CN103044567A - Extraction and separation method of phyllanthus urinaria polyose - Google Patents
Extraction and separation method of phyllanthus urinaria polyose Download PDFInfo
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Abstract
The invention discloses an extraction and separation method of phyllanthus urinaria polyose PULP III, which comprises the following steps of removing dust by washing a whole phyllanthus urinaria plant with distilled water, drying, grinding, degreasing with petroleum ether, removing micromolecular sugar with ethanol, leaching medicine dregs with water, conducting centrifugal separation on leached liquid, concentrating filtrate, conducting ethanol precipitation with ethanol, conducting deproteinization, washing, purification and dialysis on an ethanol precipitate, drying, obtaining phyllanthus urinaria fine polyose, conducting deproteinization and dialysis, treating with an ion exchange column, eluting with appropriate NaCl gradient solutions in sequence, conducting track detection by a phenol-sulfuric acid method, obtaining an elution curve, collecting a third main peak component, concentrating, dialysing with redistilled water for 48h, freeze-drying, and obtaining a pure phyllanthus urinaria polyose PULP III component. According to the method, the pure and unique phyllanthus urinaria polyose component is obtained by separating and purifying crude phyllanthus urinaria polyose.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of extraction and separation method of Common Leafflower Herb polysaccharide PULP III.
Background technology
Common Leafflower Herb (
Phyllanthus urinariaL
.), have another name called Herba Phyllanthi Urinariae, silk tree grass, yin-yang grass, belong to Euphorbiaceae (
Euphorbi aceae) the dry herb of phyllanthus plant Common Leafflower Herb, at promoting diuresis to remove toxic substance, the calming liver and clearing heat of being widely used among the people.1988, the people such as India scholar Thyagarajan
[2]First passage experimental results show that Phyllanthusamarus can make 59% patient's hepatitis B surface antigen (HBsAg) turn out cloudy, and this experimental result has caused the concern of Chinese scholars to Common Leafflower Herb.Studies show that in a large number
]Common Leafflower Herb has hepatitis B virus resisting, protecting liver, lowering enzymes, anti-hepatic fibrosis, prevents the effect of liver injury and anticancer change, and toxic side effect is low, is a kind of deeply crude drug of the treatment hepatitis B of exploitation that is worth.
Contain the number of chemical composition in the Common Leafflower Herb, document had been reported lignanoid, terpene, flavones, the matter of mixing, alkaloid etc., comprises that Octadecane, dehydrogenation chebulic acid, forulic acid, gallic acid, brevifolin carboxylic acid, Succinic Acid, methoxyl group are mixed to spend acid, camellia element, plain, the short leaf bush acid of heroubill formicester, short leaf Soviet Union art phenolic acid second fat, corilagin, dehydrogenation chebulic acid three formicesters, polysaccharide etc.
Polysaccharide compound has the multiple biological activitys such as immunomodulatory, antitumor, antiviral, anti-oxidant, anti-inflammatory, anti-peptic ulcer, anticoagulation, hypoglycemic, reducing blood-fat, radioprotective, antithrombotic, Ivy extract, antitoxin thing damage.
Hepatitis B (HBV) is global public health problem, according to global health organization (WHO) data, there are 3.5～4.0 hundred million Patients with Hepatitis B Virus Infections in the whole world, and wherein annual have nearly 1,000,000 patients to die from liver failure, liver cirrhosis and liver cancer that the HBV infection causes
[62]If be applied at present drug main Interferon, rabbit and the nucleoside analog for the treatment of hepatitis B, yet these medicines can only obtain result for the treatment of in a way, most patient is not reached the purpose of curing fully, and can occur the phenomenon of knock-on after the drug withdrawal.So develop efficient, low toxicity, the treating hepatitis B new drug of " knock-on " is not an instant problem.
In the process of seeking the treating hepatitis B medicine, the herbal polysaccharide composition more and more is subject to people's attention.Common Leafflower Herb has hepatitis B virus resisting, protect the liver, the effect such as antitumor generally confirmed, and relevant scholar is also to wherein several chemical ingredientss, done structure and bioactive research such as gallic acid, flavonoid and Common Leafflower Herb element etc., but to wherein polysaccharide researches seldom.The extraction of carrying out polysaccharide component in the Common Leafflower Herb separates, Structural Identification and bioactive detection, be conducive to further determine that Common Leafflower Herb is used for the treatment of the effective substance of hepatitis B, develop better this resources of medicinal plant of Common Leafflower Herb, lay theoretical basis for seeking the treating hepatitis B new drug.
Summary of the invention
The object of the present invention is to provide a kind of extraction and separation method of Common Leafflower Herb polysaccharide PULP III, by the separation and purification to the Common Leafflower Herb Crude polysaccharides, obtained pure single Common Leafflower Herb polysaccharide component.
For achieving the above object, the present invention adopts following technical scheme:
The extraction and separation method of Common Leafflower Herb polysaccharide PULP III of the present invention comprises the steps:
1) Common Leafflower Herb herb distilled water wash removes soil, after the drying, pulverizes, and uses petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, the concentrated rear ethanol alcohol precipitation that adds certain volume of filtrate is got in the vat liquor centrifugation, gets pure hypostasis and is thick total polysaccharides; Thick total polysaccharides is through 2) except albumen, 3) washing, removal of impurities and 4) dialysis, be drying to obtain the smart total polysaccharides (PULP) of Common Leafflower Herb, polysaccharide content is more than 95%.5) each component separation and purification of total polysaccharides: through deproteinated, the smart total polysaccharides (PULP) of Common Leafflower Herb after the dialysis treatment, cross ion exchange column, successively with suitable NaCl gradient solution wash-out, the phenolsulfuric acid method follow the tracks of to detect, and gets elution curve, the elution peak that can obviously declare out four peak shape symmetries from the curve, represent respectively four components, be designated as respectively PULP I, PULP II, PULP III and PULP IV according to the order that goes out the peak.Wherein the 2nd, the 3rd two elution peaks are larger, collect the 3rd main peak component, and are concentrated, redistilled water dialysis 48h, and lyophilize gets Common Leafflower Herb polysaccharide PULP III pure component.
Described Common Leafflower Herb polysaccharide PUIP III is the khaki color chip solid, is that a class does not contain N, S element acidic polysaccharose, and molecular-weight average is 418793.5 relatively; PULP III monose forms and ratio is rhamnosyl (Rha): pectinose (Ara): seminose (Man): glucose (Glc) is 0.27:0.28:0.1:0.35; Main chain is comprised of Rha, Ara, Man and Glc.Each monose connects by the pyranose glycosidic bond, and glycosidic link is connected to the master with 1 → 6, also has simultaneously 1 → 4 and 1 → 3 mode of connection.
Above-mentioned steps 1) optimum condition is the Phyllanthus urinaria of pulverizing, and exceeds medicinal material 0.5～1cm with sherwood oil submergence medicinal material and liquid level, 80 ℃ with normal pressure power refluxing extraction 3 times, each 2 hours, discard phegma.The dregs of a decoction continue to press same method 80 ℃ of ℃ of lower refluxing extraction 3 times with 50～100% ethanol, each 2 hours, discard phegma.The dregs of a decoction are used 90 ℃ of floodings 3 times again, and each 2 hours, merge 3 vat liquors and carry out centrifugation, centrifugal speed 4000r/min gets filtrate and is concentrated into original volume 1/4, adds 95% ethanol of 4 times of volumes, leaves standstill 24h, and is centrifugal, gets pure hypostasis and is Crude polysaccharides.
