycemic activity better.In addition, in experimentation, also to observe in hedysarum polybotys saccharide the mental status that 5 administration groups all obviously can improve diabetes rat " three-many-one-little " symptom and rat, MET does not then have.

This experiment on the impact of tissue of experimental diabetic mice blood sugar, shows it all to insulin-dependent by inquiring into 4 different componentss in hedysarum polybotys saccharide 3 and 3---the blood glucose in diabetic mice caused by tetraoxypyrimidine raises significant restraining effect; Carbohydrate tolerance test shows: 4 components can improve Diabetic mouse glucose tolerance in hedysarum polybotys saccharide 3 and in 3, suppress the rising of postprandial blood sugar quickly, may have stronger repair ability to beta Cell of islet.

In hedysarum polybotys saccharide 3 and 3,4 components all obviously can improve the mental status of diabetes rat " three-many-one-little " symptom and rat, show that it is while reduction diabetes B rat blood sugar, by regulating Organism of Rats immunologic function, thus the life quality of diabetes rat can be improved on the whole.

Six, the research of 4 component reducing blood lipid in hedysarum polybotys saccharide 3 and 3

6.1 experiment reagents and instrument

6.1.1 experimental drug and reagent

HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 are all mixed with 7.5g/l solution, preserve under being placed in 4 DEG C of conditions;

Total cholesterol test kit Zhongsheng Beikong Biological Science & Technology Co., Ltd.;

Triglyceride test kit Zhongsheng Beikong Biological Science & Technology Co., Ltd.;

High density lipoprotein cholesterol test kit Zhongsheng Beikong Biological Science & Technology Co., Ltd.;

Glucophage metformin hydrochloride tablet (lot number: 0706066)

6.1.2 laboratory apparatus:

Abbott Laboratories Abbott Alcyon 300 automatic clinical chemistry analyzer

6.1.3Wistar rat, SPF level, body weight 180 ~ 220g, ♀, laboratory animal occupancy permit number:

SCXK (sweet) 2004-0006) provided by Gansu Chinese of Traditional Chinese Medicine's scientific experiment center.

6.1.4 dosage choice and feeding

HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 dosage is 0.075g/kg.bw.Per os gives once a day, and continuous gavage surveyed indices after 30 days.If Normal group and model group (0g/kg.bw) and N1,N1-Dimethylbiguanide group, replace tested material with water (sterilizing).

High lipid food formula is: 7.88% basal feed, 1% cholesterol, 10% yolk powder, 10% lard .02% cholate.

6.2 experimental technique

6.2.1 the grouping of rat and raising

Under experimental situation, rat feeding basal feed, observes 10 days.Fasting 16 hours, gets hematometry serum total cholesterol (TC), triglyceride level (TG) and high density lipoprotein cholesterol (HDL-C).Be divided at random according to basic value level: blank group, high fat control group, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 seven groups.

Adopt preventative high fat animal model experiment method.The polysaccharide soln of HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group gavage same dose while giving high lipid food; Normal group gives chow diet, the water of gavage respective volume while high fat control group gives high lipid food.To tested material after 30 days, fasting 16 hours, measures each group of rat blood serum total cholesterol, triglyceride level and high density lipoprotein cholesterol.

6.2.2 serum total cholesterol determination method

Rat femoral is taken a blood sample, and collect serum Abbott Laboratories' Abbott Alcyon 300 automatic clinical chemistry analyzer and measure, result is with molL ^-1represent.

6.2.5 Data Processing in Experiment:

Experimental data statistic software SPSS software carries out variance analysis statistics.

6.3 results and discussion

6.3.1HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group is on the impact of SD Serum TC

In table 11HPS-3 and 3,4 components are on the impact of rat model of type 2 diabetes mellitus reducing blood lipid

Note: contrast with blank group ^*p < 0.05 and model group contrast ^△p < 0.05 and MET group contrast ^☆p > 0.05

From table 11, rat model of type 2 diabetes mellitus model dysbolism of blood fat shows that TC, TG content increases, and HDL-C content reduces, and LDL-C content raises.HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 improve diabetes rat dysbolism of blood fat: (1) reduces the content of TC, TG in blood plasma.Compare with diabetes B model group, all have statistical significance (p < 0.05).(2) content of HDL-C is made to increase (P > 0.05); The content of LDL-C is reduced, compares with model group, each dosage group of administration group all has statistical significance (p < 0.05).HPS-3 also improves dysbolism of blood fat while improving diabetes B rat model hyperglycemia, and without dose-dependently.

