CN103059157A - 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 that relates 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, at promoting diuresis to remove toxic substance, the calming liver and clearing heat of being widely used among the people.1988, people's first passages such as India scholar Thyagarajan experimental results show that Phyllanthusamarus can make 59% patient's hepatitis B surface antigen (HBsAg) turn out cloudy, and this experimental result has caused the concern of Chinese scholars to Common Leafflower Herb.Studies show that in a large number
]Common Leafflower Herb has hepatitis B virus resisting, protecting liver, lowering enzymes, anti-hepatic fibrosis, prevents the effect of liver injury and anticancer change, and toxic side effect is low, is a kind of deeply crude drug of the treatment hepatitis B of exploitation that is worth.
Contain the number of chemical composition in the Common Leafflower Herb, document had been reported lignanoid, terpene, flavones, the matter of mixing, alkaloid etc., comprises that Octadecane, dehydrogenation chebulic acid, forulic acid, gallic acid, brevifolin carboxylic acid, Succinic Acid, methoxyl group are mixed to spend acid, camellia element, plain, the short leaf bush acid of heroubill formicester, short leaf Soviet Union art phenolic acid second fat, corilagin, dehydrogenation chebulic acid three formicesters, polysaccharide etc.
Polysaccharide compound has the multiple biological activitys such as immunomodulatory, antitumor, antiviral, anti-oxidant, anti-inflammatory, anti-peptic ulcer, anticoagulation, hypoglycemic, reducing blood-fat, radioprotective, antithrombotic, Ivy extract, antitoxin thing damage.
Hepatitis B (HBV) is global public health problem, according to global health organization (WHO) data, there are 3.5～4.0 hundred million Patients with Hepatitis B Virus Infections in the whole world, and wherein annual have nearly 1,000,000 patients to die from liver failure, liver cirrhosis and liver cancer that the HBV infection causes.If be applied at present drug main Interferon, rabbit and the nucleoside analog for the treatment of hepatitis B, yet these medicines can only obtain result for the treatment of in a way.
In the process of seeking the treating hepatitis B medicine, the herbal polysaccharide composition more and more is subject to people's attention.Common Leafflower Herb has hepatitis B virus resisting, protect the liver, the effect such as antitumor generally confirmed, and relevant scholar is also to wherein several chemical ingredientss, done structure and bioactive research such as gallic acid, flavonoid and Common Leafflower Herb element etc., but to wherein polysaccharide researches seldom.The extraction of carrying out polysaccharide component in the Common Leafflower Herb separates, Structural Identification and bioactive detection, be conducive to further determine that Common Leafflower Herb is used for the treatment of the effective substance of hepatitis B, develop better this resources of medicinal plant of Common Leafflower Herb, lay theoretical basis for seeking the treating hepatitis B new drug.
Summary of the invention
The extraction and separation method that the purpose of this invention is to provide 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) the Common Leafflower Herb herb is pulverized after with the distilled water cleaning, drying, uses petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, the vat liquor centrifugation is got clear liquid and is added the ethanol alcohol precipitation after concentrated, and pure hypostasis is thick total polysaccharides; Thick total polysaccharides is through 2) except albumen, 3) alcohol precipitation, washing and 4) dialyse, be drying to obtain the smart total polysaccharides of Common Leafflower Herb; 5) each component separation and purification of polysaccharide: the smart total polysaccharides of Common Leafflower Herb is crossed ion exchange column, with NaCl solution gradient wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, and draws elution curve, occurs successively four peaks on the elution curve, represent respectively four polysaccharide fractions, be designated as respectively PULP I, PULP II, PULP III and PULP IV according to the order that goes out the peak, collect the 2nd main peak component, concentrated, the redistilled water dialysis, lyophilize gets Common Leafflower Herb polysaccharide PULP II pure component.
