CN103044567B - 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 the extraction and separation method of a kind 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, be widely used in promoting diuresis to remove toxic substance, calming liver and clearing heat among the people.1988, people's first passage experiments such as India scholar Thyagarajan proved that Phyllanthusamarus can make the hepatitis B surface antigen of 59% patient (HBsAg) turn out cloudy, and this experimental result causes the concern of Chinese scholars to Common Leafflower Herb.Large quantity research shows
]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 crude drug being worth the treatment hepatitis B of deeply exploitation.
Containing number of chemical composition in Common Leafflower Herb, what document had been reported has lignanoid, terpene, flavones, mix matter, alkaloid etc., comprises Octadecane, dehydrogenation chebulic acid, forulic acid, gallic acid, brevifolin carboxylic acid, succinic acid, methoxyl group mix and spend acid, camellia element, heroubill plain, short leaf bush acid formicester, short leaf Soviet Union art phenolic acid second fat, corilagin, dehydrogenation chebulic acid three formicester, polysaccharide etc.
Polysaccharide compound has the multiple biological activity 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, wherein has nearly 1,000,000 patients to die from HBV every year and infects the liver failure, liver cirrhosis and the liver cancer that cause.If be applied to drug main Interferon, rabbit and the nucleoside analog for the treatment of hepatitis B at present, but these medicines can only obtain result for the treatment of in a way, most patient are not reached to the object of curing completely, and there will be the phenomenon of knock-on after drug withdrawal.So developing efficient, low toxicity, not " knock-on " treating hepatitis B new drug is an instant problem.
In the process finding treating hepatitis B medicine, herbal polysaccharide composition is more and more subject to people's attention.Common Leafflower Herb has hepatitis B virus resisting, protect the liver, the effect such as antitumor is generally confirmed, and relevant scholar is also to several chemical compositions wherein, as gallic acid, flavonoid and Common Leafflower Herb element etc. has done structure and bioactive research, but little to polysaccharide researches wherein.Carry out the extraction and isolation to polysaccharide component in Common Leafflower Herb, Structural Identification and bioactive detection, being conducive to determining that Common Leafflower Herb is used for the treatment of the effective substance of hepatitis B further, developing this resources of medicinal plant of Common Leafflower Herb better, laying theoretical basis for finding treating hepatitis B new drug.
Summary of the invention
The object of the present invention is to provide the extraction and separation method of a kind of Common Leafflower Herb polysaccharide PULP III, by the separation and purification to Common Leafflower Herb Crude polysaccharides, obtain 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 drying, pulverizes, with petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, vat liquor centrifugation, gets the ethanol alcohol precipitation adding certain volume after filtrate concentrates, and gets alcohol hypostasis and is thick total polysaccharides; Thick total polysaccharides is through 2) removing protein, 3) washing, removal of impurities and 4) dialysis, be drying to obtain Common Leafflower Herb essence total polysaccharides (PULP), polysaccharide content is more than 95%.5) each Component seperation purifying of total polysaccharides: through deproteinated, Common Leafflower Herb essence total polysaccharides (PULP) after dialysis treatment, cross ion exchange column, successively with suitable NaCl gradient solution wash-out, phend-sulphuric acid tracing detection, obtains elution curve, obviously can declare out the elution peak of four peak shape symmetries from curve, represent four components respectively, be designated as PULP I, PULP II, PULP III and PULP IV respectively according to the order going out peak.Wherein the 2nd, the 3rd two elution peaks are comparatively large, collect the 3rd main peak component, concentrated, and redistilled water dialysis 48h, lyophilize obtains Common Leafflower Herb polysaccharide PULP III pure component.
Described Common Leafflower Herb polysaccharide PUIP III is khaki color chip solid, and be that a class does not contain N, S element acidic polysaccharose, Relative average molecular weight is 418793.5; PULP III monose composition and ratio thereof are rhamnosyl (Rha): pectinose (Ara): seminose (Man): glucose (Glc) is 0.27:0.28:0.1:0.35; Main chain is made up of Rha, Ara, Man and Glc.Each monose is connected by pyranose glycosidic bond, and glycosidic link connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously.
Above-mentioned steps 1) optimum condition is the Phyllanthus urinaria pulverized, and with sherwood oil submergence medicinal material and liquid level exceeds medicinal material 0.5 ~ 1cm, 80 DEG C and normal pressure power refluxing extraction 3 times, each 2 hours, discards phegma.The dregs of a decoction continue to press same method refluxing extraction 3 times at 80 DEG C DEG C with 50 ~ 100% ethanol, each 2 hours, discard phegma.90 DEG C of floodings 3 times used again by the dregs of a decoction, each 2 hours, merge 3 vat liquors and carry out centrifugation, centrifugal speed 4000r/min, get filtrate and be concentrated into original volume 1/4, add 95% ethanol of 4 times of volumes, leave standstill 24h, centrifugal, get alcohol hypostasis and are Crude polysaccharides.
Step 2) namely removing protein use Sevag method deproteinated: alcohol hypostasis distilled water step 1) obtained redissolves to obtain Crude polysaccharides solution, the preferred system adding chloroform and propyl carbinol mixed solution in Crude polysaccharides solution is Crude polysaccharides solution: chloroform and propyl carbinol mixed solution are 3:1 with volume ratio metering, mixed solution=chloroform: propyl carbinol=4:1(volume ratio metering), magnetic agitation 30min, sufficient standing layering, discards precipitation.Until interface is without white precipitate after repeating 7 ~ 8 times.
Step 3) is washed, and the optimum condition of removal of impurities is the 95% alcohol settling 24h adding 4 times of volumes in the polysaccharide soln after removing protein, centrifugal, abandons filtrate, precipitation successively with dehydrated alcohol, acetone, ether cleaning, repeat 3 times.
Step 4) is dialysed, dry: namely after alcohol precipitation washing, filter residue adds appropriate distilled water, dialysis after fully redissolving, and dialysis is carried out under magnetic stirring, and sample liquid and dialyzate (distilled water) volume ratio optimum condition are that 1:20,8h change a water, dialysis 72h.After dialysis, sugar soln is 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, processed by DEAE-52 cellulose wadding before using, its optimum condition uses distilled water immersion 48h, and period 3h changes a water, and remove suspended impurity with decantation, then carry out alkali-acid-alkali process.After first soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, then is washed till neutrality with distilled water after HCl solution soaking 30 min of 0.5 mol/L, and after finally soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, obtains OH
-fiber type element.Wet method dress post (column dimension is 500 × 50mm), distilled water balance 48h, avoids bubble and fault-layer-phenomenon in dress post process.