Step 2) namely use Sevag method deproteinated except albumen: the pure hypostasis that step 1) is obtained redissolves to get Crude polysaccharides solution with distilled water, the preferred system that adds chloroform and propyl carbinol mixed solution in Crude polysaccharides solution is Crude polysaccharides solution: chloroform and propyl carbinol mixed solution are 3:1 with the volume ratio metering, mixed solution=chloroform: propyl carbinol=4:1(volume ratio metering), magnetic agitation 30min, the sufficient standing layering discards precipitation.After repeating 7～8 times until interface is without white precipitate.
Step 3) washing, the optimum condition of removal of impurities are the 95% ethanol precipitation 24h that adds 4 times of volumes in except the polysaccharide soln behind the albumen, and be centrifugal, abandons filtrate, and precipitation is cleaned, repeated 3 times with dehydrated alcohol, acetone, ether successively.
The step 4) dialysis, drying: i.e. filter residue adds an amount of distilled water after the alcohol precipitation washing, dialysis after fully redissolving, dialysis is carried out under magnetic agitation, and sample liquid and dialyzate (distilled water) volume ratio optimum condition is 1:20, and 8h changes water one time, dialysis 72h.Dialyse complete after with sugar soln put into freeze drier dry Common Leafflower Herb total polysaccharides (PULP), total polysaccharides of the present invention (PULP) content is more than 95% after measured.
The ion exchange column of step 5) is DEAE-52 anionite-exchange resin, before using the DEAE-52 cellulose wadding is processed, its optimum condition use distilled water immersion 48h, during 3h change water one time, and remove suspended impurity with decantation, then carry out alkali-acid-alkali and process.Be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L first, be washed till neutrality with distilled water behind HCl solution soaking 30 min of 0.5 mol/L again, be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L at last, obtain OH
-The fiber type element.(column dimension is 500 * 50mm) to wet method dress post, and distilled water balance 48h avoids bubble and fault-layer-phenomenon in the dress post process.
The NaCl gradient solution of the gradient scope of the amount of substance concentration of the suitable NaCl of step 5)=0～1mol/L.
The gradient solution of the amount of substance concentration of the preferred NaCl of step 5) is respectively 0,0.1,0.25,0.5,0.75mol/L.
Step 5) is successively with suitable NaCl gradient solution wash-out, and the operation of optimizing is the amount of substance concentration eluant solution 8 hours of every kind of NaCl, and flow velocity 4mL/min collects elutriant with pipe, and every 10min collects a pipe.
The described elution curve of step 5) is for take the Guan Xu (pipe number) that collects as X-coordinate, and the absorbance of solution is that the ordinate zou drawing obtains.
Common Leafflower Herb polysaccharide PUIP III of the present invention has certain anti-oxidant, anti-hepatitis B virus activities.
Beneficial effect of the present invention: by the separation and purification to the Common Leafflower Herb Crude polysaccharides, obtained pure single Common Leafflower Herb polysaccharide component.By the research to the structural analysis of the pure component PULP III of polysaccharide and antioxidation in vitro, antiviral activity experiment, having proved conclusively the Common Leafflower Herb polysaccharide is the effective constituent of phyllanthus for treating hepatitis B, and this anti-HBV effect mechanism for research Common Leafflower Herb polysaccharide provides theoretical basis; Simultaneously, elementary composition to the physico-chemical property of Common Leafflower Herb polysaccharide fraction PULP III, functional group, initial analysis has been carried out in monose proportion of composing and glycosidic link position, for the later on further further investigation of Common Leafflower Herb polysaccharide molecule structure is provided fundamental basis.From now on can be on this basis, in conjunction with the structure activity relationship of polysaccharide, further exploitation lays the foundation take the Common Leafflower Herb polysaccharide as raw-material anti-hepatic-B virus medicine.
Description of drawings
Fig. 1 is the glucose typical curve.
Fig. 2 is polysaccharide gradient elution curve.
Fig. 3 is the rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg.
Embodiment
The present invention is described in detail below in conjunction with embodiment:
The extraction of Common Leafflower Herb polysaccharide
Common Leafflower Herb herb distilled water wash removes soil, after 50 ℃ of cryodryings, pulverizes, and seals for subsequent use.Its powder is yellow-green colour.
80 ℃ of backflow degreasings of sherwood oil → 80 ℃ of backflows of 95% ethanol are taken off small molecular sugar → 90 ℃ water refluxing extraction 3 times, it is centrifugal to merge 3 filtrate 4000r/min, be concentrated into 95% ethanol of 1/4 volume → 4 times of volumes of adding, leave standstill 24h, centrifugal, distilled water redissolution → sevag method is except albumen (chloroform: propyl carbinol=4:1, polysaccharide soln: mixed solution=3:1), magnetic agitation 30min, the sufficient standing layering repeats 7～8 operations and can remove free protein → add the 95% ethanol precipitation 24h of 4 times of volumes → use successively dehydrated alcohol except the polysaccharide soln behind the albumen, acetone, ether cleans, triplicate → dialysis 72h → lyophilize gets Common Leafflower Herb total polysaccharides (PULP).
The preparation of glucose reference liquid and sample test liquid
Precision takes by weighing dextrose standard sample (being dried to weight under 105 ℃ no longer changes) 10mg, behind dissolved in distilled water, places the 100mL volumetric flask, is settled to scale with distilled water, shakes up, and makes the glucose reference liquid of 0.1mg/mL, and is for subsequent use.
Precision takes by weighing the Common Leafflower Herb polysaccharide 10mg that is dried to constant weight, behind dissolved in distilled water, places 100mL volumetric flask adding distil water to be settled to scale, shakes up, and the sample test liquid of making 0.1mg/mL is for subsequent use.
Determining of absorbing wavelength
Get each 1 mL of glucose reference liquid and sample solution, add respectively 1mL 5% phenol solution (get 5g and heavily steam phenol adding distil water constant volume in the brown volumetric flask of 100mL), after shaking up, the unsettled vertical adding 5mL vitriol oil, put and heat 15min in the boiling water bath, then put in the cooling bath and cool off, full wavelength scanner in 400～600 nm scopes is determined absorbing wavelength.
The glucose standard curve making
Accurate glucose reference liquid 0.2,0.4,0.6,0.8, the 1mL of drawing is in 10mL tool plug scale test tube, adding successively water, to make final volume be 1mL, blank is 1mL water, then adding 1mL 5% phenol solution shakes up, add rapidly the 5mL vitriol oil (unsettled vertical adding), put and heat 15min in the boiling water bath, then put in the cooling bath and cool off, measure absorbancy in the 490nm place.Take absorbancy as Y-axis, glucose quality is X-axis, the drawing standard curve, and calculate regression equation.
The calculating of sugar content
The accurate test liquid 1mL that draws measures absorbancy in the 490nm place.
Calculate sugared content according to formula:
Sugar content=C/(C
0* V) * 100%
C: the glucose micrograms that is checked in by typical curve
C
0: the concentration of sample solution (0.1 mg/mL)
V: the sample solution volume (1.0mL) of using during mensuration
The extraction yield of polysaccharide
The quality of the extraction yield=polysaccharide of polysaccharide/raw-material quality * 100 %
The maximum absorption band wavelength of sample solution and glucose reference liquid is all located about 490 nm, so choose 490 nm as the measurement of the polysaccharide content wavelength.
Take absorbancy as Y-axis, glucose micrograms (μ g) is X-axis, and the drawing standard curve such as Fig. 1, can find out that glucose amount and absorbancy have good linear relationship in 20～100 μ g scopes.
The absorbance A that records sample liquid is 0.388, is 28.27 μ g according to the micrograms of its corresponding glucose of regression equation calculation.Get sugared content according to formula:
Sugar content=C/(C
0* V) * 100%=28.27/ (0.1 * 1) * 100%=28.27%
The extraction yield of polysaccharide==1.5/100 * 100 %=1.5%
Petroleum ether degreasing is passed through in this experiment, slough the small-molecule substances such as oligose with 95% ethanol again, then from Common Leafflower Herb, isolate polysaccharide with water extraction and alcohol precipitation method, and employing sevag method deproteinated, the phenolsulfuric acid method is measured its content, its maximum absorption wavelength is 490nm, and polysaccharide extract rate is 1.5%, and sugared content is 28.27%.