6.4 conclusion

Experiment adopts preventative hyperlipidemia model experimental method, per os gives rat HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 is after 30 days, compare with model control group: HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group not only can reduce the content (P < .005) of serum serum triglyceride and reduce the content (P < .005) of serum total cholesterol, and the content (P < .005) of energy increasing serum high density lipoprotein cholesterol, decision method according to hypolipemic function can be reached a conclusion: HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 has prevention and reduces the effect of hyperlipidemia.

Seven. 4 component strengthening immunity functional study in hedysarum polybotys saccharide 3 and 3

7.1 materials and methods

7.1.1HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 are all mixed with 7.5g/l solution, preserve under being placed in 4 DEG C of conditions;

7.1.2 on the impact of mouse immune organ weight: see the table 3 in four.

7.1.3 on the impact of Phagocytosis By The Peritoneal Macrophages In Mice

Get Kunming mouse 70, body weight 19-21g, male and female half and half, be divided into contrast and experimental group at random.Administration group gavage gives HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 that concentration is 7.5g/l, and control group gives tap water.Gavage volume 100mg/kg body weight.Every day 1 time, successive administration 14 days.Drug withdrawal every mouse peritoneal on the same day injects 5% chicken red blood cell normal saline suspension 0.5ml.Animal is put to death after 20 hours.Injection 2.5mlHank liquid rinses abdominal cavity, and get 0.2ml and rinse drop on slide glass, 37 DEG C of temperature incubate 30 minutes.Wash away not adherent cell with physiological saline, methyl alcohol is fixed, and a Ji-Rui Shi mixes dye liquor dyeing.Calculate the cell count of 200 macrophage phagocytic chicken red blood cells under mirror, calculate phagocytic percentage, and be calculated as follows phagocytic index.

Phagocytic index=by chicken red blood cell sum/100 scavenger cells engulfed

7.1.4 the inhibiting impact of red corpuscle, oligoleukocythemia and immune organ is caused on endoxan

Kunming mouse 70, body weight 19-21g, male and female half and half, are divided into 6 groups at random.Blank group, endoxan group, HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group, in 1,3,6 days each 1 time of intraperitoneal injection of cyclophosphamide 50mg/kg, administration group gavage 100mg/kg body weight, every day gastric infusion 1 time, continuous 7 days, and in each 1 time of 1,3 and 6 day abdominal injection ring phosphorus phthalein amine 50mg/kg.After last administration 1 hour, tail point counted red, white corpuscle under getting blood mirror, and put to death mouse and claim Thymus and spleen weight in wet base.

8.1 result

In table 12 HPS-3 and 3,4 components are on the impact of Phagocytosis By The Peritoneal Macrophages In Mice

* P < 0.05 * * P < 0.01 is compared with control group

Table 12 shows, the phagocytic function of Turnover of Mouse Peritoneal Macrophages strengthens, and phagocytic rate is increased to 62% by control group 27%.

In table 13 HPS-3 and 3,4 components cause red corpuscle to ring phosphorus phthalein amine, leukocyte count reduces and the impact of immunosuppressive action:

Table 13 shows, cyclic phosphoric acid amine can cause red corpuscle, leukocyte count reduces and spleen, atrophy of thymus gland. compared with ring phosphorus phthalein amine group, HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 can make red corpuscle, quantity of leucocyte significantly increases, and Thymus and spleen weight is increased.

8.2 discuss

HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 promote the effect of Phagocytosis By The Peritoneal Macrophages In Mice, in addition, HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 also can increase immune organ weight, suppress mouse red blood cell, oligoleukocythemia and atrophy of immune organ that ring phosphorus phthalein amine causes.Prompting HPS-3-A HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 not only can increase non-specific immune function, also can resist the toxic action of chemotherapeutic.