The NaCl gradient solution of the gradient scope=0 ~ 1mol/L of the substance withdrawl syndrome of the comparatively suitable NaCl of step 5).
The gradient solution of the substance withdrawl syndrome 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 comparatively optimized is the substance withdrawl syndrome eluant solution 8 hours of often kind of NaCl, and flow velocity 4mL/min, collects elutriant with pipe, and every 10min collects a pipe.
Elution curve described in step 5) be for the Guan Xu (pipe number) collected for X-coordinate, the absorbance of solution is that ordinate zou is drawn and obtained.
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 Common Leafflower Herb Crude polysaccharides, obtains pure single Common Leafflower Herb polysaccharide component.By the research of the structural analysis of the pure component PULP III to polysaccharide and antioxidation in vitro, antiviral activity experiment, confirm the effective constituent that Common Leafflower Herb polysaccharide is phyllanthus for treating hepatitis B, this is that the anti-HBV effect mechanism studying Common Leafflower Herb polysaccharide provides theoretical basis; Meanwhile, to the physico-chemical property of Common Leafflower Herb polysaccharide fraction PULP III, elementary composition, functional group, initial analysis has been carried out in monose proportion of composing and glycosidic link position, for the further further investigation of later Common Leafflower Herb polysaccharide molecule structure is provided fundamental basis.From now on can on this basis, in conjunction with the structure activity relationship of polysaccharide, exploitation is that raw-material anti-hepatic-B virus medicine lays the foundation with Common Leafflower Herb polysaccharide further.
Accompanying drawing explanation
Fig. 1 is glucose standard curve.
Fig. 2 is polysaccharide gradient elution curve.
Fig. 3 is the rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail:
the extraction of Common Leafflower Herb polysaccharide
Common Leafflower Herb pre-treatment
Common Leafflower Herb herb distilled water wash removes soil, after 50 DEG C of cryodryings, pulverizes, seals for subsequent use.Its powder is yellow-green colour.
The extracting method of Common Leafflower Herb polysaccharide
Sherwood oil 80 DEG C backflow degreasing → 95% ethanol 80 DEG C of de-small molecular sugar → 90 of backflow DEG C water refluxing extraction 3 times, merge 3 filtrate 4000r/min centrifugal, be concentrated into 95% ethanol of 1/4 volume → add, 4 times of volumes, leave standstill 24h, centrifugal, distilled water redissolution → sevag method removing protein (chloroform: propyl carbinol=4:1, polysaccharide soln: mixed solution=3:1), magnetic agitation 30min, sufficient standing layering, repeat 7 ~ 8 operations can remove free protein → removing protein after polysaccharide soln add 95% alcohol settling 24h of 4 times of volumes → use dehydrated alcohol successively, acetone, ether cleans, in triplicate → dialysis 72h → lyophilize obtains Common Leafflower Herb total polysaccharides (PULP).
The mensuration of Common Leafflower Herb total polysaccharides content
The preparation of glucose standard and sample test liquid
Precision takes dextrose standard sample (being dried to weight no longer to change at 105 DEG C) 10mg, after dissolving, is placed in 100mL volumetric flask, is settled to scale with distilled water, shake up, make the glucose standard of 0.1mg/mL with distilled water, for subsequent use.
Precision takes the Common Leafflower Herb polysaccharide 10mg being dried to constant weight, and after dissolving with distilled water, be placed in 100mL volumetric flask adding distil water and be settled to scale, shake up, the sample test liquid making 0.1mg/mL is for subsequent use.
The determination of absorbing wavelength
Get glucose standard and each 1 mL of sample solution, add 1mL 5% phenol solution (get 5g and heavily steam phenol adding distil water constant volume in the brown volumetric flask of 100mL) respectively, after shaking up, unsettledly vertically add the 5mL vitriol oil, put in boiling water bath and heat 15min, then put in cooling bath and cool, full wavelength scanner within the scope of 400 ~ 600 nm, determines absorbing wavelength.
Glucose standard curve makes
Accurate draw glucose standard 0.2,0.4,0.6,0.8,1mL is in 10mL tool plug scale test tube, adding water successively makes final volume be 1mL, blank is 1mL water, then add 1mL 5% phenol solution to shake up, add rapidly the 5mL vitriol oil (unsettled vertically add), put in boiling water bath and heat 15min, then put in cooling bath and cool, measure absorbancy in 490nm place.Take absorbancy as Y-axis, glucose quality is X-axis, drawing standard curve, and calculates regression equation.
The calculating of sugar content
Accurate absorption test liquid 1mL, measures absorbancy in 490nm place.
According to formulae discovery sugar content:
Sugar content=C/(C
0× V) × 100%
C: the glucose micrograms checked in by typical curve
C
0: the concentration (0.1 mg/mL) of sample solution
V: the sample solution volume (1.0mL) during mensuration
The extraction yield of polysaccharide
Quality/raw-material quality × 100 % of the extraction yield=polysaccharide of polysaccharide
Results and analysis
Measure the determination of wavelength
The maximum absorption band wavelength of sample solution and glucose standard is all located at about 490 nm, so choose 490 nm as measurement of the polysaccharide content wavelength.
Glucose standard curve
Take absorbancy as Y-axis, glucose micrograms (μ g) is X-axis, drawing standard curve, as Fig. 1, can find out that glucose amount and absorbancy have good linear relationship within the scope of 20 ~ 100 μ g.
Sugar content
The absorbance A recording sample liquid is 0.388, is 28.27 μ g according to the micrograms of its corresponding glucose of regression equation calculation.Sugared content is obtained according to formula:
Sugar content=C/(C
0× V) × 100%=28.27/ (0.1 × 1) × 100%=28.27%
The extraction yield of polysaccharide
Extraction yield==1.5/100 × 100 %=1.5% of polysaccharide
Conclusion
Petroleum ether degreasing is passed through in this experiment, the small-molecule substances such as oligose are sloughed again with the ethanol of 95%, then from Common Leafflower Herb, polysaccharide is isolated with water extraction and alcohol precipitation method, and adopt sevag method deproteinated, phend-sulphuric acid measures its content, its maximum absorption wavelength is 490nm, and polysaccharide extract rate is 1.5%, and sugared content is 28.27%.
the separation and purification of Common Leafflower Herb polysaccharide
DEAE-52 post is separated
DEAE-52 filler pre-treatment
DEAE-52 cellulose wadding distilled water immersion 48h, period 4 ~ 5h change a water, and remove suspended impurity with decantation, then carry out alkali-acid-alkali process.After first soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, then is washed till neutrality with distilled water after HCl solution soaking 30 min of 0.5 mol/L, and after finally soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, obtains OH
-fiber type element.Wet method dress post (column dimension is 500 × 50mm), distilled water balance 48h, avoids bubble and fault-layer-phenomenon in dress post process.