The Common Leafflower Herb Separation and purification
Post separates
The DEAE-52 filler pre-treatment
DEAE-52 cellulose wadding distilled water immersion 48h, during 4～5h change water one time, and remove suspended impurity with decantation, then carry out alkali-acid-alkali and process.Be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L first, be washed till neutrality with distilled water behind HCl solution soaking 30 min of 0.5 mol/L again, be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L at last, obtain OH
-The fiber type element.(column dimension is 500 * 50mm) to wet method dress post, and distilled water balance 48h avoids bubble and fault-layer-phenomenon in the dress post process.
Cross the separation and purification of DEAE-52 chromatography column
Take by weighing 640mg Common Leafflower Herb total polysaccharides and be dissolved in (8 mg/mL) in the 80mL redistilled water, excessively loading behind the 0.45 μ m filter membrane.Use successively 0,0.1,0.25,0.5,0.75mol/L NaCl gradient elution, every 10min collects a pipe, flow velocity 4mL/min; The phenolsulfuric acid method is followed the tracks of and detected, and collects each main peak component, and is concentrated, redistilled water dialysis 48h, and lyophilize gets each component of Common Leafflower Herb polysaccharide.
SephadexG-200 dextran gel filtration method
Pre-treatment: the SephadexG-200 filler adds an amount of distilled water immersion 24h, during 4～5h change water one time, and remove suspended impurity with decantation, then ultrasonic degas is not until there is bubble to occur in the coagulant liquid.(column dimension is 500 * 25mm) to wet method dress post, and is for subsequent use behind NaCl solution equilibria 48 h with 0.05 mol/L.
Column chromatography: each set of dispense of collecting is above made the sample liquid of 25mg/mL, and loading 2mL is with the NaCl wash-out of 0.05 mol/L.Automatically Fraction Collector is collected, and every pipe 5 mL, per 30 min collect a pipe, and the phenolsulfuric acid method is followed the tracks of and detected.
The HPLC high performance liquid chromatography
Each fraction polysaccharide behind the purifying is mixed with the sample liquid of 1mg/mL, crosses sample introduction behind the filter membrane of 0.45 μ m.
High-efficient liquid phase chromatogram condition:
Chromatographic column: PolySep-SEC 4000 Part No:00H-3144-K0 Column Size:300 * 7.8mm;
Detector: differential detector; Column temperature: 30 ℃; Moving phase: distilled water; Flow velocity: 0.8 mL/min; Sample size: 20 μ L.
Column chromatography for separation result
Through deproteinated, the smart polysaccharide (PULP) of Common Leafflower Herb after the dialysis treatment is crossed the DEAE-52 ion exchange column, uses successively 0,0.1,0.25,0.5,0.75mol/L NaCl solution gradient wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, and gets as shown in Figure 2 middle elution curve.As seen from Figure 2, PULP can obviously isolate the elution peak of four peak shape symmetries after the DEAE-52 column chromatography for separation, represent respectively four components, is designated as PULP I, PULP II, PULP III and PULP IV.Collecting the larger component PULP III of content is further analyzed.
Post and HPLC Purity
SephadexG-200 post result
Get Common Leafflower Herb polysaccharide fraction PULP III and cross the SephadexG-200 gel column, the NaCl wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, make elution curve with absorbance and wash-out pipe number, the elution curve of PULP III on Sephadex G-200 post is symmetrical simple spike, illustrates that PULP III component is the polysaccharide fraction of the relative homogeneous of molecular weight.
HPLC method Purity
When utilizing the HPLC method to detect the purity of Common Leafflower Herb polysaccharide fraction PULP III, the result shows, polysaccharide fraction PULP III after the separation and purification of DEAE-52 cellulose column, relatively more symmetrical in high performance liquid phase collection of illustrative plates superiors type, and calculating its purity through area normalization method reaches more than 95%, figure result matches with Sephadex G-200 gel chromatography, illustrates that purity is higher, can be used for doing Structural Identification.
Obtain four components after the Common Leafflower Herb polysaccharide process DEAE-52 cellulose column separation and purification behind the water extract-alcohol precipitation deproteinated, be designated as respectively PULP I, PULP II, PULP III and PULP IV.Collect the larger component PULP III of content, carry out Purity with SephadexG-200 gel filtration chromatography method and two kinds of methods of high performance liquid chromatography (HPLC), prove the relative homogeneous of its component, can further do structural analysis.
The physico-chemical property of III and structural analysis
State, dissolubility test
The polysaccharide fraction PULP III that takes by weighing after an amount of separation and purification is dissolved in pure water, dehydrated alcohol, acetone, ether and ethyl acetate equal solvent, observes its solvability.
Ninhydrin reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, add 1.0mL 0.5% ninhydrin reagent, boiling water bath 5min observes colour-change after the cooling, if red-purple occurs, then proof has protein, on the contrary then without.With distilled water and calf serum solution in contrast.
The Molish reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, drip two Molish reagent, shake up.The inclination test tube adds about 1mL vitriol oil along tube wall, careful vertically after, examine the colour-change of two-layer liquid level intersection behind 2～3min, if there is red-purple to occur, then proving has glucide.With distilled water and starch solution in contrast.
The phenolsulfuric acid reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, add the phenol of 1mL 5%, add the 5mL vitriol oil again, vibration if be orange-yellow, illustrates to contain sugar.Take distilled water as contrast.
The IKI reaction
Get the sample solution of 1.0mL 1.0mg/mL, drip 250 μ L Wagner's reagents after, if aobvious blue, illustrate that this polysaccharide is non-starch based.With the negative contrast of distilled water, 0.5% the positive contrast of starch indicating liquid.
Sulfuric acid-carbazole reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, in ice-water bath, in pipe, add the 6mL vitriol oil, shake up and be placed on 20min in 85 ℃ of water-baths, taking-up is cooled to room temperature, adds the carbazole liquid of 0.2mL 0.1%, keeps 2h under the room temperature, if there is the red-purple material to occur, illustrating and contain uronic acid, is acid sugar, with the negative contrast of distilled water.
The Fehling reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, add excessive fehling reagent, boil 2 min, if produce red red copper oxid precipitation, then proof contains reducing sugar.
Ferric chloride test
Get 1.0mg/mL sample solution 1.0mL in test tube, regulating the pH value is 4～5, adds 1% ferric chloride aqueous solutions, if blue, blackish green or bluish voilet occurs, then proof contains tannin class or phenols.
The sulfate qualitative test
Get 2mg polysaccharide sample, add 1.0mL 1mol/L HCl, then 110 ℃ of airtight hydrolysis 3h drip the bariumchloride of 0.5mL-gelatin reagent, if the generation of adularescent precipitation illustrates and contains sulfate.
The method determining molecular weight
High-efficient liquid phase chromatogram condition
Chromatographic column: PolySep-SEC 4000 Part No:00H-3144-K0 Column Size:300 * 7.8mm;
Detector: differential detector; Column temperature: 30 ℃; Moving phase: distilled water; Flow velocity: 0.8 mL/min; Sample size: 20 μ L.
The preparation of sample solution
Polysaccharide fraction PULP III is mixed with the solution that concentration is 1mg/mL, behind centrifugal 10 min of 10000r/min, crosses 0.45 μ m filter membrane, for subsequent use.
