CN103059157B - Phyllanthus urinaria L polysaccharide component extraction and separation method - Google Patents
Phyllanthus urinaria L polysaccharide component extraction and separation method Download PDFInfo
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Abstract
The invention relates to a Phyllanthus urinaria L polysaccharide component PULP II extraction and separation method. The method comprises the following steps: 1, washing whole Phyllanthus urinaria L with distilled water, drying, crushing, degreasing with petroleum ether, removing micro-molecular sugars with ethanol, extracting the obtained herbal residues with water, centrifuging the obtained extract liquid, taking the obtained supernatant, and adding ethanol for ethanol precipitation to obtain ethanol precipitation substances which are crude polysaccharides; 2, removing proteins from the crude polysaccharides; 3, carrying out alcohol precipitation, and washing; 4, dialyzing and drying to obtain refined Phyllanthus urinaria L total polysaccharides; and 5, separating and purifying all components of the polysaccharides, and collecting the second main peak component to obtain he pure Phyllanthus urinaria L polysaccharide PULP II component. The pure and single Phyllanthus urinaria L polysaccharide component is obtained through separating and purifying the crude Phyllanthus urinaria L polysaccharides. Researches on the structural analysis and the in-vitro anti-oxidation and antiviral activity experiments of the pure polysaccharide component PULP II prove that the Phyllanthus urinaria L polysaccharides are effective components of Phyllanthus urinaria L for treating hepatitis B, so theoretical bases are provided for researching the anti-hepatitis B virus effect mechanism of the Phyllanthus urinaria L polysaccharides.
Description
Technical field
A kind of extraction and separation method relating to Common Leafflower Herb polysaccharide component PUIP II of the present invention.
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.
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 this invention is to provide the extraction and separation method of a kind of Common Leafflower Herb polysaccharide component PUIP II.
The extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II of the present invention, comprises the steps:
1) pulverize after the cleaning, drying of Common Leafflower Herb herb distilled water, with petroleum ether degreasing, after ethanol takes off small molecular sugar, dregs of a decoction flooding, vat liquor centrifugation, get after clear liquid concentrates and add ethanol alcohol precipitation, alcohol hypostasis is thick total polysaccharides; Thick total polysaccharides is through 2) removing protein, 3) alcohol precipitation, washing and 4) dialyse, be drying to obtain Common Leafflower Herb essence total polysaccharides; 5) each Component seperation purifying of polysaccharide: Common Leafflower Herb essence total polysaccharides crosses ion exchange column, use NaCl solution gradient elution, phend-sulphuric acid tracing detection, draws elution curve, elution curve occurs successively four peaks, represent four polysaccharide fractions respectively, be designated as PULP I, PULP II, PULP III and PULP IV respectively according to the order going out peak, collect the 2nd main peak component, concentrated, redistilled water is dialysed, and lyophilize obtains Common Leafflower Herb polysaccharide PULP II pure component.
The Common Leafflower Herb herb powder that step 1) is pulverized, by sherwood oil refluxing extraction 2 ~ 4 times at 70 ~ 90 DEG C, each 1 ~ 3 hour, discard phegma, the dregs of a decoction continue by 50 ~ 100% ethanol 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 obtained, each 1 ~ 3 hour, merge 3 vat liquors and carry out centrifugation, centrifugal speed 2000 ~ 6000r/min, get clear liquid and be concentrated into original volume 1/4, add 90 ~ 100% ethanol of 4 times of volumes, leave standstill 24h, centrifugal, get alcohol hypostasis and be Crude polysaccharides.
Step 2) described removing protein adopts Sevag method deproteinated: alcohol hypostasis distilled water step 1) obtained redissolves to obtain Crude polysaccharides solution, chloroform and propyl carbinol mixed solution is added in Crude polysaccharides solution, Crude polysaccharides solution: chloroform and propyl carbinol mixeding liquid volume are 1 ~ 6:1 ~ 3, chloroform in described chloroform and propyl carbinol mixed solution: propyl carbinol volume ratio=1 ~ 4:1 ~ 4, stir, sufficient standing layering, discards precipitation, until interface is without white precipitate after repeating 1 ~ 10 time.
Alcohol precipitation, washing described in step 3): 75 ~ 100% ethanol adding 3 ~ 6 times of volumes in the polysaccharide soln after removing protein, leave standstill 12 ~ 36h, centrifugal, and precipitation with dehydrated alcohol, acetone, ether cleaning, repeats 2 ~ 5 times successively.