Cross the separation and purification of DEAE-52 chromatography column
Take 640mg Common Leafflower Herb total polysaccharides and be dissolved in (8 mg/mL) in 80mL redistilled water, excessively loading after 0.45 μm of filter membrane.Use 0 successively, 0.1,0.25,0.5,0.75mol/L NaCl gradient elution, every 10min collects a pipe, flow velocity 4mL/min; Phend-sulphuric acid tracing detection, collects each main peak component, concentrated, and redistilled water dialysis 48h, lyophilize obtains each component of Common Leafflower Herb polysaccharide.
Purity
SephadexG-200 dextran gel filtration method
The pre-treatment of SephadexG-200: SephadexG-200 filler adds appropriate distilled water immersion 24h, period 4 ~ 5h changes a water, and removes suspended impurity with decantation, and then ultrasonic degas is not until have bubble to occur in coagulant liquid.Wet method dress post (column dimension is 500 × 25mm), for subsequent use after balancing 48 h by the NaCl solution of 0.05 mol/L.
SephadexG-200 column chromatography: the sample liquid each component of collecting being mixed with 25mg/mL above, loading 2mL, with the NaCl wash-out of 0.05 mol/L.Automatic Fraction Collector is collected, often pipe 5 mL, and every 30 min collect a pipe, phend-sulphuric acid tracing detection.
HPLC high performance liquid chromatography
Each fraction polysaccharide after purifying is mixed with the sample liquid of 1mg/mL, sample introduction after the filter membrane of mistake 0.45 μm.
High-efficient liquid phase chromatogram condition:
Chromatographic column: PolySep-SEC 4000 Part No:00H-3144-K0 Column Size:300 × 7.8mm;
Detector: Composition distribution; Column temperature: 30 DEG C; Moving phase: distilled water; Flow velocity: 0.8 mL/min; Sample size: 20 μ L.
Results and analysis
DEAE-52 column chromatography for separation result
Through deproteinated, Common Leafflower Herb essence polysaccharide (PULP) after dialysis treatment, crosses DEAE-52 ion exchange column, use 0 successively, 0.1,0.25,0.5,0.75mol/L NaCl solution gradient elution, phend-sulphuric acid tracing detection, obtains elution curve as shown in Figure 2.As seen from Figure 2, PULP, after DEAE-52 column chromatography for separation, obviously can isolate the elution peak of four peak shape symmetries, represents four components respectively, is designated as PULP I, PULP II, PULP III and PULP IV.The component PULP III collecting content larger is further analyzed.
SephadexG-200 post and HPLC Purity
SephadexG-200 post result
Get Common Leafflower Herb polysaccharide fraction PULP III and cross SephadexG-200 gel column, NaCl wash-out, phend-sulphuric acid tracing detection, elution curve is made 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 relatively homogeneous polysaccharide fraction of molecular weight.
HPLC method Purity
When utilizing HPLC method to detect the purity of Common Leafflower Herb polysaccharide fraction PULP III, result shows, polysaccharide fraction PULP III after the separation and purification of DEAE-52 cellulose column, more symmetrical in HPLC-UV detection superiors type, and calculate its purity through area normalization method and reach more than 95%, match with Sephadex G-200 gel chromatography figure result, illustrate that purity is higher, can be used for doing Structural Identification.
Conclusion
Common Leafflower Herb polysaccharide after water extract-alcohol precipitation deproteinated obtains four components after the separation and purification of DEAE-52 cellulose column, is designated as PULP I, PULP II, PULP III and PULP IV respectively.Collect the component PULP III that content is larger, carry out Purity by SephadexG-200 gel filtration chromatography method and high performance liquid chromatography (HPLC) two kinds of methods, prove that its component is relatively homogeneous, structural analysis can be done further.
the physico-chemical property of III and structural analysis
State, dissolubility test
Take the polysaccharide fraction PULP III after appropriate separation and purification and be dissolved in pure water, dehydrated alcohol, acetone, ether and ethyl acetate equal solvent, observe 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 cooling, if there is red-purple, then prove have protein, otherwise then without.With distilled water and calf serum solution in contrast.
Molish reacts
Get 1.0mg/mL sample solution 1.0mL in test tube, drip two Molish reagent, shake up.Inclination test tube, adds the about 1mL vitriol oil along tube wall, careful vertically after, examine the colour-change of two-layer liquid level intersection after 2 ~ 3min, if there is red-purple to occur, then proof has glucide.With distilled water and starch solution in contrast.
Phenolsulfuric acid reacts
Get 1.0mg/mL sample solution 1.0mL in test tube, add the phenol of 1mL 5%, then add the 5mL vitriol oil, vibration, if in orange-yellow, illustrate sugary.Take distilled water as contrast.
IKI reacts
Get the sample solution of 1.0mL 1.0mg/mL, after dripping 250 μ L Wagner's reagents, if not aobvious blue, illustrate that this polysaccharide is non-starch class.Take distilled water as negative control, the starch indicating liquid of 0.5% is positive control.
Sulfuric acid-carbazole reaction
Get 1.0mg/mL sample solution 1.0mL in test tube, in pipe, the 6mL vitriol oil is added in ice-water bath, shake up and be placed on 20min in 85 DEG C of water-baths, taking-up is cooled to room temperature, adds the carbazole liquid of 0.2mL 0.1%, keeps 2h under room temperature, if there is red-purple material to occur, illustrating containing uronic acid, is acid sugar, take distilled water as negative control.
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 prove containing reducing sugar.
Ferric chloride test
Get 1.0mg/mL sample solution 1.0mL in test tube, adjust ph is 4 ~ 5, adds the ferric chloride aqueous solutions of 1%, if there is blue, blackish green or bluish voilet, then proves containing tannin class or phenols.
Sulfate qualitative test
Get 2mg polysaccharide sample, add 1.0mL 1mol/L HCl, 110 DEG C of airtight hydrolysis 3h, then drip the bariumchloride-gelatin reagent of 0.5mL, if adularescent precipitation generates, illustrate containing sulfate.