Nine. 4 component radiation-resisting functional researchs in hedysarum polybotys saccharide 3 and 3

9.1 experimental section

9.1.1 experiment material

Healthy Male Kunming strain mice, 20 ± 2g, in 6 ~ 7 weeks ages of mouse, provided by Lanzhou University's GLP Experimental Animal Center, totally 90 × 2 batches, often organize 15, be divided into 6 groups at random, blank group (not irradiating not administration), experiment contrast group (irradiation model group, only irradiate not administration), HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group (100mg/kg).

8.1.2. experimental technique

Animal dis motility rate measures: male and healthy mouse 90, after laboratory adaptability feeds three days, is divided into 7 groups at random, often organizes 15.Normal group and radiation control group give normal saline, and all on an empty stomach gavage in morning, every day 1 time, each 0.5mL, weighs for every two days, according to body weight adjustment dosage, period animal freely ingest and drink water.Carry out x-ray source irradiation after each group of continuous gavage 10d, after radiation, continue gavage 10d, observe 30d survival rate and survival time after irradiating.

Peripheral blood cells number, thymus gland index and spleen index measure: healthy male mice 90, after laboratory adaptability feeds three days, is divided into 7 groups at random, often organize 15.Blank group and radiation control group give equivalent distilled water, all in gavage on an empty stomach in morning, every day 1 time, each 0.5mL, every day weighs, and record also adjusts dosage according to body weight, period animal freely ingest and drink water.X-ray bombardment is carried out after each group of continuous gavage 10d, in pre-irradiation, irradiate after the 2nd, 5,7,11,16d, cut the blood sampling of tail point respectively, peripheral blood leucocyte and lymphocyte number, oxyphorase, thrombocyte, reticulocyte is surveyed with automatic blood cell analysis machine (Japanese Sysmex, KX-21N) after adding diluent.After irradiating, 15d weighs Mouse Weight, and cervical dislocation is won the Thymus and spleen of mouse, after blotting residual blood, weighed, respectively divided by Mouse Weight, then be multiplied by 10, obtain Thymus and Spleen index with filter paper after putting to death.

Illuminate condition: carry out disposable full-body exposure with 6MHz x-ray radiation source (being provided by Lanzhou ground force Zong Yuan) to mouse, absorbed dose rate is 1.0Gy/min, and absorption dose is 8Gy, and spacing is 50cm, observes mouse survival rate; Absorbed dose rate is 1.0Gy/min, and absorption dose is 4Gy, and spacing is 50cm, and mouse carries out disposable full-body exposure observation to the impact of mouse hemopoietic function.

Statistical procedures:

Data represent with mean ± standard deviation (X ± S), and group difference significance adopts t inspection, adopts SPSS statistical software,

Application LSD method compares between two.The variance analysis of peripheral blood cells number, significance (p < 0.01,0.05) is as effective foundation.

Meter software, application LSD method compares between two.The variance analysis of peripheral blood cells number, significance (p < 0.01,0.05) is as effective foundation.

9.2 results and discussion

9.2.1 on the impact of Mouse Weight:

In table 14.HPS-3 and 3,4 components are on the impact (g, x ± s) of Mouse Weight

^*p < 0.05 compared with irradiation model group, ^*p < 0.01 compared with blank group.

Result shows: within after irradiating 2nd ~ 5 days, respectively organize body weight except blank group and all obviously decline, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 dosage group and model group all have significant difference.Along with the oral time lengthening of dosage group, body weight also slowly increases thereupon, illustrates that HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 all can increase Mouse Weight after irradiation.

9.2.2HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 affected by according to mouse x-ray bombardment mouse survival rate 30 days:

In table 15.HPS-3 and 3,4 components are on the impact of irradiating by 8Gy X-each group of mouse survival rate

^*p < 0.05 compared with irradiation model group, ^*p < 0.01 compared with irradiation model group.