Standard control solution
The dextran standard series (T-10, T-20, T-110, T-500, T-2000) of various known molecular amounts is mixed with the solution that concentration is 1mg/mL, and treatment process is with sample solution, and is for subsequent use.
The drafting of typical curve
With the dextran standard of various known molecular amounts by small molecules to macromole successively average sample introduction 3 times, record corresponding retention time t
RAnd color atlas, with retention time t
RBe X-coordinate, the logarithmic value of molecular weight is ordinate zou drawing standard curve.
The mensuration of PULP III relative molecular mass
With each sample introduction of sample solution of preparing above three times, the HPLC chromatographic condition is the same with standard substance, records corresponding retention time t
RAnd color atlas, the regression equation calculation molecular weight that obtains according to typical curve.
The drafting of typical curve
Adopt the sulfuric acid carbazole method to measure glucuronic acid content.The glucal acid solution 25mL of preparation 0.2mg/mL measures 0.25,0.5,1.0,1.5,2.0, the 2.5mL mentioned solution successively, and constant volume is made the standardized solution of 5,10,20,30,40,50 μ g/mL and put in 4 ℃ of refrigerators for subsequent use in the 10mL volumetric flask.
Measure 5mL borax sulphuric acid soln (0.025 mol/L) in tool plug test tube, then ice bath carefully adds 1mL glucuronic acid standardized solution acid solution layer top, with distilled water as blank, the limit shakes up the cooling of (shake up gently first, rear acutely rock) limit ice bath.Then test tube is heated 10min in boiling water bath, add 0.2mL carbazole solution (0.125% ethanol solution) after being cooled to room temperature, shake up, boiling water heating 15min, be cooled to room temperature, measure the light absorption value at 530nm place, take the concentration of glucuronic acid as X-coordinate, light absorption value ordinate zou drawing standard curve.
The mensuration of glucuronic acid content in the sample
Polysaccharide fraction PULP III is made into the solution of 5 μ g/mL, operates the samely, measure it at the light absorption value at 530nm place.
Protein and nucleic acid determination
Polysaccharide fraction PULP III behind the purifying is mixed with the polysaccharide solution of 1mg/mL, see that in the interscan of 200～400nm scope it is at 260nm (nucleic acid) with the UV ultraviolet spectrophotometer, 280nm(Pr) locate whether absorption peak is arranged, to be determined with existing without protein and nucleic acid.
, H, N ultimate analysis
Take by weighing respectively the PULP III sample 2.5mg of two parts of complete dryinies, adopt Elementar Vario EL III elemental analyser to measure the content of C, H, N.
IR) detection
Get 2mg polysaccharide sample, with behind Potassium Bromide (KBr) compressing tablet at 400～4000cm
-1Do Infrared spectroscopy in the wavelength region.
Adopt the vapor-phase chromatography of sugared nitrile acetic ester derivative to carry out the monose composition measuring.Can determine the monose moiety of sample according to appearance time, according to the proportion of composing that goes out peak area and can determine monose.
The monose aldoononitrile acetate takes by weighing respectively rhamnosyl (Rha), wood sugar (Xyl), semi-lactosi (Gal), glucose (Glc), pectinose (Ara); each 10 mg of seminose (Man); add respectively the 10mg oxammonium hydrochloride; 0.5 after the vibration of mL pyridine; 90 ℃ of water-bath 30 min; and frequently vibration; after taking-up is cooled to room temperature; add 0.5 mL diacetyl oxide; acidylate 30 mim in 90 ℃ of water-baths; the product that obtains namely is sugared nitrile acetic ester derivative, directly injects GC and analyzes.
The polysaccharide aldoononitrile acetate takes by weighing polysaccharide 10mg, adds 2mol/L trifluoroacetic acid 5mL, behind 110 ℃ of vacuum sealing tube hydrolysis 4h, under 40 ℃, be evaporated to driedly, then add 4mL methyl alcohol, rotary evaporated to dryness, repeat 3～4 times, to remove trifluoroacetic acid.Then add 10 mg oxammonium hydrochlorides and 0.5 mL pyridine; put into 90 ℃ of heating in water bath for reaction 30 min; and vibration is chilled to room temperature after taking out frequently; add 0.5 mL acetic anhydride; continue reaction 30 min and carry out acetylize in 90 ℃ of water-baths, reaction product can directly be carried out gas chromatographic analysis
Chromatographic condition:
Chromatographic column: OV1701(30.0 m * 0.32 μ m * 0.25 μ m) capillary column;
Injector temperature: 250 ℃; (FID) detector temperature: 240 ℃;
Nebulizer gas pressure: 0.4 MPa; Temperature programming: 150 ℃ of lower 4 min that keep, then rise to 200 ℃ with 10 ℃/min, keep 8 min, rise to 280 ℃ with 15 ℃/min again, keep 8 min.
Get 20mg PULP III polysaccharide fraction, carry out the part acid hydrolysis with 2mL 0.05mol/L trifluoroacetic acid, centrifugal behind the hydrolysis 16h, precipitation is carried out GC and is analyzed.Add the methyl alcohol rotary evaporation in the supernatant liquor, repeat 3～4 times and remove trifluoroacetic acid, after then water redissolves, dialysis tubing (molecular retention amount 3500) dialysis.After dialysis finishes, carry out GC after the outer part vacuum-drying of bag and analyze; Part adds the alcohol chromatography of 4 times of volumes in bag, and supernatant part and alcohol precipitation part are carried out the GC analysis after the vacuum-drying respectively.
The smith degraded
The making of sodium periodate typical curve
Get 6 dry test tubes, according to the form below operation production standard curve:
Get respectively the 0.1mL mentioned solution, be settled to 25 mL, measure optical density(OD) at 223 nm places.Then take sodium periodate concentration as X-coordinate, optical density value is ordinate zou, the drawing standard curve.
Sample preparation
Accurately take by weighing sugared sample 25 mg, be dissolved in 15 mmol/L NaIO
4In the solution, put in the 25 mL volumetric flasks, constant volume shakes up.In the dark reaction under 4 ℃, respectively 0,6,12,24,36,48,60h ... after 0.1 mL that takes a sample successively, place the brown volumetric flask of 25mL, be settled to scale with distilled water, namely dilute 250 times, do blank with distilled water, measure the absorbancy at 223 nm places, until absorbance stable after, add ethylene glycol and place 20min, destroy excessive Periodic acid termination reaction.According to the optical density(OD) stationary value that records, according to the sodium periodate typical curve, can calculate the consumption of Periodic acid.
Formic acid is measured
Getting the reaction soln that the above-mentioned ethylene glycol of 2mL was processed, is under the condition of indicator in tetrabromo-mcresolsulfonphthalein, with NaOH solution (take Potassium Hydrogen Phthalate as the demarcating liquid) titration of 0.01mol/L, calculates the growing amount of formic acid.
The Smith degraded
Polysaccharide sample solution dialysis 48h after the ethylene glycol processing is evaporated to about 10mL under 40 ℃, adds 70mg NaBH under the room temperature
4After, place the dark place to stir 24h, so that the reduction polysaccharide aldehyde.Behind the 24h, add among 50% HAc and remaining NaBH
4, to pH be 6～7 rear dialysis 48h (flowing water and distilled water respectively 24h).After dialysis is finished, add the H of 1 mol/L of equal volume
2SO
4, vacuum sealing tube, then 100 ℃ of Water Under solution 8h add BaCO
3Regulate pH to 6, filter with quantitative paper, filtrate vacuum concentration evaporate to dryness carries out GC and analyzes after the acetylize.
The PULP III is the khaki color chip solid, and is soluble in water, is insoluble to ethanol, acetone and other organic solvent.Molish reaction, phenolsulfuric acid reaction, sulfuric acid-carbazole reaction positive, ninhydrin reaction, IKI reaction, the Fehling reaction, ferric chloride reaction and sulfate reaction are negative.