HPLC 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: Composition distribution; Column temperature: 30 DEG C; 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, after centrifugal 10 min of 10000r/min, crosses 0.45 μm of 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 amount is mixed with the solution that concentration is 1mg/mL, the same sample solution for the treatment of process, for subsequent use.
The drafting of typical curve
By the dextran standard of various known molecular amount by small molecules to macromole successively average sample introduction 3 times, record corresponding retention time t
rand color atlas, with retention time t
rfor X-coordinate, the logarithmic value of molecular weight is ordinate zou drawing standard curve.
The mensuration of PULP III relative molecular mass
By each sample introduction of sample solution for preparing above three times, HPLC chromatographic condition is the same with standard substance, records corresponding retention time t
rand color atlas, according to the regression equation calculation molecular weight that typical curve obtains.
The mensuration of glucuronic acid content
The drafting of typical curve
Sulfuric acid carbazole method is adopted to measure glucuronic acid content.The glucal acid solution 25mL of preparation 0.2mg/mL, measure 0.25 successively, 0.5,1.0,1.5,2.0, the above-mentioned solution of 2.5mL, constant volume is in 10mL volumetric flask, and the standardized solution making 5,10,20,30,40,50 μ g/mL is put in 4 DEG C of refrigerators for subsequent use.
Measure 5mL borax sulphuric acid soln (0.025 mol/L) in tool plug test tube, ice bath, then carefully adds above acid solution layer, using distilled water as blank by 1mL glucuronic acid standardized solution, (first shake up gently, rear acutely rock) limit ice bath cooling while shake up.Then test tube is heated 10min in boiling water bath, 0.2mL carbazole solution (ethanol solution of 0.125%) is added 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, with the concentration of glucuronic acid for X-coordinate, light absorption value ordinate zou drawing standard curve.
The mensuration of glucuronic acid content in sample
Polysaccharide fraction PULP III is made into the solution of 5 μ g/mL, operates the same, measure its light absorption value at 530nm place.
Protein and nucleic acid determination
Polysaccharide fraction PULP III after purifying is mixed with the polysaccharide solution of 1mg/mL, see that it is at 260nm (nucleic acid) with UV ultraviolet spectrophotometer in the interscan of 200 ~ 400nm scope, 280nm(Pr) whether place has absorption peak, to determine whether the existence of protein and nucleic acid.
C, H, N element analysis
Take the PULP III sample 2.5mg of two parts of complete dryinies respectively, adopt Elementar Vario EL III elemental analyser to measure the content of C, H, N.
The detection of infrared spectra (IR)
Get 2mg polysaccharide sample, with after Potassium Bromide (KBr) compressing tablet at 400 ~ 4000cm
-1infrared spectroscopy is done in wavelength region.
Monosaccharide composition analysis
The vapor-phase chromatography of sugared nitrile acetic ester derivative is adopted to carry out monose composition measuring.The monose moiety of sample can be determined, according to the proportion of composing going out peak area and can determine monose according to appearance time.
Monose aldoononitrile acetate takes rhamnosyl (Rha), wood sugar (Xyl), semi-lactosi (Gal), glucose (Glc), pectinose (Ara) respectively; each 10 mg of seminose (Man); add 10mg oxammonium hydrochloride respectively; after 0.5 mL pyridine vibration; 90 DEG C of water-bath 30 min; and frequently vibrate; after taking-up is cooled to room temperature; add 0.5 mL diacetyl oxide; acidylate 30 mim in 90 DEG C of water-baths; namely the product obtained is sugared nitrile acetic ester derivative, directly injects GC and analyzes.
Polysaccharide aldoononitrile acetate takes polysaccharide 10mg, adds 2mol/L trifluoroacetic acid 5mL, after 110 DEG C of vacuum sealing tube hydrolysis 4h, be evaporated to dry at 40 DEG C, then add 4mL methyl alcohol, rotary evaporated to dryness, repeat 3 ~ 4 times, to remove trifluoroacetic acid.Then 10 mg oxammonium hydrochlorides and 0.5 mL pyridine is added; put into 90 DEG C 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; in 90 DEG C of water-baths, continue reaction 30 min carry out acetylize, reaction product directly can carry out gas chromatographic analysis
Chromatographic condition:
Chromatographic column: OV1701(30.0 m × 0.32 μm × 0.25 μm) capillary column;
Injector temperature: 250 DEG C; (FID) detector temperature: 240 DEG C;
Nebulizer gas pressure: 0.4 MPa; Temperature programming: keep 4 min at 150 DEG C, then rises to 200 DEG C with 10 DEG C/min, keeps 8 min, then rises to 280 DEG C with 15 DEG C/min, keeps 8 min.
Partial acid hydrolysis
Get 20mg PULP III polysaccharide fraction, carry out Partial acid hydrolysis with 2mL 0.05mol/L trifluoroacetic acid, centrifugal after hydrolysis 16h, precipitation carries out GC analysis.Add methyl alcohol rotary evaporation in supernatant liquor, repeat 3 ~ 4 removing trifluoroacetic acids, after then redissolving with water, dialysis tubing (molecular retention amount 3500) is dialysed.After dialysis terminates, after the outer part vacuum-drying of bag, carry out GC analysis; In bag, part adds the alcohol chromatography of 4 times of volumes, and supernatant fraction and alcohol precipitation part carry out GC analysis after vacuum-drying respectively.
Periodate oxidation and smith degraded
The making of sodium periodate typical curve
Get 6 dry test-tubes, according to the form below operation production standard curve:
Get the above-mentioned solution of 0.1mL respectively, be settled to 25 mL, measure optical density(OD) at 223 nm places.Then with sodium periodate concentration for X-coordinate, optical density value is ordinate zou, drawing standard curve.
Sample preparation
Accurately take sugared sample 25 mg, be dissolved in 15 mmol/L NaIO
4in solution, put in 25 mL volumetric flasks, constant volume shakes up.In the dark react at 4 DEG C, respectively 0,6,12,24,36,48,60h ... after sample 0.1 mL successively, be placed in 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 recorded, according to sodium periodate typical curve, the consumption of Periodic acid can be calculated.
Formic acid measures
Getting the reaction soln of the above-mentioned ethylene glycol process of 2mL, is under the condition of indicator in tetrabromo-mcresolsulfonphthalein, with NaOH solution (being demarcate liquid with the Potassium Hydrogen Phthalate) titration of 0.01mol/L, calculates the growing amount of formic acid.