Each group of mouse (except blank group) same day after radiation exposure, mouse appetite decline activity reduces, and body weight all declines, and occurs One's spirits are drooping depressed successively, auricle and afterbody pale, there is blood cake in some animals auricle, irradiation group is more obvious.But As time goes on mouse rejuvenates gradually, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group recovers very fast, about two weeks; Mouse hair color is glossy and denser, lessly occurs the sparse loose phenomenon of fur.After irradiation, it is fluffy and disorderly by hair that irradiation group mouse starts appearance, sparse tarnish, and food consumption declines, dead successively from the 8th day.Irradiation group is compared with experimental group, and the death time, comparatively early death toll was more.As can be seen from Table 4, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group all can improve the survival rate of irradiation mouse.HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 amount group compares with irradiation group, and dead mouse mean survival time extends all to some extent, can significant prolongation mean survival time.

Survival rate and survival time are the substantially the most objective indexs reflecting radiation resistance, because radiation-resistant final purpose is exactly to improve survival rate and extending the survival time.Experimental result shows that HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 group is irradiated mouse to X-and had good radioprotective effect; can increase the body weight of mouse, mean survival time and 30d survival rate, namely HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 have provide protection to x-ray irradiation mouse.

9.2.3 HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 are on the impact of irradiated mice peripheral blood leucocyte and lymphocyte quantity:

In table 16.HPS-3 and 3,4 components are on the impact (10 of leukocyte counts after mouse pre-irradiation ⁹/ L, X ± S)

^*compared with irradiation model group, P < 0.05 ^*compared with irradiation model group, P < 0.01

The impact (10 that in table 17.HPS-3 and 3,4 components count mouse irradiation peripheral lymphocyte ⁹/ L, X ± S)

^*compared with irradiation model group, P < 0.05 ^*compared with irradiation model group, P < 0.01

After median dose 4.0Gry actinism, what mainly suffer major injury is take marrow as the hemocytopoietic organ of representative.Irradiation makes hemopoietic function suppressed, occurs the early radiation effect of hemopoietic system: hemopoietic stem cell reduces, and breeds ineffective, and hematopoieticmicroenviron-ment regulation and control imbalance, Hemopoietic factor network adjustment is disorderly, causes pancytopenia.

As shown in Table 16, each administration group more all significance can improve irradiation murine interleukin quantity at the 16th day with model group.From table 17, each administration group can significantly improve peripheral blood lymphocyte quantity.The change of leukocyte count and peripheral white blood cell is basically identical.To sum up, each administration group peripheral blood leucocyte and lymphocyte number have raising in various degree.

9.2.4HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3-are on the impact of mouse spleen index and thymus index:

In table 18.HPS-3 and 3,4 components are on the impact of radiation murine thymus gland, index and spleen index

^*p < 0.05 compared with irradiation model group, ^*p < 0.01 compared with irradiation model group.

Thymus gland is the important immune organ of body, plays an important role in lymphocytic ripening process.Radiation damage can make atrophy of thymus gland, and thymic weight alleviates, and the change of its function adjoint, cause immature lymphocyte showed increased in body, thus affect the immunologic function of body, so general using thymus gland as the index of studies on immune function.

Compare with model group, the thymus index of each administration group all has significant difference, and spleen is immune organ and the hemopoietic tissue of mouse, and each administration group index and spleen index and Normal group there are no significant difference, but has significant difference with model group.These results suggest that HPS-3-A, HPS-3-B, HPS-3-C ,-D, HPS-3 all can improve the organ index of immune organ thymus gland, spleen, have promoter action to the recovery of mouse immune damage.This experimental result shows, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, HPS-3 all have better protecting effect to the radiation injury of X-induced by X-ray.

Accompanying drawing explanation

Fig. 1 is the HPGPC figure of 4 components in HPS-3;

Fig. 2 is HPS-3-A differential figure (1) and an angle of 90 degrees laser peak color atlas (2);

Fig. 3 is HPS-3-B differential figure (1) and an angle of 90 degrees laser peak color atlas (2);

Fig. 4 is HPS-3-C differential figure (1) and an angle of 90 degrees laser peak color atlas (2);

Fig. 5 is HPS-3-D differential figure (1) and an angle of 90 degrees laser peak color atlas (2);

Fig. 6 is the mass profile of 4 components in HPS-3;

Fig. 7 is the rootmean-square rotation radius distribution plan of 4 components in HPS-3;