The molecular weight of III
Color atlas and t according to each standard specimen
R, try to achieve logarithmic value log Mw and the t of molecular weight
RThe regression equation of relation is y(log Mw)=-0.5487x(t
R)+9.3541 (R
2=0.9995).The retention time of PULP III is 6.801 min, and the relative molecular weight of trying to achieve sample P ULP III according to regression equation is 418793.5.
The regression equation that sulfate-carbazole is measured glucuronic acid content is: y=0.0146x+0.0917 (R
2=0.9994).
Recording the light absorption value of Common Leafflower Herb polysaccharide fraction PULP III at the 530nm place is 0.75, and according to regression equation calculation as can be known, glucuronic acid content is 45.1%.
Protein and nucleic acid in the III molecule
The ultraviolet absorpting spectrum of PULP III shows without absorbing without protein and nucleic acid, so exist without protein and nucleic acid in the PULP III.
The III ultimate analysis
PULP III results of elemental analyses is shown in table 2-1:
Table 2-1 results of elemental analyses
2-1 finds out that the N percentage composition in conjunction with front ultraviolet absorption spectroscopy result, can not contain amino in the interpret sample below 7% in the polysaccharide, is non-aminosugar by table.Because if be aminosugar, then the content of N element at least should be more than 7%.
The infrared absorption pattern of III
Infared spectrum shows that the PULP III is at 3100～3500 cm
-1, 2800～2900 cm
-1, 1400～1530 cm
-1, 1000～1100 cm
-1There is the charateristic avsorption band of obvious polysaccharide at the place.And the PULP III is at 1010～1100 cm
-1Three strong absorption peaks are arranged, illustrated that the pyranose glycosidic bond exists.
The monose of III forms
According to acetolysis step and GC condition, the GC collection of illustrative plates of bioassay standard monose mixed derivative, the retention time of obtaining various standard monose and mixing monose.
The retention time of table 2-2 standard sugar and PULP III hydrolysis derivative
2-2 can find out according to table, and the monose in the PULP III forms and ratio is that Rha:Ara:Man:Glc is 0.27:0.28:0.1:0.35.
III part acid hydrolysis result
The analysis (GC) of PULP III part acid hydrolysis products shows:
(1) the PULP III mainly is comprised of Rha, Ara, Man and Glc;
(2) contain Rha, Ara, Man and Glc in the centrifugal precipitation that obtains after the acid hydrolysis of PULP III part, illustrate that PULP III main chain mainly is made of Rha, Ara, Man and Glc.
III Periodic acid and Smith degradation results
The periodate oxidation result
NaIO
4The regression equation of concentration and light absorption value relation is after the oxidation: the y(absorbance)=and 0.0416x(NaIO
4Concentration)-0.0188, R
2=0.999.
The PULP III is at the 120h place, and it is stable that absorbance reaches.According to regression equation, the periodate oxidation analytical results sees Table 2-3.
Table 2-3 PULP III periodate oxidation is analyzed
2-3 analyzes according to table, draws the periodate oxidation result of PLUP III: (1) has formic acid to generate explanation necessarily has 1 → 6 to connect; (2) consumption of Periodic acid is all greater than two times of formic acid growing amount, explanation may exist and only consumes Periodic acid and do not generate the glycosidic link of formic acid type and exist not and the Periodic acid glycosidic link type that reacts, namely may also have 1 → 2,1 → 4 or 1 → 2,4 to connect existence and 1 → 3 connection.
PULP III Smith degradation results
PULP III periodate oxidation product carries out the Smith degraded, and interpretation of result sees Table 2-4.
The gas chromatographic analysis result of table 2-4 Smith degraded product
The Smith degradation results shows that there is rhamnosyl, pectinose and the glucose of different content in PULP III (1), illustrates to exist not by the of bonding of periodate oxidation (1 → 3 mode of connection); (2) there is tetrahydroxybutane in the degraded product, and do not have glycerine, illustrate and all contain 1 → 4 glycosidic link.
Comprehensive above-mentioned periodate oxidation and Smith degradation results are analyzed, and the mode of connection of PULP III glycosidic link is connected to the master with 1 → 6, also has simultaneously 1 → 4 and 1 → 3 mode of connection.
(1) physico-chemical property of PULP III and molecular structure Preliminary Analysis Results:
By physico-chemical property, glucuronic acid content is measured, and ultimate analysis, infrared and ultraviolet, HPLC analysis-by-synthesis show in the PULP III it is that a class does not contain N, S element acidic polysaccharose, and molecular-weight average is 418793.5 relatively;
The IR collection of illustrative plates shows that the PULP III has the charateristic avsorption band of polysaccharide, and at 1010～1100 cm
-1Three strong absorption peaks are arranged, show to have the pyranose glycosidic bond;
Be comprised of and part acid hydrolysis interpretation of result monose, PULP III monose forms and ratio is that Rha:Ara:Man:Glc is 0.27:0.28:0.1:0.35; Main chain mainly is comprised of Rha, Ara, Man and Glc.
Comprehensive periodate oxidation and Smith degradation results are analyzed, and PULP III glycosidic link is connected to the master with 1 → 6, also has simultaneously 1 → 4 and 1 → 3 mode of connection.About the configuration of glycosidic link, await to use NMR
1H spectrum and
13The C spectrum is further studied.
The antioxidation activity in vitro preliminary study of Common Leafflower Herb polysaccharide PUIP III
Laboratory reference Oyaizu method is slightly done a little and is changed.Get the polysaccharide soln of 1 mL different concns (1,2,3,4,5mg/mL) in tool plug test tube, then add respectively 2.5 mL 0.2 mol/L pH, 6.6 phosphate buffer solutions and 1% potassium ferricyanide solutions, ice bath cools off rapidly behind 50 ℃ of water-bath 20 min, the trichoroacetic acid(TCA) solution that adds 2.5 mL 10%, shake up, centrifugal (3000 r/min, 10 min).Get supernatant liquor 2.5 mL, add 2.5 mL distilled waters and 0.5 mL, 0.1% FeCl
3, shake up, react 10 min after, measure the absorbancy at 700 nm places.
OH) mensuration of clearance rate
In the Fenton reaction system, H
2O
2With Fe
2+Mix Chan Sheng ﹒ OH.You Yu ﹒ OH reactive behavior is strong, and the survival time is very short, adds Whitfield's ointment, and just Bu Zhuo and is created on the coloring matter that there is strong absorption at 510 nm places Dao ﹒ OH effectively.Simultaneously, if in this system, add with Whitfield's ointment have Competition can Qing Chu ﹒ OH material, then the growing amount of coloring matter tails off, the absorbancy step-down, absorbancy is lower, proves that the ability of this material Qing Chu ﹒ OH is stronger.
The FeSO that in tool plug test tube, adds respectively 2 mL, 9 mmol/L
4The Whitfield's ointment ethanolic soln of solution, 2 mL, 9 mmol/L, the polysaccharide soln of different concns (1,2,3,4,5mg/mL) adds the H of 2 mL 8.8mmol/L at last
2O
2Solution starts reaction, reacts 30 min under the room temperature, measures the absorbancy at 510 nm places, replaces polysaccharide soln to do blank with distilled water.
Free radical scavenging activity calculation formula: P=(A
0-A
i)/A
0* 100%
A
0: blank absorbency, A
i: the sample absorbancy
Draw 0.4 mL yolk suspension [V(yolk): V(PBS)=1:25], the polysaccharide soln of 1 mL different concns (1,2,3,4,5mg/mL), the FeSO of 0.4 mL, 25 mmol/L
4, in tool plug test tube, PBS solution to the cumulative volume that adds 0.1 mol/LpH7.45 is 4.0 mL, 37 ℃ of constant temperature water baths, 15 min that vibrate.The TCA that adds 1.0 mL 20% after taking out, leave standstill 10 min after, centrifugal (3500 r/min, 10 min).Draw 4.0 mL supernatant liquors, add the thiobarbituricacidα-of 2.0 mL 0.8%, jump a queue, boiling water bath 15 min after the cooling, do reference with PBS, measure the light absorption value at 532 nm places.