Smith degrades
The polysaccharide sample solution dialysis 48h after ethylene glycol process, at 40 DEG C, be evaporated to about 10mL, under room temperature, add 70mg NaBH
4after, be placed in dark place and stir 24h, to reduce polysaccharide aldehyde.After 24h, add in 50% HAc and remaining NaBH
4, be the 48h (flowing water and distilled water 24h respectively) that dialyses after 6 ~ 7 to pH.After having dialysed, add the H of 1 mol/L of same volume
2sO
4, vacuum sealing tube, 100 DEG C of Water Under solution 8h, then add BaCO
3regulate pH to 6, filter with quantitative paper, filter vacuum concentrates evaporate to dryness, carries out GC analysis after acetylize.
Results and analysis
Physico-chemical property
PULP III is khaki color chip solid, soluble in water, is insoluble to ethanol, acetone and other organic solvent.Molish reaction, phenolsulfuric acid reaction, sulfuric acid-carbazole reaction positive, the reaction of ninhydrin reaction, IKI, the Fehling reaction, ferric chloride reaction and sulfate reaction are for negative.
The molecular weight of PULP III
According to color atlas and the t of 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 measures 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 530nm place is 0.75, and according to regression equation calculation, glucuronic acid content is 45.1%.
Protein and nucleic acid in PULP III molecule
The ultraviolet absorpting spectrum display of PULP III absorbs without without protein and nucleic acid, therefore exists without protein and nucleic acid in PULP III.
PULP III ultimate analysis
PULP III results of elemental analyses is as shown in table 2-1:
Table 2-1 results of elemental analyses
Found out by table 2-1, in polysaccharide, N percentage composition is below 7%, and in conjunction with ultraviolet absorption spectroscopy result above, can not contain amino in interpret sample, be non-amino sugar.Because if be aminosugar, then the content of N element at least should more than 7%.
The infrared absorption pattern of PULP III
Infared spectrum shows, 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 place.And PULP III is at 1010 ~ 1100 cm
-1there are three strong absorption peaks, illustrated that pyranose glycosidic bond exists.
The monose composition of PULP III
According to acetolysis step and GC condition, the GC collection of illustrative plates of bioassay standard monose mixed derivative, obtains the retention time of various standard monose and mixing monose.
The retention time of table 2-2 standard sugar and PULP III hydrolysis derivative
Can find out according to table 2-2, the monose composition in PULP III and ratio thereof are Rha:Ara:Man:Glc is 0.27:0.28:0.1:0.35.
PULP III Partial acid hydrolysis result
The analysis (GC) of PULP III partial acid hydrolysis products shows:
(1) PULP III forms primarily of Rha, Ara, Man and Glc;
(2) contain Rha, Ara, Man and Glc in the centrifugal precipitation obtained after PULP III Partial acid hydrolysis, illustrate that PULP III main chain is formed primarily of Rha, Ara, Man and Glc.
PULP III Periodic acid and Smith degradation results
Periodate oxidation result
NaIO
4after oxidation, the regression equation of concentration and light absorption value relation is: y(absorbance)=0.0416x(NaIO
4concentration)-0.0188, R
2=0.999.
PULP III is at 120h place, and absorbance reaches stable.According to regression equation, periodate oxidation analytical results is in Table 2-3.
The analysis of table 2-3 PULP III periodate oxidation
Analyze according to table 2-3, draw 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 there is the glycosidic link type that not react with Periodic acid, namely may also have 1 → 2,1 → 4 or 1 → 2,4 connect existence and 1 → 3 connects.
PULP III Smith degradation results
PULP III periodate oxidation product carries out Smith degraded, and interpretation of result is in Table 2-4.
The gas chromatographic analysis result of table 2-4 Smith degraded product
Smith degradation results shows, PULP III (1) exists the rhamnosyl of different content, pectinose and glucose, illustrates and exists not by the of bonding of periodate oxidation (1 → 3 mode of connection); (2) there is tetrahydroxybutane in degraded product, and there is not glycerine, illustrate all containing 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 connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously.
Conclusion
(1) physico-chemical property of PULP III and molecular structure Preliminary Analysis Results:
By physico-chemical property, glucuronic acid content measures, ultimate analysis, infrared and ultraviolet, and be that a class does not contain N, S element acidic polysaccharose in the bright PULP III of HPLC summary analysis, Relative average molecular weight is 418793.5;
IR collection of illustrative plates display PULP III has the charateristic avsorption band of polysaccharide, and at 1010 ~ 1100 cm
-1there are three strong absorption peaks, show to there is pyranose glycosidic bond;
Be made up of and Partial acid hydrolysis interpretation of result monose, PULP III monose composition and ratio thereof are Rha:Ara:Man:Glc is 0.27:0.28:0.1:0.35; Main chain forms primarily of Rha, Ara, Man and Glc.
Comprehensive periodate oxidation and Smith degradation results are analyzed, and PULP III glycosidic link connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously.About the configuration of glycosidic link, need to use NMR
1h spectrum and
13c spectrum is further studied.
the antioxidation activity in vitro preliminary study of Common Leafflower Herb polysaccharide PUIP III
The mensuration of polysaccharide reducing power
Laboratory reference Oyaizu method, slightly does and changes a little.Get 1 mL different concns (1,2,3,4,5mg/mL) polysaccharide soln in tool plug test tube, then 2.5 mL 0.2 mol/L pH 6.6 phosphate buffer solution and 1% potassium ferricyanide solutions are added respectively, after 50 DEG C of water-bath 20 min, ice bath cools rapidly, add the solution of trichloroacetic acid of 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, after reacting 10 min, measure the absorbancy at 700 nm places.
Polysaccharide external hydroxyl radical free radical (﹒ OH) mensuration of clearance rate
In Fenton reaction system, H
2o
2with Fe
2+mixing Chan Sheng ﹒ OH.You Yu ﹒ OH reactive behavior is strong, and the survival time is very short, adds Whitfield's ointment, just can Bu Zhuo Dao ﹒ OH effectively, and is created on the coloring matter that there is strong absorption at 510 nm places.Meanwhile, what to have a Competition with Whitfield's ointment if add in this system can the material of Qing Chu ﹒ OH, then the growing amount of coloring matter tails off, absorbancy step-down, and absorbancy is lower, proves that the ability of this material Qing Chu ﹒ OH is stronger.