Fig. 8 is the graph of a relation between Rg and the Mw of HPS-3-A;

Fig. 9 is the graph of a relation between Rg and the Mw of HPS-3-A;

Figure 10 is the graph of a relation between Rg and the Mw of HPS-3-A;

Figure 11 is the graph of a relation between Rg and the Mw of HPS-3-A;

Figure 12 is that in hedysarum polybotys saccharide 3,4 component TLC scheme (1.HPS-3-A; 2.HPS-3-B; 3. mix monose standard substance (Rf value is ascending is followed successively by semi-lactosi, glucose, pectinose, wood sugar, rhamnosyl); 4.HPS-3-C; 5.HPS-3-D);

The gas chromatogram of Figure 13 mixing monose standard substance;

The gas chromatogram of Figure 14 HPS-3-A;

The gas chromatogram of Figure 15 HPS-3-B;

The gas chromatogram of Figure 16 HPS-3-C;

The gas chromatogram of Figure 17 HPS-3-D.

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.

(1) extraction of hedysarum polybotys saccharide 3

After red stilbene pulverizing medicinal materials, with 95% alcohol degreasing 1 ~ 3h.5 ~ 20 times of water yields are extracted 1 ~ 5 time at 40 ~ 100 DEG C, each 1 ~ 3h.United extraction liquid, is condensed into certain volume.First be 0 ~ 70% alcohol settling with final concentration by concentrated solution, filter, precipitation; Supernatant liquor final concentration after above-mentioned precipitation is 20% ~ 80% ethanol redeposition; Supernatant liquor after redeposition is 30% ~ 90% alcohol settling with final concentration again, filters, and pellet frozen is dry, obtains hedysarum polybotys saccharide 3 (HPS-3).

(2) separation and purification of hedysarum polybotys saccharide 3

HPS-3 is through Sevag method deproteinated, and hydrogen peroxide for decoloration, the HPS-3 solution after decolouring is concentrated into certain volume, is 70% ~ 90% alcohol settling 1 ~ 3 time with final concentration, lyophilize.After HPS-3 water dissolution after decolouring, cross DEAE-cellulose 52 column chromatography (2.6cm × 70cm), respectively with the NaAc-HAc buffering salt wash-out containing 0 ~ 2M different concns NaCl, phend-sulphuric acid spike, obtains 4 components.

Above 4 components cross Sephadex G-100 (2.6cm × 90cm), Sephadex 6-75 gel column (2.6cm × 90cm) respectively repeatedly, use distilled water wash-out, finally obtain HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D 4 components.

HPS-3 crosses DEAE-cellulose 52 column chromatography, and the pH value of elute soln need keep about 5.2.Adopt containing 0,0.1, the NaAc-HAc buffering salt wash-out of 0.3M 3 kinds of different concns NaCl.Before loading, for insoluble impurity centrifugal segregation.

(3) conformational analysis of 4 components in hedysarum polybotys saccharide 3

Through HPGPC qualification, HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D are homogeneous components, adopt Ultrahydrogel 1000,500 Coupled columns gel chromatography, and moving phase is the 0.1MNaNO containing 0.02%NaN3 ₃solution, flow velocity is 0.8ml/min, column temperature: 40 DEG C; The light source gas of MALLS is helium and neon, wavelength: 690nm.The refractive index of moving phase gets 1.330, and polysaccharide refractive index increment in the solution (dn/dc) is 0.135, and the instrument(al)constant of calibration laser is 8.1104 × 10 ^-6(V cm) ^-1, calibration differential refraction detector instrument(al)constant is 2.2697 × 10 ^-4(V cm) ^-1.

Precision takes sample HPS-3-A, HPS-3-B, HPS-3-C, HPS-3-D, is mixed with 5mg/ml solution by moving phase, considers membrane filtration, carry out GPC-MALLS analysis through 0.22 μm.