The Common Leafflower Herb herb powder that step 1) is pulverized, with sherwood oil 70～90 ℃ of lower refluxing extraction 2～4 times, each 1～3 hour, discard phegma, the dregs of a decoction continue with 50～100% ethanol 80～95 ℃ of lower refluxing extraction 2～4 times, each 1～3 hour, discard phegma, the dregs of a decoction that obtain are used 60～95 ℃ of floodings 3 times again, each 1～3 hour, merge 3 vat liquors and carry out centrifugation, centrifugal speed 2000～6000r/min gets clear liquid and is concentrated into original volume 1/4,90～100% ethanol that add 4 times of volumes, leave standstill 24h, centrifugal, get pure hypostasis and be Crude polysaccharides.
Step 2) describedly adopt Sevag method deproteinated except albumen: the pure hypostasis that step 1) is obtained redissolves to get Crude polysaccharides solution with distilled water, in Crude polysaccharides solution, add chloroform and propyl carbinol mixed solution, Crude polysaccharides solution: chloroform and propyl carbinol mixeding liquid volume are 1～6:1～3, chloroform in described chloroform and the propyl carbinol mixed solution: propyl carbinol volume ratio=1～4:1～4, stir, the sufficient standing layering discards precipitation, repeat 1～10 time after until the interface without white precipitate.
The described alcohol precipitation of step 3), washing: add 75～100% ethanol of 3～6 times of volumes in except the polysaccharide soln behind the albumen, leave standstill 12～36h, centrifugal, precipitation is cleaned with dehydrated alcohol, acetone, ether successively, repeats 2～5 times.
The described dialysis of step 4), drying: the polysaccharide of getting after alcohol precipitation, the washing adds distilled water, use distill water dialysis after fully redissolving, dialysis is carried out under magnetic agitation, sample liquid and distilled water volume ratio are 1:10～30,4～8h changes water one time, dialysis 12～120h, dialyse complete after with sugar soln 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 the described NaCl solution of 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) is used different concns NaCl gradient elution successively, every kind of concentration NaCl eluant solution 8 hours, and flow velocity 4mL/min collects elutriant with pipe, and every 10min collects a pipe, draws elution curve.The Guan Xuwei X-coordinate of described elution curve to collect, the absorbance of solution are that the ordinate zou drafting forms.
Common Leafflower Herb polysaccharide PUIP II of the present invention is the khaki color powder, and easily the moisture absorption is that a class does not contain N, S element acidic polysaccharose, and molecular-weight average is 579962.6 relatively; PULP II monose forms and ratio is rhamnosyl (Rha): pectinose (Ara): seminose (Man): glucose (Glc) is 0.11:0.36:0.08:0.45; Main chain is comprised of Rha, Ara and Glc.Each monose connects by the furanose glycosidic bond, and glycosidic link is connected to the master with 1 → 6, also has simultaneously 1 → 4 and 1 → 3 mode of connection.
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 the Common Leafflower Herb Crude polysaccharides, obtained pure single Common Leafflower Herb polysaccharide component.By the research to the structural analysis of the pure component PULP II of polysaccharide and antioxidation in vitro, antiviral activity experiment, having proved conclusively the Common Leafflower Herb polysaccharide is the effective constituent of phyllanthus for treating hepatitis B, and this anti-HBV effect mechanism for research Common Leafflower Herb polysaccharide provides theoretical basis; Can on this basis, in conjunction with the structure activity relationship of polysaccharide, further lay the foundation take the Common Leafflower Herb polysaccharide as raw-material anti-hepatic-B virus medicine for exploitation from now on.
Description of drawings
Fig. 1 is the glucose typical curve.
Fig. 2 is polysaccharide gradient elution curve.
Fig. 3 is the rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg.
Embodiment
The present invention is described in detail below in conjunction with embodiment:
The extraction of embodiment 1 Common Leafflower Herb polysaccharide
1 materials and methods
1.1 material (summary)
1.2 experimental technique
1.2.1 Common Leafflower Herb pre-treatment
Common Leafflower Herb herb distilled water wash removes soil, after 50 ℃ of cryodryings, pulverizes, and seals for subsequent use.Its powder is yellow-green colour.