The FeSO of 2 mL 9 mmol/L is added respectively in tool plug test tube
4the Whitfield's ointment ethanolic soln of solution, 2 mL 9 mmol/L, different concns (1,2,3,4,5mg/mL) polysaccharide soln, finally add the H of 2 mL 8.8mmol/L
2o
2solution starts reaction, reacts 30 min, measure the absorbancy at 510 nm places under room temperature, 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: sample absorbance
Anti peroxidation of lipid ability
Draw 0.4 mL yolk suspension [V(yolk): V(PBS)=1:25], 1 mL different concns (1,2,3,4,5mg/mL) polysaccharide soln, the FeSO of 0.4 mL 25 mmol/L
4, in tool plug test tube, PBS solution to the cumulative volume adding 0.1 mol/LpH7.45 is 4.0 mL, and 37 DEG C of constant temperature water baths vibrate 15 min.The TCA of 1.0 mL 20% is added after taking-up, after leaving standstill 10 min, 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 cooling, does reference with PBS, measures the light absorption value at 532 nm places.
The inhibiting rate of sample to lipovitellinin lipid peroxidation represents the ability of the anti-oxidant activity of sample, that is:
Inhibiting rate I=(A
0-A)/A
0× 100%
A
0the absorbancy of-control tube; The absorbancy of A-sample
Results and analysis
The reducing power of PULP III
Experimental result shows that 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 with concentration and strengthens.
The external hydroxyl radical free radical clearance rate of PULP III
Experimental result shows that PULP III polysaccharide soln under each concentration has certain Scavenging activity to external hydroxyl radical free radical, and in the concentration range of 1 ~ 5mg/mL, and its clearance rate increases with concentration and strengthens.During 5mg/mL, the clearance rate of PULP III is 50.7%%.
PULP III anti peroxidation of lipid ability
Experimental result shows that PULP III polysaccharide soln under each concentration has certain restraining effect to LPO, and there is dose-effect relationship in the concentration range of 1 ~ 5mg/mL, and its inhibiting rate increases with concentration and strengthens.During 5mg/mL, the inhibiting rate of PULP III is 23.7%.
Conclusion
Common Leafflower Herb polysaccharide PULP III has certain reducing power, the outer hydroxyl radical free radical ability of purged body 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, reaches 50.7%, be respectively 23.7% to the inhibiting rate of LPO to the clearance rate of external hydroxyl free.This illustrates that Common Leafflower Herb polysaccharide has certain anti-oxidant activity and its anti-oxidant activity is mainly manifested on the outer hydroxyl radical free radical of purged body.
common Leafflower Herb polysaccharide PUIP III effect on hepatitics B virus in vitro is studied
This test adopts current most widely used effect on hepatitics B virus in vitro medicaments sifting model HepG2.2.2.15 cell model, PUIP III and HepG2.2.2.15 cytosis is obtained from, Common Leafflower Herb extraction and isolation, get its cell conditioned medium liquid, measure the titre change of its hepatitis B surface antigen (HBsAg), e antigen (HBeAg) respectively, judge that whether this medicine is effective, and adopt mtt assay to measure the cytotoxicity of each several part.These two indexs comprehensive, In Vitro Anti hepatitis B pharmacodynamics test is carried out to Common Leafflower Herb polysaccharide PUIP III, and select clinically the certified acyclovir (ACV) with anti-HBV effect as positive control medicine, to evaluate the effect of each position hepatitis B virus resisting, screen efficient part or effective constituent with this.
The recovery of cell
Basic step: the warm water allocating 37 DEG C ~ 40 DEG C; From liquid nitrogen container, take out HepG2.2.2.15 cell cryopreservation tube, drop into immediately in the warm water of 37 DEG C ~ 40 DEG C and rock fast, until frozen storing liquid melts completely, melting process completes in 1-2min; Cell cryopreservation suspension is moved into centrifuge tube, adds about 5mL nutrient solution, blow even gently; By centrifugal for cell suspension 800r-1000r/min 5min, abandon supernatant liquor; Add complete culture solution to cell precipitation, pressure-vaccum beats gently, and cell suspension is moved into culturing bottle, fills up nutrient solution and cultivates, and is placed in 37 DEG C, 5%CO
2cell culture incubator is cultivated.
The Secondary Culture of cell
HepG2.2.2.15 cell is placed in 5%CO
2, 37 DEG C of cultivations.Substratum is DMEM, adds the foetal calf serum of 10%, 3% L-glutaminate 1%, G418 200ug/mL, penicillin 100u/mL, Streptomycin sulphate 100u/mL.2.2.15, after cell covers with culturing bottle, first digest 10-30 second with EDTA, then digest 3-10min with 0.25% pancreatin 37 DEG C, add nutrient solution piping and druming, 1:3 or 1:4 goes down to posterity, and within 8 days, covers with, and adopts the numeration of cell count plate, is mixed with 10
5individual/mL inoculating cell culture plate, the every hole 0.1mL of 96 orifice plate; 24 orifice plates every hole 1mL, 5%CO
2, 37 DEG C of cultivations are tested for 24 hours.
Method for cell count
General blood cell counting plate, counts by white blood cell count(WBC) method.
Get cell suspension 1mL to be diluted to add physiological saline and do 5 times of dilutions, count with the multiple of 10 × 10, central authorities are red blood cell count(RBC) use, the large lattice in corner are that white blood cell count(WBC) is used, complete cell is only added up during counting, if the cell bunched up counts by a cell, in a grid, if there is cell to be positioned on 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 after counting to calculate the cell count in every mL suspension, the area due to each grid in tally is 0.1cm
2, height is 0.01cm, and volume is 0.0001cm
3.Every mL cell count calculates by formula 1:
Every mL cell count=(n/4) × 10000 × 5 (1)
In formula: n is four large lattice total cellular score
MTT colorimetry surveys cell survival rate
HepG2.2.2.15 cell cultures in 96 well culture plates, every hole 80 μ l nutrient solution; 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 room temperature about 10min(or flat board is placed in micro vibrator concussion), crystallisate is dissolved; Microplate reader measures photoabsorption, and mensuration wavelength is 570nm.
Mtt assay surveys drug toxicity
By HepG2.2.2.15 cell by 10
5individual/mL is inoculated in 96 well culture plates, 0.1 mL/ hole, and medicine to be measured is made into several different concentration by secondary daily nutrient solution respectively, adds cell hole, and every hole concentration adds 3 holes, 0.1mL/ hole, and establishes without medicine cell controls and positive control drug.Cultivate 4 days after dosing, abandon supernatant MTT and dye, every hole adds the MTT serum-free medium 0.1mL of 1mg/mL, hatches 4 hours, abandons supernatant, after adding 0.04mol/L hydrochloric acid Virahol 0.1mL dissolving, by 570nm wavelength colorimetric estimation OD value, and experiment repetition 3 times.