Sample represents the ability of the anti-oxidant activity of sample to the inhibiting rate of lipovitellinin lipid peroxidation, that is:
Inhibiting rate I=(A
0-A)/A
0* 100%
A
0The absorbancy of-control tube; The absorbancy of A-sample
The reducing power of III
Experimental result shows that the PULP III polysaccharide soln under each concentration all has certain reducing power, and in the concentration range of 1～5mg/mL, and its reducing power increases and strengthens with concentration.
The external hydroxyl radical free radical clearance rate of III
Experimental result shows that the PULP III polysaccharide soln under each concentration has certain removing ability to external hydroxyl radical free radical, and in the concentration range of 1～5mg/mL, and its clearance rate increases and strengthens with concentration.During 5mg/mL, the clearance rate of PULP III is 50.7%%.
III anti peroxidation of lipid ability
Experimental result shows that the PULP III polysaccharide soln under each concentration has certain restraining effect to LPO, and has dose-effect relationship in the concentration range of 1～5mg/mL, and its inhibiting rate increases and strengthens with concentration.During 5mg/mL, the inhibiting rate of PULP III is 23.7%.
Common Leafflower Herb polysaccharide PULP III has certain reducing power, removes external hydroxyl radical free radical ability and anti peroxidation of lipid ability, and strengthens along with the increase of polysaccharide concentration.When 5mg/mL, the reducing power of PULP III is 0.940, and the clearance rate of external hydroxyl free is reached 50.7%, and the inhibiting rate of LPO is respectively 23.7%.This explanation Common Leafflower Herb polysaccharide has certain anti-oxidant activity and its anti-oxidant activity is mainly manifested on the external hydroxyl radical free radical of removing.
The research of Common Leafflower Herb polysaccharide PUIP III effect on hepatitics B virus in vitro
Present most widely used effect on hepatitics B virus in vitro medicaments sifting model HepG2.2.2.15 cell model is adopted in this test, extract to separate from, Common Leafflower Herb and obtain PUIP III and HepG2.2.2.15 cytosis, get its cell conditioned medium liquid, the titre of measuring respectively its hepatitis B surface antigen (HBsAg), e antigen (HBeAg) changes, judge whether this medicine is effective, and adopt mtt assay to measure the cytotoxicity of each several part.Comprehensive these two indexs, Common Leafflower Herb polysaccharide PUIP III is carried out In Vitro Anti hepatitis B pharmacodynamics test, and select and certifiedly clinically have the acyclovir (ACV) of anti-HBV effect as the positive control medicine, to estimate the effect of each position hepatitis B virus resisting, screen efficient part or effective constituent with this.
The recovery of cell
Basic step: allocate 37 ℃ ~ 40 ℃ warm water; Take out the HepG2.2.2.15 cell cryopreservation tube from liquid nitrogen container, drop into immediately in 37 ℃ ~ 40 ℃ the warm water and rock fast, until frozen storing liquid melts fully, melting process is finished in 1-2min; The cell cryopreservation suspension is moved into centrifuge tube, add about 5mL nutrient solution, blow gently even; With the centrifugal 5min of cell suspension 800r-1000r/min, abandon supernatant liquor; Add complete culture solution to cell precipitation, pressure-vaccum is beaten evenly gently, and cell suspension is moved into culturing bottle, fills up nutrient solution and cultivates, and places 37 ℃, 5%CO
2Cell culture incubator is cultivated.
The cultivation of going down to posterity of cell
The HepG2.2.2.15 cell places 5%CO
2, 37 ℃ of cultivations.Substratum is DMEM, adds 10% foetal calf serum, 3% L-glutaminate 1%, G418 200ug/mL, penicillin 100u/mL, Streptomycin sulphate 100u/mL.2.2.15 after cell covers with culturing bottle, digest 10-30 second with EDTA first, with 37 ℃ of digestion of 0.25% pancreatin 3-10min, add nutrient solution piping and druming again, 1:3 or 1:4 go down to posterity, and cover with in 8 days, adopt the numeration of cell count plate, are mixed with 10
5Individual/mL inoculating cell culture plate, the every hole 0.1mL of 96 orifice plates; The every hole 1mL of 24 orifice plates, 5%CO
2, cultivate for 37 ℃ and tested in 24 hours.
Method for cell count
Generally use blood cell counting plate, count by the white blood cell count(WBC) method.
Getting cell suspension 1mL to be diluted adds physiological saline and does 5 times of dilutions, multiple with 10 * 10 is counted, central authorities use for red blood cell count(RBC), four jiaos of large lattice are that white blood cell count(WBC) is used, only add up complete cell during counting, if the cell that bunches up is counted by a cell, in a grid, if there is cell to be positioned on the line, general meter is reached the standard grade to disregard and is rolled off the production line, and counts left line and disregards right line, and counting error is no more than ± and 5%, need to calculate the cell count in every mL suspension behind the counting, because the area of each grid in the tally is 0.1cm
2, height is 0.01cm, volume is 0.0001cm
3Every mL cell count is calculated by formula 1:
Every mL cell count=(n/4) * 10000 * 5 (1)
In the formula: n is four large lattice total cellular score
The MTT colorimetry is surveyed cell survival rate
The HepG2.2.2.15 cell cultures is in 96 well culture plates, every hole 80 μ l nutrient solutions; Add 20 μ lMTT, continue to cultivate 3-4h; Suck 100 μ l nutrient solutions, add equivalent 0.04-0.1mol/L hydrochloric acid aqueous isopropanol, under the room temperature about 10min(or flat board placed the micro vibrator concussion), crystallisate is dissolved; Measure photoabsorption in microplate reader, the mensuration wavelength is 570nm.
Mtt assay is surveyed drug toxicity
The HepG2.2.2.15 cell is pressed 10
5Individual/mL is inoculated in 96 well culture plates, 0.1 mL/ hole, and inferior daily nutrient solution is made into respectively several different concentration with medicine to be measured, adds cell hole, and every hole concentration adds 3 holes, the 0.1mL/ hole, and establish without the contrast of medicine cell and positive control drug.Cultivated 4 days after the dosing, abandon supernatant and dye with MTT, every hole adds the MTT serum-free medium 0.1mL of 1mg/mL, hatches 4 hours, abandons supernatant, and after the adding 0.04mol/L hydrochloric acid Virahol 0.1mL dissolving, with 570nm wavelength colorimetric estimation OD value, experiment repeats 3 times.
Survey HBsAg, HBeAg secreting law
With HepG2.2.2.15 cell 10
5/ mL is inoculated in 24 porocyte culture plates, every hole 1.0mL, totally 10 holes, 3rd, collected supernatant 250 μ l in 5,8,11,13 days, nutrient solution was supplied commercial weight to the 13 days, and culture supernatant is frozen in-20 ℃, concentrate at last and press the test kit specification sheets, measure HBsAg, HBeAg with the ELISA method.
Medicine is to the HBV inhibition test
The HepG2.2.2.15 cell is pressed 10
5/ mL is inoculated in 24 orifice plates, and every hole 1.0mL abandons nutrient solution next day, carries out doubling dilution take the half toxic concentration of the HepG2.2.2.15 cell of medicine to be measured as initial concentration with nutrient solution, and other establishes without medicine contrast and positive control drug, is incubated at 37 ℃, 5%CO
2In the incubator.Collected supernatant in per 4 days, and change and add the original content liquid and continue to cultivate, the supernatant-20 collected is ℃ frozen, collected in the 12nd day, to concentrate with the ELISA method and measure HBsAg, HBeAg, experiment repeats 3 times.