Dialysis described in step 4), drying: the polysaccharide got after alcohol precipitation, washing adds distilled water, distill water dialysis is used after abundant redissolution, dialysis is carried out under magnetic stirring, sample liquid and distilled water volume ratio are 1:10 ~ 30,4 ~ 8h changes a water, dialysis 12 ~ 120h, after dialysis, sugar soln is put into freeze drier dry Common Leafflower Herb essence total polysaccharides, polysaccharide content is more than 95%.
The ion exchange column of step 5) adopts DEAE-52 anionite-exchange resin.
The concentration gradient scope of NaCl solution described in step 5) is 0 ~ 3mol/L.Preferably, the concentration gradient of described NaCl solution be respectively 0,0.1,0.25,0.5,0.75mol/L.
Step 5) uses different concns NaCl gradient elution successively, often kind of concentration NaCl solution wash-out 8 hours, and flow velocity 4mL/min, collects elutriant with pipe, and every 10min collects a pipe, draws elution curve.Described elution curve is with the Guan Xuwei X-coordinate collected, and the absorbance of solution is that ordinate zou drafting forms.
Common Leafflower Herb polysaccharide PUIP II of the present invention is khaki color powder, the easy moisture absorption, and be that a class does not contain N, S element acidic polysaccharose, Relative average molecular weight is 579962.6; PULP II monose composition and ratio thereof are rhamnosyl (Rha): pectinose (Ara): seminose (Man): glucose (Glc) is 0.11:0.36:0.08:0.45; Main chain is made up of Rha, Ara and Glc.Each monose is connected by furanose glycosidic bond, and glycosidic link connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously.
Common Leafflower Herb polysaccharide PUIP II of the present invention, has certain anti-oxidant, anti-hepatitis B virus activities.
Advantage of the present invention is: 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 II 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; From now on can on this basis, in conjunction with the structure activity relationship of polysaccharide, further for exploitation is that raw-material anti-hepatic-B virus medicine lays the foundation with Common Leafflower Herb polysaccharide.
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 embodiment 1 Common Leafflower Herb polysaccharide
1 materials and methods
1.1 materials (summary)
1.2 experimental technique
1.2.1 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.
1.2.2 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).
1.2.3 the mensuration of Common Leafflower Herb total polysaccharides content
1.2.3.1 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.
1.2.3.2 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.
1.2.3.3 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.
1.2.3.4 the calculating of sugared content
Accurate absorption test liquid 1mL, by " 1.2.3.3 " with method operation, 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
1.2.3.5 the extraction yield of polysaccharide
Quality/raw-material quality × 100 % of the extraction yield=polysaccharide of polysaccharide
2. results and analysis
2..1 the determination of wavelength is measured
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.
2. 2 glucose standard curve
Take absorbancy as Y-axis, glucose micrograms (g) is X-axis, drawing standard curve, as Fig. 1, can find out that, within the scope of 20 ~ 100 g, glucose amount and absorbancy have good linear relationship.
2.3 sugared content
The absorbance A recording sample liquid is 0.388, is 28.27 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 2.4 polysaccharide
Extraction yield==1.5/100 × 100 %=1.5% of polysaccharide
3 conclusions
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 embodiment 2 Common Leafflower Herb polysaccharide
1 materials and methods
1.1 materials (summary)
1.2. experimental technique
1.2.1 DEAE-52 post is separated
1.2.1.1 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.
1.2.1.2 the separation and purification of DEAE-52 chromatography column is crossed
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.
1.2.2. Purity
1.2.2.1 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.
1.2.2 2 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.
Chromatographic 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.
2 results and analysis
2.1 DEAE-52 column chromatography for separation results
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 II collecting content larger is further analyzed.
2.2 SephadexG-200 posts and HPLC Purity
2.2.1 SephadexG-200 post result
Get Common Leafflower Herb polysaccharide fraction PULP II 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 II on Sephadex G-200 post is symmetrical simple spike, illustrates that PULP II component is the relatively homogeneous polysaccharide fraction of molecular weight.
2.2.2 HPLC method Purity
When utilizing HPLC method to detect the purity of Common Leafflower Herb polysaccharide fraction PULP II, result shows, polysaccharide fraction PULP II 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.
3 conclusions
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 II 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 embodiment 3 PULP II and structural analysis
1, physico-chemical property
PULP II is khaki color powder, easy moisture absorption.Soluble in water, be 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.
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 II of HPLC summary analysis, Relative average molecular weight is 579962.6.
2, the infrared absorption pattern of PULP II
Infared spectrum shows, PULP II 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 II is at 1010 ~ 1100 cm
-1there are two strong absorption peaks, illustrated that furan type glycosidic link exists.