Survey HBsAg, HBeAg secreting law
By HepG2.2.2.15 cell 10
5/ mL is inoculated in 24 porocyte culture plates, every hole 1.0mL, totally 10 holes, 3rd, within 5,8,11,13 days, collect supernatant 250 μ l, nutrient solution supplied commercial weight to the 13rd day, and culture supernatant is frozen in-20 DEG C, finally concentrate and press test kit specification sheets, measure HBsAg, HBeAg by ELISA method.
Medicine is to HBV inhibition test
By HepG2.2.2.15 cell by 10
5/ mL is inoculated in 24 orifice plates, every hole 1.0mL, and next day abandons nutrient solution, carries out doubling dilution with the half toxic concentration of the HepG2.2.2.15 cell of medicine to be measured for initial concentration nutrient solution, separately establishes without medicine contrast and positive control drug, is incubated at 37 DEG C, 5%CO
2in incubator.Within every 4 days, collect supernatant, and change and add original content liquid and continue to cultivate, by frozen for the supernatant-20 DEG C collected, collected in the 12nd day, concentrated ELISA method measures HBsAg, HBeAg, tests repetition 3 times.
ELISA method surveys HBsAg, HBeAg
(1) each for test kit component is taken out from box, balance to room temperature (18 ~ 25 DEG C).
(2) concentrated cleaning solution fully shakes up before preparing and should fully melt if any crystal, and concentrated cleaning solution distilled water or deionized water use after pressing 1:19 dilution.
(3) micropore lath is fixed on support, numbers according to the order of sequence.
(4) every hole adds sample 50 μ l to be measured, if each 2 holes of yin, yang contrast, 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 except), abundant mixing shrouding afterwards, be placed in 37 DEG C of environment and hatch 30min;
(6) plate is washed by hand; Discard liquid in hole, the full each hole of washings storage, leaves standstill and dries after 5 seconds, pat dry after repeating 5 times;
(7) every hole adds developer A liquid, each 50 μ l of B liquid, fully mixes, shrouding, puts in 37 DEG C of environment and hatch 15min;
(8) every hole adds stop buffer 50 μ l, mixing;
(9) use microplate reader reading, get wavelength 450nm, first use blank empty school zero.Then each hole OD value is read.(negative control OD value calculates as 0.05 lower than 0.05, calculates by actual OD value higher than 0.05.）
Data analysis
Mtt assay can obtain after surveying drug toxicity:
（2）
The poisonous concentration TC of half is calculated according to Red-Meuench method
50can obtain
（3）
In formula: B---
A——
C——A-B
Adopt ELISA method, after color reaction completes, the automatic microplate reader of use reads OD value, calculating inhibiting rate, being greater than 50% for there being restraining effect with inhibiting rate.Drug inhibition antigen percentage is calculated according to determination data; Drug inhibition antigen medium effective concentration IC
50, drug level when namely HBsAg and HBeAg inhibiting rate is 50%; It is effective for selecting therapeutic index TI, TI to be greater than more than 1 person, illustrates that medicine is effective, but have certain toxicity between 1 ~ 2; Be greater than 2 effects better, toxicity is less; Index is larger, then curative effect is better, and safety range is larger.Statistical procedures, between employing t checks and organizes, mean compares, and data statistic analysis is by SPSS11.5 software processes, and P<0.05 represents that difference has significance.
（4）
（5）
In formula: B---
A——
C——A-B
（6）
Interpretation of result
The rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Under the culture condition described in test method, this is tested HepG2.2.2.15 cell used and started in the 3rd day to have measured HBsAg, HBeAg secretion, peaks, weaken gradually later at the 8th day, last till the 13rd day, the rule of HBsAg, HBeAg secretion as shown in Figure 3.
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 is cultivated, the display of MTT test-results has restraining effect to 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%), it can thus be appreciated that the TC of ACV
0for 0.3125mg/mL.TC can be obtained 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 HepG2.2.2.15 cell cultures
Positive drug (ACV) suppresses the effect of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Different concns ACV process cultivation collecting cell supernatant liquor after 4 days and 8 days, ELISA method surveys HBsAg, HBeAg level display 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 the results are shown in Table 3-2 and 3-3. to the restraining effect of HBsAg, HBeAg in HepG2.2.2.15 cell cultures
Show 3-2 ACV in HepG2.2.2.15 cell cultures to the restraining effect of HBsAg
Show 3-3 ACV in HepG2.2.2.15 cell cultures to the restraining effect of HBeAg
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 time IC
50be respectively 0.58mg/mL and 0.49mg/mL.The therapeutic index TI of ACV to HBsAg, HBeAg can be obtained according to formula (6) and be respectively 7.86 and 9.31.Concrete outcome is in Table 3-4.
Show 3-4 ACV in HepG2.2.2.15 cell cultures to the IC of HBsAg and HBeAg
50and TI
The effect on hepatitics B virus in vitro effect of Common Leafflower Herb polysaccharide PUIP III
The toxicity of Common Leafflower Herb polysaccharide PUIP III pair of HepG2.2.2.15 cell
After different concns Common Leafflower Herb polysaccharide PUIP III treatment group is cultivated, the display of MTT test-results has restraining effect to 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%), it can thus be appreciated that the TC of Common Leafflower Herb polysaccharide PUIP III
0for 3.12mg/mL.TC can be obtained 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 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 processes cultivation collecting cell supernatant liquor after 4 days and 8 days, ELISA method surveys HBsAg, HBeAg level display Common Leafflower Herb polysaccharide PUIP III couple of HBsAg, HBeAg all has restraining effect, and along with the increase of 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 the results are shown in Table 3-6 and 3-7 to the restraining effect of HBsAg, HBeAg in HepG2.2.2.15 cell cultures
Show 3-6 Common Leafflower Herb polysaccharide PUIP III in HepG2.2.2.15 cell cultures to the restraining effect of HBsAg
Show 3-7 Common Leafflower Herb polysaccharide PUIP III in HepG2.2.2.15 cell cultures to the restraining effect of HBeAg
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 time IC
50be respectively 3.82mg/mL and 14.60mg/mL.The therapeutic index TI that can obtain Common Leafflower Herb polysaccharide PUIP III couple of HBsAg, HBeAg according to formula (6) respectively 11.89 for and 3.11.Concrete outcome is in Table 3-8.