The ELISA method is surveyed HBsAg, HBeAg
(1) each component of test kit is taken out from box, balance is to room temperature (18 ~ 25 ℃).
(2) fully shake up if any crystal and should fully melt before the concentrated cleaning solution preparation, concentrated cleaning solution uses after pressing the 1:19 dilution with distilled water or deionized water.
(3) the micropore lath is fixed in support, according to the order of sequence numbering.
(4) every hole adds sample 50 μ l to be measured, establishes each 2 hole of yin, yang contrast, and every hole adds each 50 μ l of yin, yang contrast, and establishes blank one hole;
(5) every hole adds enzymic-labelled antibody 50 μ l(except blank), shrouding behind the abundant mixing, place 37 ℃ of environment to hatch 30min;
(6) the manual plate of washing; Discard liquid in the hole, full each hole of washings storage is left standstill and was dried afterwards in 5 seconds, pats dry after repeating 5 times;
(7) every hole adds developer A liquid, each 50 μ l of B liquid, abundant mixing, and shrouding is put in 37 ℃ of environment and is hatched 15min;
(8) every hole adds stop buffer 50 μ l, mixing;
(9) use the microplate reader reading, get wavelength 450nm, use first blank empty school zero.Then read each hole OD value.(negative control OD value is lower than 0.05 as 0.05 calculating, is higher than 0.05 by actual OD value calculating.）
Data analysis
Mtt assay can get after surveying drug toxicity:
Calculate the poisonous concentration TC of half according to the Red-Meuench method
50Can get
In the formula: B---
A——
C——A-B
Adopt the ELISA method, the automatic microplate reader of usefulness read OD value after color reaction was finished, the calculating inhibiting rate, with inhibiting rate greater than 50% for restraining effect is arranged.Calculate medicine according to determination data and suppress the antigen percentage; Medicine suppresses antigen medium effective concentration IC
50, the drug level when namely HBsAg and HBeAg inhibiting rate are 50%; Select therapeutic index TI, TI is effectively greater than 1 above person, and the explanation medicine is effective between 1 ~ 2, but certain toxicity is arranged; Better greater than 2 effects, toxicity is less; Index is larger, and then curative effect is better, and safety range is larger.Statistical procedures, mean compares between employing t check work group, and data statistic analysis is by the SPSS11.5 software processes, and P＜0.05 expression difference has significance.
In the formula: B---
A——
C——A-B
Interpretation of result
The rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Under the described culture condition of test method, the used HepG2.2.2.15 cell of this test began to have measured HBsAg, HBeAg secretion in the 3rd day, peaked at the 8th day, weakened gradually later on, lasted till that the rule of HBsAg, HBeAg secretion as shown in Figure 3 the 13rd day.
The effect on hepatitics B virus in vitro test-results of positive drug (ACV)
Positive drug (ACV) is to the toxicity of HepG2.2.2.15 cell
After different concns ACV process to cultivate, the MTT test-results shows had restraining effect to the HepG2.2.2.15 Growth of Cells, and its effect has certain concentration dependent, and concentration is larger, and restraining effect is more obvious.Cell inhibitory rate when concentration is 0.3125mg/mL is 3.51%, and the cell survival rate under this concentration is 96.49% (〉 95%), the TC of hence one can see that ACV
0Be 0.3125mg/mL.Can get TC according to formula (3)
50Be 4.56 mg/mL.The cytotoxic assay of ACV the results are shown in Table 3-1.
The toxicity of table 3-1 ACV in the HepG2.2.2.15 cell cultures
Positive drug (ACV) suppresses the effect of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Different concns ACV processes and cultivates collecting cell supernatant liquor after 4 days and 8 days, the ELISA method is surveyed HBsAg, the HBeAg level shows that ACV all has restraining effect to HBsAg, HBeAg, and along with the increase of ACV concentration, its restraining effect strengthens, inhibiting rate increases, and demonstrates certain dose-effect relationship.And along with the passing restraining effect of incubation time also strengthens gradually.Different concns ACV restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures the results are shown in Table 3-2 and 3-3.
Show 3-2 ACV restraining effect to HBsAg in the HepG2.2.2.15 cell cultures
Show 3-3 ACV restraining effect to HBeAg in the HepG2.2.2.15 cell cultures
The therapeutic index (TI) of positive drug (ACV) In Vitro Anti B-type hepatitis
According to formula (5), we can obtain ACV suppressed at the 8th day HepG2.2.2.15 emiocytosis HBsAg, HBeAg the time IC
50Be respectively 0.58mg/mL and 0.49mg/mL.Can obtain ACV according to formula (6) the therapeutic index TI of HBsAg, HBeAg is respectively 7.86 and 9.31.Concrete outcome sees Table 3-4.
Show 3-4 ACV IC to HBsAg and HBeAg in the HepG2.2.2.15 cell cultures
50And TI
The effect on hepatitics B virus in vitro effect of Common Leafflower Herb polysaccharide PUIP III
Common Leafflower Herb polysaccharide PUIP III is to the toxicity of HepG2.2.2.15 cell
After different concns Common Leafflower Herb polysaccharide PUIP III treatment group was cultivated, the MTT test-results shows had restraining effect to the HepG2.2.2.15 Growth of Cells, and its effect has certain concentration dependent, and concentration is larger, and restraining effect is more obvious.Cell inhibitory rate when concentration is 3.12mg/mL is 1.52%, and the cell survival rate under this concentration is 98.48% (〉 95%), the TC of Common Leafflower Herb polysaccharide PUIP III that hence one can see that
0Be 3.12mg/mL.Can get TC according to formula 1-2
50Be 45.41 mg/ml.The cytotoxic assay of Common Leafflower Herb polysaccharide PUIP III the results are shown in Table 3-5.
The toxicity of table 3-5 Common Leafflower Herb polysaccharide PUIP III in the HepG2.2.2.15 cell cultures
Common Leafflower Herb polysaccharide PUIP III suppresses the effect of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Different concns Common Leafflower Herb polysaccharide PUIP III is processed and is cultivated collecting cell supernatant liquor after 4 days and 8 days, the ELISA method is surveyed HBsAg, the HBeAg level shows that Common Leafflower Herb polysaccharide PUIP III all has restraining effect to HBsAg, HBeAg, and the increase along with Common Leafflower Herb polysaccharide PUIP III concentration, its restraining effect strengthens, inhibiting rate increases, and demonstrates certain dose-effect relationship.And along with the passing restraining effect of incubation time also strengthens gradually.Different concns Common Leafflower Herb polysaccharide PUIP III restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures the results are shown in Table 3-6 and 3-7
Show 3-6 Common Leafflower Herb polysaccharide PUIP III restraining effect to HBsAg in the HepG2.2.2.15 cell cultures
Show 3-7 Common Leafflower Herb polysaccharide PUIP III restraining effect to HBeAg in the HepG2.2.2.15 cell cultures
The therapeutic index (TI) of Common Leafflower Herb polysaccharide PUIP III In Vitro Anti B-type hepatitis
According to formula (5), we can obtain Common Leafflower Herb polysaccharide PUIP III suppressed at the 8th day HepG2.2.2.15 emiocytosis HBsAg, HBeAg the time IC
50Be respectively 3.82mg/mL and 14.60mg/mL.According to formula (6) can obtain Common Leafflower Herb polysaccharide PUIP III to the therapeutic index TI of HBsAg, HBeAg respectively 11.89 for and 3.11.Concrete outcome sees Table 3-8.