Be made up of and Partial acid hydrolysis interpretation of result monose, PULP II monose composition and ratio thereof are Rha:Ara:
Man:Glc is 0.11:0.36:0.08:0.45; Main chain forms primarily of Rha, Ara and Glc.
Comprehensive periodate oxidation and Smith degradation results are analyzed, and PULP II glycosidic link connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously.
The antioxidation activity in vitro preliminary study of embodiment 4 Common Leafflower Herb polysaccharide PUIP II
1 materials and methods
1.1 materials and reagent (summary)
1.2 experimental technique
1.2.1 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.
1.2..2 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
1.2.3 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
2 results and analysis
The reducing power of 2.1 PULP II
Experimental result shows that PULP II 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 2.2 PULP II
Experimental result shows that PULP II 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 II is 35.5%.
2.3 PULP II anti peroxidation of lipid abilities
Experimental result shows that PULP II polysaccharide soln under each concentration all 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 II is 10.7%.
3 conclusions
Common Leafflower Herb polysaccharide PULP II 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 II is 0.538, reaches 35.5%, be respectively 10.7% 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.
Embodiment 5 Common Leafflower Herb polysaccharide PUIP II 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 II 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 II, 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.
1 materials and methods
1.1 materials and reagent (summary)
1.2 experimental technique
1.2.1 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.
1.2.2 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.After 2.2.15 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.
1.2.3 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
1.2.4 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.
1.2.5 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.
1.2.6 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.
1.2.7 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.
1.2.8 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.）
1.3 data analysis
1.3.1 mtt assay can obtain after surveying drug toxicity:
1.3.2 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 between 1 and 2, but has certain toxicity; 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.
2 interpretations of result
The rule of 2.1 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 2.2 positive drugs (ACV)
2.2.1 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 2-1.
The toxicity of table 2-1 ACV in HepG2.2.2.15 cell cultures
Show 2-2 ACV in HepG2.2.2.15 cell cultures to the restraining effect of HBsAg
2.2.2 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 2-2 and 2-3 to the restraining effect of HBsAg, HBeAg in HepG2.2.2.15 cell cultures
Show 2-3 ACV in HepG2.2.2.15 cell cultures to the restraining effect of HBeAg
2.2.3 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 2-4.
Show 2-4 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 2.3 Common Leafflower Herb polysaccharide PUIP II
2.3.1 the toxicity of Common Leafflower Herb polysaccharide PUIP II pair of HepG2.2.2.15 cell
After different concns Common Leafflower Herb polysaccharide PUIP II 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 0.156mg/mL is 5.76%, and the cell survival rate under this concentration is 94.24% (≈ 95%), it can thus be appreciated that the TC of Common Leafflower Herb polysaccharide PUIP II
0<0.156mg/mL.Maximal percentage inhibition <50% within the scope of experimental concentration, therefore can only TC be calculated
50>10mg/mL.The cytotoxic assay of Common Leafflower Herb polysaccharide PUIP II the results are shown in Table 2-5.
The toxicity of table 2-5 Common Leafflower Herb polysaccharide PUIP II in HepG2.2.2.15 cell cultures
2.3.2 Common Leafflower Herb polysaccharide PUIP II suppresses the effect of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Different concns Common Leafflower Herb polysaccharide PUIP II processes cultivation collecting cell supernatant liquor after 4 days and 8 days, ELISA method surveys HBsAg, HBeAg level display Common Leafflower Herb polysaccharide PUIP II couple of HBsAg, HBeAg all has restraining effect, and along with the increase of Common Leafflower Herb polysaccharide PUIP II 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 II the results are shown in Table 2-6 and 2-7 to the restraining effect of HBsAg, HBeAg in HepG2.2.2.15 cell cultures.
Show 2-6 Common Leafflower Herb polysaccharide PUIP II in HepG2.2.2.15 cell cultures to the restraining effect of HBsAg
Show 2-7 Common Leafflower Herb polysaccharide PUIP II in HepG2.2.2.15 cell cultures to the restraining effect of HBeAg
2.3.3 the therapeutic index (TI) of Common Leafflower Herb polysaccharide PUIP II 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 1.59mg/mL and 0.49mg/mL.The therapeutic index TI difference >6.29 that can obtain Common Leafflower Herb polysaccharide PUIP III couple of HBsAg, HBeAg according to formula (6) is and >20.41.Concrete outcome is in Table 2-8.
Show 2-8 Common Leafflower Herb polysaccharide PUIP II in HepG2.2.2.15 cell cultures to the IC of HBsAg and HBeAg
50and TI
3 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 II HepG2.2.2.15 cell
0) <0.156 mg/mL; Poisonous concentration (the TC of half
50) >10 mg/mL.