HPS-3-A is spherical conformation, and weight-average molecular weight is 3 ~ 20KDa, and rootmean-square rotation radius Rg is 20 ~ 70nm, and polydispersity coefficient Mw/Mn is 1.000 ~ 1.300; HPS-3-B is highly-branched degree structure, and weight-average molecular weight is 10 ~ 200KDa, Rg be 10 ~ 25nm, Mw/Mn is 1.005 ~ 1.410; HPS-3-C conformation is a random ball of string, and weight-average molecular weight is 15 ~ 200KDa, Rg be 10 ~ 23.5nm, Mw/Mn is 1.100 ~ 1.350; HPS-3-D is highly-branched degree structure, and weight-average molecular weight is 11 ~ 150KDa, Rg be 11 ~ 25nm, Mw/Mn is 1.800 ~ 3.000.

GPC-MALLS analyzes, and sample is in complete dissolved state in the solution, and sample concentration is about 5mg/ml.Adopt 0.02%NaN ₃0.1MNaNO ₃solution is moving phase.

(4) in hedysarum polybotys saccharide 3, the monose of 4 components forms

1. thin-layer chromatography condition

Developer: phthalic acid 1.6g is dissolved in water saturated propyl carbinol 100mL, adds aniline 0.93g (being equivalent to 0.9mL).Developping agent: V (ethyl acetate): V (Glacial acetic acid): V (methyl alcohol): V (water)=7: 2: 2: 1.Join before use.Thin layer plate: silica-gel plate, uses 0.3molL ^-1naH ₂pO ₄cMC-Na solution bed board, 105 DEG C of activation 0.5h.

2. GC conditions

Temperature programming: keep 4min at 120 DEG C, then with 5 DEG C of min ^-1rise to 190 DEG C, keep 4min, then with 3 DEG C of min ^-1rise to 210 DEG C, keep 10min.

Injector temperature: 250 DEG C; Detector temperature: 240 DEG C; Nebulizer gas pressure: 0.5Mpa

Adopt the molar ratio of monose in chromatographic peak area normalization method working sample, determine the mixed polysaccharide that HPS-3-A is made up of pectinose, seminose, glucose, semi-lactosi, the molar ratio of each monose composition of HPS-3-A is: glucose: semi-lactosi :=70 ~ 95: 1 ~ 10, HPS-3-B, HPS-3-C, HPS-3-D are all then the mixed polysaccharide by pectinose, seminose, glucose, semi-lactosi, rhamnosyl, and the molar ratio of HPS-3-B monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=10 ~ 55: 5 ~ 50: 0 ~ 15: 0 ~ 18; The molar ratio of HPS-3-C monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=12 ~ 40: 15 ~ 65: 0 ~ 13: 0 ~ 3; The molar ratio of HPS-3-D monose composition is: pectinose: semi-lactosi: glucose: rhamnosyl=15 ~ 60: 13 ~ 55: 0 ~ 10: 0 ~ 25.5.

(5) determination of polysaccharide of 4 components in hedysarum polybotys saccharide 3

The preparation of standardized solution: each 20mg of various monose accurately taking drying, is dissolved in 100mL distilled water respectively.It is 40,60 that reference liquid is diluted to concentration respectively, 80,100,120,150 μ gm L ^-1solution, get each 0.2mL of this solution (sugary 8 ~ 30 μ g, as follows) be placed in 10mL tool plug test tube, after adding 5% phenol solution 0.5mL mixing, add rapidly the 2.5mL vitriol oil, mixing, 15min is incubated in 60 DEG C of water-baths, cool immediately, survey absorbancy at 490nm place, using distilled water as blank.

In HPS-3-A, polysaccharide content is 50 ~ 100%; In HPS-3-B, polysaccharide content is 50 ~ 99%; In HPS-3-C, polysaccharide content is 50 ~ 98%; In HPS-3-D, polysaccharide content is 50 ~ 98.5%.

(6) the saccharide portion chemical structure research of 4 components in hedysarum polybotys saccharide 3

The structure of 4 fraction polysaccharide parts in Partial acid hydrolysis, the hedysarum polybotys saccharide 3 that methylated in conjunction with technical study such as compounds GC-MS is adopted to be studied.