Show 3-8 Common Leafflower Herb polysaccharide PUIP III in HepG2.2.2.15 cell cultures to the IC of HBsAg and HBeAg
50and TI
Discuss
This test have employed mtt assay and carries out cytotoxicity detection, the maximal non-toxic concentration (TC of result display 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 in HepG2.2.2.15 cell cultures to the restraining effect of HBsAg, HBeAg, in 7 concentration that result display Common Leafflower Herb polysaccharide PUIP III sets under non-toxic concn, except minimum concentration, other each concentration group all shows obvious restraining effect compared with cell controls group, and along with the increase of Common Leafflower Herb polysaccharide PUIP III concentration, its restraining effect strengthens, and inhibiting rate increases, and demonstrates certain dose-effect relationship.Be at present therapeutic index (TI) for evaluating the result for the treatment of index of medicine clinically, it is drug ineffective as TI<1, as 1<TI<2, medicine poor efficiency is poisonous, the effective low toxicity of medicine as TI>2.It can thus be appreciated that, the effective low toxicity of therapeutic index (TI is respectively 11.89 and 3.11) of Common Leafflower Herb polysaccharide PUIP III couple of HBsAg, HBeAg; The effective low toxicity of the therapeutic index of positive control medicine acyclovir to HBsAg, HBeAg (TI is respectively 7.86 and 9.31).
In sum, Common Leafflower Herb polysaccharide PUIP III all has certain restraining effect to HBsAg, HBeAg in HepG2.2.2.15 cell cultures.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (6)
1. an extraction and separation method of Common Leafflower Herb polysaccharide PULP III, is characterized in that: described method comprises the steps:
1) Common Leafflower Herb herb distilled water wash removes soil, after drying, pulverizes, with petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, vat liquor centrifugation, gets after filtrate concentrates and adds ethanol alcohol precipitation, get alcohol hypostasis and be thick total polysaccharides; Thick total polysaccharides is through 2) removing protein, 3) washing, removal of impurities and 4) dialysis, be drying to obtain Common Leafflower Herb essence total polysaccharides, polysaccharide content is more than 95%; 5) each Component seperation purifying of polysaccharide: through deproteinated, Common Leafflower Herb essence total polysaccharides after dialysis treatment, cross ion exchange column, use NaCl gradient solution wash-out successively, phend-sulphuric acid tracing detection, obtains elution curve, obviously can declare out the elution peak of four peak shape symmetries from curve, represent four components respectively, be designated as PULP I, PULP II, PULP III and PULP IV respectively according to the order going out peak; Wherein the 2nd, the 3rd two elution peaks are comparatively large, collect the 3rd main peak component, concentrated, redistilled water dialysis 48h, and lyophilize obtains Common Leafflower Herb polysaccharide PULP III pure component;
The ion exchange column of step 5) is DEAE-52 anionite-exchange resin, and by DEAE-52 cellulose wadding distilled water immersion 48h before using, period 4 ~ 5h changes a water, and removes suspended impurity with decantation, then carries out alkali-acid-alkali process; After first soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, then is washed till neutrality with distilled water after HCl solution soaking 30 min of 0.5 mol/L, and after finally soaking 30 min by the NaOH solution of 0.5 mol/L, distilled water is washed till neutrality, obtains OH
-fiber type element; Wet method dress post, column dimension is 500 × 50mm, distilled water balance 48h, avoids bubble and fault-layer-phenomenon in dress post process;
The substance withdrawl syndrome of the NaCl gradient solution of step 5) is respectively 0,0.1,0.25,0.5,0.75mol/L;
Step 5) uses NaCl gradient solution wash-out successively, the substance withdrawl syndrome eluant solution of often kind of NaCl 8 hours, and flow velocity 4mL/min, collects elutriant with pipe, and every 10min collects a pipe.
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, with sherwood oil submergence medicinal material and liquid level exceeds medicinal material 0.5 ~ 1cm, 70 ~ 90 DEG C and normal pressure power refluxing extraction 2 ~ 4 times, each 1 ~ 3 hour, discard phegma; Dregs of a decoction continuation massfraction be the ethanolic soln of 50 ~ 100% by same method refluxing extraction 2 ~ 4 times at 80 ~ 95 DEG C, each 1 ~ 3 hour, discard phegma; 60 ~ 95 DEG C of floodings 3 times used again by the dregs of a decoction, 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 adding 4 times of volumes is the ethanolic soln of 90 ~ 100%, leaves standstill 24h, centrifugal, get alcohol 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 removing protein use Sevage method deproteinated: alcohol hypostasis distilled water step 1) obtained redissolves to obtain Crude polysaccharides solution, chloroform and propyl carbinol mixed solution is added, wherein Crude polysaccharides solution: the volume ratio of chloroform and propyl carbinol mixed solution is 1 ~ 6:1 ~ 3 in Crude polysaccharides solution; Chloroform in mixed solution: the volume ratio of propyl carbinol is 1 ~ 4:1 ~ 4; Magnetic agitation 20 ~ 40min, sufficient standing layering, discards precipitation; Until interface is without white precipitate after repeating 1 ~ 10 time.
4. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: step 3) is washed, removal of impurities: the massfraction adding 3 ~ 6 times of volumes in the polysaccharide soln after removing protein is the ethanolic soln precipitation 12 ~ 36h of 75 ~ 100%, centrifugal, abandon filtrate, precipitation with dehydrated alcohol, acetone, ether cleaning, repeats 2 ~ 5 times successively.
5. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, it is characterized in that: step 4) is dialysed, dry: namely after alcohol precipitation washing, filter residue adds appropriate distilled water, dialyse after abundant redissolution, dialysis is carried out under magnetic stirring, the volume ratio of sample liquid and dialyzate and distilled water is 1:10 ~ 30, and 4 ~ 8h changes a water, dialysis 12 ~ 120h; After dialysis, sugar soln is put into freeze drier dry Common Leafflower Herb essence total polysaccharides, content is more than 95%.
6. the extraction and separation method of Common Leafflower Herb polysaccharide PULP III according to claim 1, is characterized in that: step 5) elution curve is the Guan Xuwei X-coordinate collected, and the absorbance of solution is that ordinate zou drawing obtains.
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| CN111647095B (en) * | 2020-06-30 | 2022-02-22 | 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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| "多糖的提取和分析方法";刘婷 等;《化工时刊》;20080325;第22卷(第3期);第66-70页 * |
| "楮头红多糖的提取、结构及其活性研究";朱庆银;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20071215(第6期);第21-23页，第31-33页 * |
| "正交试验优选叶下珠中多糖的提取工艺";周宇 等;《化工时刊》;20090825;第23卷(第8期);第32页 * |
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