This test adopts elisa technique to analyze Common Leafflower Herb polysaccharide PUIP II 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 II 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 II 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 of Common Leafflower Herb polysaccharide PUIP II couple of HBsAg, HBeAg (TI be respectively >=6.29 and 20.41); 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 II 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 component PUIP II, comprises the steps:
1) pulverize after the cleaning, drying of Common Leafflower Herb herb distilled water, with petroleum ether degreasing, after ethanol takes off small molecular sugar, dregs of a decoction flooding, vat liquor centrifugation, get after clear liquid concentrates and add ethanol alcohol precipitation, alcohol hypostasis is thick total polysaccharides; Thick total polysaccharides is through 2) removing protein, 3) alcohol precipitation, washing and 4) dialyse, be drying to obtain Common Leafflower Herb essence total polysaccharides; 5) each Component seperation purifying of polysaccharide: Common Leafflower Herb essence total polysaccharides crosses ion exchange column, use NaCl solution gradient elution, phend-sulphuric acid tracing detection, draw elution curve, elution curve there are successively four peaks, represent four polysaccharide fractions respectively, be designated as PULP I, PULP II, PULP III and PULP IV respectively according to the order going out peak, collect the 2nd main peak component, concentrated, redistilled water is dialysed, and lyophilize obtains Common Leafflower Herb polysaccharide PULP II pure component, i.e. Common Leafflower Herb polysaccharide component PUIP II;
Described Common Leafflower Herb polysaccharide PUIP II is khaki color powder, the easy moisture absorption, and be that a class does not contain N, S element acidic polysaccharose, Relative average molecular weight is 579962.6; PULP II monose composition and ratio thereof are rhamnosyl Rha: pectinose Ara: seminose Man: glucose Glc is 0.11:0.36:0.08:0.45; Main chain is made up of Rha, Ara and Glc; Each monose is connected by furanose glycosidic bond, and glycosidic link connects based on 1 → 6, also there is 1 → 4 and 1 → 3 mode of connection simultaneously;
The ion exchange column of step 5) adopts DEAE-52 anionite-exchange resin;
The concentration gradient of described NaCl solution is respectively 0,0.1,0.25,0.5,0.75mol/L;
Step 5) uses different concns NaCl gradient elution successively, often kind of concentration NaCl solution wash-out 8 hours, and flow velocity 4mL/min, collects elutriant with pipe, and every 10min collects a pipe, draws elution curve.
2. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that the Common Leafflower Herb herb powder that step 1) is pulverized, by sherwood oil refluxing extraction 2 ~ 4 times at 70 ~ 90 DEG C, each 1 ~ 3 hour, discard phegma, the dregs of a decoction continue by 50 ~ 100% ethanol 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 obtained, each 1 ~ 3 hour, merge 3 vat liquors and carry out centrifugation, centrifugal speed 2000 ~ 6000r/min, get clear liquid and be concentrated into original volume 1/4, add 90 ~ 100% ethanol of 4 times of volumes, leave standstill 24h, centrifugal, get alcohol hypostasis and be Crude polysaccharides.
3. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that step 2) described removing protein adopts Sevag method deproteinated: alcohol hypostasis distilled water step 1) obtained redissolves to obtain Crude polysaccharides solution, chloroform and propyl carbinol mixed solution is added in Crude polysaccharides solution, Crude polysaccharides solution: chloroform and propyl carbinol mixeding liquid volume are 1 ~ 6:1 ~ 3, chloroform in described chloroform and propyl carbinol mixed solution: propyl carbinol volume ratio=1 ~ 4:1 ~ 4, stir, sufficient standing layering, discard precipitation, until interface is without white precipitate after repeating 1 ~ 10 time.
4. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that alcohol precipitation described in step 3), washing: 75 ~ 100% ethanol adding 3 ~ 6 times of volumes in the polysaccharide soln after removing protein, leave standstill 12 ~ 36h, centrifugal, precipitation with dehydrated alcohol, acetone, ether cleaning, repeats 2 ~ 5 times successively.
5. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that dialysis described in step 4), drying: the polysaccharide got after alcohol precipitation, washing adds distilled water, distill water dialysis is used after abundant redissolution, dialysis is carried out under magnetic stirring, sample liquid and distilled water volume ratio are 1:10 ~ 30,4 ~ 8h changes a water, dialysis 12 ~ 120h, after dialysis, sugar soln is put into freeze drier dry Common Leafflower Herb essence total polysaccharides.
6. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, is characterized in that described elution curve is with the Guan Xuwei X-coordinate collected, and the absorbance of solution is that ordinate zou drafting forms.
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