Hedysarum polybotys saccharide 3-A (is abbreviated as p) glucose by α-D-(1 → 4) pyrans and forms main chain, 1 branch is had in O-6 position every 3 ~ 7 saccharide residues, wherein branch contains → 1) Glc, → 1) Gal (4 →, every 8 ~ 12 saccharide residues have 1 sulfate, and sulfate is in O-6 position; Hedysarum polybotys saccharide 3-B is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 7 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →, with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 13 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-C is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has comparatively higly branched chain, every 4 ~ 9 saccharide residues have 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →; With → 1) Rha (2 →, also have non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 11 ~ 16 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi; Hedysarum polybotys saccharide 3-D is formed main chain with β-D-(1 → 4) semi-lactosi and β-D-(1 → 4) galacturonic acid, wherein also has a large amount of side chain, every 1 ~ 5 saccharide residue has 1 shorter or long-chain branch in O-6 position, side chain has → 1) Ara (5 →, → 1) Ara (3,5 → with → 1) Rha (2 →, also has non-reducing end α-D-(1 →) Glucopyranose in addition, Ara (1 →, every 15 ~ 19 saccharide residues have 1 sulfate, and sulfate is present in the O-6 position of β-D-(1 → 4) semi-lactosi.

(7) amino acid analysis of 4 components in hedysarum polybotys saccharide 3

First the glycoconjugate sample prepared taking certain mass is dissolved in the hydrochloric acid soln of 0.5ml6mol/L, after moving into hydrolysis pipe tube sealing, at 110 DEG C of hydrolysis 20 ~ 24h, cuts tube sealing, after draining, carries out amino acid analysis with amino acidanalyser.

Owing to not containing protein in HPS-3-A, therefore aminoacids content is that 0.HPS-3-B is mainly containing Val (37.5%) and Gly (22.3%), HPS-3-C is mainly containing Asp (19.8%), Glu (17.6%), Gly (15.7%) three seed amino acid, HPS-3-D then mainly contains Gly (24.6%), Glu (17.3%), Asp (13.1%) three seed amino acid.

(8) analysis of polysaccharide and albumen mode of connection

Getting 4 each 10mg of component in hedysarum polybotys saccharide 3 is dissolved in 5ml 0.1mol/L NaOH solution, and 30 DEG C of reaction 20h, measure the change of 240nm place aminoacids content before and after alkaline purification.

In hedysarum polybotys saccharide 3,4 components are after β-eliminative reaction, and HPS-3-B, HPS-3-C, HPS-3-D have obvious absorption peak at 240nm place, therefore can illustrate that three is O-glycoprotein compositions.

(9) hedysarum polybotys saccharide 3 capsule preparation

After red stilbene pulverizing medicinal materials, 5 ~ 20 times of water yields are extracted 1 ~ 5 time at 40 ~ 100 DEG C, each 1 ~ 3h.United extraction liquid, is condensed into certain volume.First be 0 ~ 70% alcohol settling with final concentration by concentrated solution, filter, precipitation; Supernatant liquor final concentration after above-mentioned precipitation is 20% ~ 80% ethanol redeposition; Supernatant liquor after redeposition is 30% ~ 90% alcohol settling with final concentration again, filters, and precipitate less than 60 DEG C dryings, adds 60%-95% ethanol particle, fill No. 1 capsule (loading amount is 0.1g-1.0g).

(10) hedysarum polybotys saccharide 3 beverage preparation

After red stilbene pulverizing medicinal materials, 5 ~ 20 times of water yields are extracted 1 ~ 5 time at 40 ~ 100 DEG C, each 1 ~ 3h.United extraction liquid, is equivalent to the medicinal extract of the red stilbene medicinal material of 0.01-1g, adds sweeting agent and correctives, make beverage in medicinal extract by every for extracting solution simmer down to 1ml liquid.

(11) hedysarum polybotys saccharide 3-A, the preparation of 3-B, 3-C, 3-D preparation

Hedysarum polybotys saccharide 3-A, 3-B, 3-C, 3-D powder, adds lubricant, disintegrating agent that pharmaceutical preparation is conventional, compressing tablet, film-making agent.

Hedysarum polybotys saccharide 3-A, 3-B, 3-C, 3-D powder, adds wetting agent, thinner that pharmaceutical preparation is conventional, granule processed.