Show 3-8 Common Leafflower Herb polysaccharide PUIP III IC to HBsAg and HBeAg in the HepG2.2.2.15 cell cultures
50And TI
Discuss
This test has been adopted mtt assay to carry out cytotoxicity and has been detected, and the result shows the maximal non-toxic concentration (TC of Common Leafflower Herb polysaccharide PUIP III HepG2.2.2.15 cell
0) 3.12 mg/mL; Poisonous concentration (the TC of half
50) 45.41mg/mL.
This test adopts elisa technique to analyze Common Leafflower Herb polysaccharide PUIP III restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures, the result shows in 7 concentration that Common Leafflower Herb polysaccharide PUIP III sets under non-toxic concn, except minimum concentration, other each concentration group is compared with the cell control group and is all shown obvious restraining effect, and the increase along with Common Leafflower Herb polysaccharide PUIP III concentration, its restraining effect strengthens, and inhibiting rate increases, and demonstrates certain dose-effect relationship.The result for the treatment of index that is used for clinically at present estimating medicine is therapeutic index (TI), is that medicine is invalid when TI＜1, and the medicine poor efficiency is poisonous when 1＜TI＜2, as TI〉the effective low toxicity of medicine 2 time.Hence one can see that, and Common Leafflower Herb polysaccharide PUIP III is to the effective low toxicity of therapeutic index (TI is respectively 11.89 and 3.11) of HBsAg, HBeAg; Positive control medicine acyclovir is to the effective low toxicity of therapeutic index (TI is respectively 7.86 and 9.31) of HBsAg, HBeAg.
In sum, Common Leafflower Herb polysaccharide PUIP III all has certain restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures.
The above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (10)
1. the extraction and separation method of a Common Leafflower Herb polysaccharide PULP III, it is characterized in that: described method comprises the steps:
1) Common Leafflower Herb herb distilled water wash removes soil, after the drying, pulverizes, and uses petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, the concentrated rear ethanol alcohol precipitation that adds of filtrate is got in the vat liquor centrifugation, gets pure hypostasis and is thick total polysaccharides; Thick total polysaccharides is through 2) except albumen, 3) washing, removal of impurities and 4) dialysis, be drying to obtain the smart total polysaccharides of Common Leafflower Herb, polysaccharide content is more than 95%; 5) each component separation and purification of polysaccharide: through deproteinated, the smart total polysaccharides of Common Leafflower Herb after the dialysis treatment, cross ion exchange column, successively with suitable NaCl gradient solution wash-out, the phenolsulfuric acid method follow the tracks of to detect, and gets elution curve, the elution peak that can obviously declare out four peak shape symmetries from the curve, represent respectively four components, be designated as respectively PULP I, PULP II, PULP III and PULP IV according to the order that goes out the peak; Wherein the 2nd, the 3rd two elution peaks are larger, collect the 3rd main peak component, and are concentrated, redistilled water dialysis 48h, and lyophilize gets Common Leafflower Herb polysaccharide PULP III pure component.
2. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: the Phyllanthus urinaria that step 1) is pulverized, exceed medicinal material 0.5～1cm with sherwood oil submergence medicinal material and liquid level, 70～90 ℃ and normal pressure power refluxing extraction 2～4 times, each 1～3 hour, discard phegma; It is 50～100% ethanolic soln by same method 80～95 ℃ of lower refluxing extraction 2～4 times that the dregs of a decoction continue with massfraction, each 1～3 hour, discards phegma; The dregs of a decoction are used 60～95 ℃ of floodings 3 times again, each 1～3 hour, merge 3 vat liquors and carry out centrifugation, centrifugal speed 2000～6000r/min, get filtrate and be concentrated into original volume 1/4, the massfraction that adds 4 times of volumes is 90～100% ethanolic soln, leaves standstill 24h, centrifugal, get pure hypostasis and be Crude polysaccharides.
3. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: step 2) namely use Sevag method deproteinated except albumen: the pure hypostasis that step 1) is obtained redissolves to get Crude polysaccharides solution with distilled water, add chloroform and propyl carbinol mixed solution, wherein Crude polysaccharides solution in Crude polysaccharides solution: the volume ratio of chloroform and propyl carbinol mixed solution is 1～6:1～3; Chloroform in the mixed solution: the volume ratio of propyl carbinol is 1～4:1～4; Magnetic agitation 20～40min, the sufficient standing layering discards precipitation; After repeating 1～10 until interface is without white precipitate.
4. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: the step 3) washing, removal of impurities: the massfraction that adds 3～6 times of volumes in except the polysaccharide soln behind the albumen is ethanolic soln precipitation 12～36h of 75～100%, centrifugal, abandon filtrate, precipitation is cleaned with dehydrated alcohol, acetone, ether successively, repeats 2～5 times.
5. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: the step 4) dialysis, dry: i.e. filter residue adds an amount of distilled water after the alcohol precipitation washing, dialysis after fully redissolving, dialysis is carried out under magnetic agitation, sample liquid and dialyzate are that the volume ratio of distilled water is that 1:10～30,4～8h changes water one time, dialysis 12～120h; Dialyse complete after with sugar soln put into freeze drier dry the smart total polysaccharides of Common Leafflower Herb, content is more than 95%.
6. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: the ion exchange column of step 5) is DEAE-52 anionite-exchange resin, use front with DEAE-52 cellulose wadding distilled water immersion 48h, 4～5h changes water one time during this time, and remove suspended impurity with decantation, then carry out alkali-acid-alkali and process; Be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L first, be washed till neutrality with distilled water behind HCl solution soaking 30 min of 0.5 mol/L again, be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L at last, obtain OH
-The fiber type element; Wet method dress post, column dimension is 500 * 50mm, distilled water balance 48h avoids bubble and fault-layer-phenomenon in the dress post process.
7. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: the amount of substance concentration of the NaCl gradient solution of step 5) is 0～3mol/L.
8. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 7 is characterized in that: the amount of substance concentration of the NaCl gradient solution of step 5) is respectively 0,0.1,0.25,0.5,0.75mol/L.
9. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: step 5) is successively with suitable NaCl gradient solution wash-out, the amount of substance concentration eluant solution of every kind of NaCl 8 hours, flow velocity 4mL/min, collect elutriant with pipe, every 10min collects a pipe.
10. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1 is characterized in that: the step 5) elution curve is the Guan Xuwei X-coordinate to collect, and the absorbance of solution is that ordinate zou is drawn and obtained.
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| CN104829737A (en) * | 2015-04-30 | 2015-08-12 | 华南理工大学 | Crude raspberry leaf polysaccharide, and preparation method and application thereof |
| CN106883303B (en) * | 2017-03-02 | 2019-09-03 | 河南中医药大学 | A kind of preparation method and application of geranium wilfordii polysaccharide and oligosaccharide |
| CN111647095A (en) * | 2020-06-30 | 2020-09-11 | 华润三九医药股份有限公司 | Polysaccharide of fraxinus chinensis, preparation method and application thereof |
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Cited By (4)
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| CN104829737A (en) * | 2015-04-30 | 2015-08-12 | 华南理工大学 | Crude raspberry leaf polysaccharide, and preparation method and application thereof |
| CN104829737B (en) * | 2015-04-30 | 2017-09-29 | 华南理工大学 | A kind of palmleaf raspberry leaf Thick many candies and preparation method and application |
| CN106883303B (en) * | 2017-03-02 | 2019-09-03 | 河南中医药大学 | A kind of preparation method and application of geranium wilfordii polysaccharide and oligosaccharide |
| CN111647095A (en) * | 2020-06-30 | 2020-09-11 | 华润三九医药股份有限公司 | Polysaccharide of fraxinus chinensis, preparation method and application thereof |
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