Hedysarum polybotys saccharide 3-A, 3-B, 3-C, 3-D powder, add wetting agent, thinner that pharmaceutical preparation is conventional, particle processed, fills No. 1 capsule (loading amount is 0.1g-1.0g).

Hedysarum polybotys saccharide 3-A, 3-B, 3-C, 3-D powder, inject and make concentration reach every ml containing 0.01mg-10mg 3-A with water, 3-B, 3-C, 3-D, be packaged in ampulla, every bottle of 1ml-5ml, and autoclaving, makes sterile injectable preparation, for intramuscular injection or intravenous injection.

Hedysarum polybotys saccharide 3-A, 3-B, 3-C, 3-D powder, injects and makes concentration reach every ml containing 0.01mg-10mg 3-A with water, 3-B, 3-C, 3-D, autoclaving, be loaded in hundred grades of environment purifications in cillin bottle, lyophilize, make lyophilized injectable powder, for intramuscular injection or intravenous injection.

The foregoing is only explanation embodiments of the present invention; be not limited to the present invention, for a person skilled in the art, within the spirit and principles in the present invention all; any amendment of doing, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (8)

1. structure is such as formula the compound shown in I, and name is called hedysarum polybotys saccharide 3-A:

（Ⅰ）

Wherein, n=1 ~ 14.

2. structure is such as formula the compound shown in II, and name is called hedysarum polybotys saccharide 3-B:

（Ⅱ）

Wherein, n=1 ~ 85.

3. structure is such as formula the compound shown in III, and name is called hedysarum polybotys saccharide 3-C:

（Ⅲ）

Wherein, n=1 ~ 93.

4. structure is such as formula the compound shown in IV, and name is called hedysarum polybotys saccharide 3-D:

（Ⅳ）

Wherein, n=1 ~ 120.

5. the method for separating and preparing of hedysarum polybotys saccharide 3-A according to claim 1, hedysarum polybotys saccharide 3-B according to claim 2, hedysarum polybotys saccharide 3-C according to claim 3 or hedysarum polybotys saccharide 3-D according to claim 4, is characterized in that: obtain through following steps:

(1) hedysarum polybotys saccharide 3 is through Sevag method deproteinated, hydrogen peroxide for decoloration, and hedysarum polybotys saccharide 3 solution after decolouring is concentrated into certain volume, is 50% ~ 90% alcohol settling 1 ~ 3 time with final concentration, lyophilize; Hedysarum polybotys saccharide 3 after decolouring, with after water dissolution, crosses DEAE-cellulose 52 column chromatography, and respectively with the NaAc-HAc buffering salt wash-out containing 0 ~ 2M different concns NaCl, the pH value of elute soln keeps 4 ~ 7, and phend-sulphuric acid spike, obtains 4 components;

(2) more than, 4 components cross Sephadex G-100, Sephadex G-75 gel column respectively repeatedly, use distilled water wash-out, obtain hedysarum polybotys saccharide 3-A, hedysarum polybotys saccharide 3-B, hedysarum polybotys saccharide 3-C, hedysarum polybotys saccharide 3-D 4 components.

6. what any one or any two of hedysarum polybotys saccharide 3-A according to claim 1, hedysarum polybotys saccharide 3-B according to claim 2, hedysarum polybotys saccharide 3-C according to claim 3, hedysarum polybotys saccharide 3-D according to claim 4 were above is combined in the application prepared in antiaging agent, hypoglycemic drug, blood lipid-lowering medicine, raising immunity of organisms medicine, antiradiation drug.

7. a pharmaceutical preparation, comprise above combinations of any one or any two of hedysarum polybotys saccharide 3-A according to claim 1, hedysarum polybotys saccharide 3-B according to claim 2, hedysarum polybotys saccharide 3-C according to claim 3, hedysarum polybotys saccharide 3-D according to claim 4, and pharmaceutically acceptable auxiliary material.

8. pharmaceutical preparation according to claim 7, is characterized in that: the formulation of described preparation is solution, syrup, granule, capsule, powder, pill, tablet, aqueous injection, lyophilisate, patch, gelifying agent, film, pill, vina, extractum or sustained-release preparation.