1.2.2 the extracting method of Common Leafflower Herb polysaccharide
80 ℃ of backflow degreasings of sherwood oil → 80 ℃ of backflows of 95% ethanol are taken off small molecular sugar → 90 ℃ water refluxing extraction 3 times, it is centrifugal to merge 3 filtrate 4000r/min, be concentrated into 95% ethanol of 1/4 volume → 4 times of volumes of adding, leave standstill 24h, centrifugal, distilled water redissolution → sevag method is except albumen (chloroform: propyl carbinol=4:1, polysaccharide soln: mixed solution=3:1), magnetic agitation 30min, the sufficient standing layering repeats 7～8 operations and can remove free protein → add the 95% ethanol precipitation 24h of 4 times of volumes → use successively dehydrated alcohol except the polysaccharide soln behind the albumen, acetone, ether cleans, triplicate → dialysis 72h → lyophilize gets Common Leafflower Herb total polysaccharides (PULP).
1.2.3 the mensuration of Common Leafflower Herb total polysaccharides content
1.2.3.1 the preparation of glucose reference liquid and sample test liquid
Precision takes by weighing dextrose standard sample (being dried to weight under 105 ℃ no longer changes) 10mg, behind dissolved in distilled water, places the 100mL volumetric flask, is settled to scale with distilled water, shakes up, and makes the glucose reference liquid of 0.1mg/mL, and is for subsequent use.
Precision takes by weighing the Common Leafflower Herb polysaccharide 10mg that is dried to constant weight, behind dissolved in distilled water, places 100mL volumetric flask adding distil water to be settled to scale, shakes up, and the sample test liquid of making 0.1mg/mL is for subsequent use.
1.2.3.2 determining of absorbing wavelength
Get each 1 mL of glucose reference liquid and sample solution, add respectively 1mL 5% phenol solution (get 5g and heavily steam phenol adding distil water constant volume in the brown volumetric flask of 100mL), after shaking up, the unsettled vertical adding 5mL vitriol oil, put and heat 15min in the boiling water bath, then put in the cooling bath and cool off, full wavelength scanner in 400～600 nm scopes is determined absorbing wavelength.
1.2.3.3 glucose standard curve making
Accurate glucose reference liquid 0.2,0.4,0.6,0.8, the 1mL of drawing is in 10mL tool plug scale test tube, adding successively water, to make final volume be 1mL, blank is 1mL water, then adding 1mL 5% phenol solution shakes up, add rapidly the 5mL vitriol oil (unsettled vertical adding), put and heat 15min in the boiling water bath, then put in the cooling bath and cool off, measure absorbancy in the 490nm place.Take absorbancy as Y-axis, glucose quality is X-axis, the drawing standard curve, and calculate regression equation.
1.2.3.4 the calculating of sugared content
The accurate test liquid 1mL that draws with the method operation, measures absorbancy by " 1.2.3.3 " in the 490nm place.
Calculate sugared content according to formula:
Sugar content=C/(C
0* V) * 100%
C: the glucose micrograms that is checked in by typical curve
C
0: the concentration of sample solution (0.1 mg/mL)
V: the sample solution volume (1.0mL) of using during mensuration
1.2.3.5 the extraction yield of polysaccharide
The quality of the extraction yield=polysaccharide of polysaccharide/raw-material quality * 100 %
2. results and analysis
2..1 measure determining of wavelength
The maximum absorption band wavelength of sample solution and glucose reference liquid is all located about 490 nm, so choose 490 nm as the measurement of the polysaccharide content wavelength.
2. 2 glucose typical curves
Take absorbancy as Y-axis, glucose micrograms () is X-axis, and the drawing standard curve such as Fig. 1, can find out that glucose amount and absorbancy have good linear relationship in 20～100 g scopes.
2.3 sugared content
The absorbance A that records sample liquid is 0.388, is 28.27 g according to the micrograms of its corresponding glucose of regression equation calculation.Get sugared content according to formula:
Sugar content=C/(C
0* V) * 100%=28.27/ (0.1 * 1) * 100%=28.27%
2.4 the extraction yield of polysaccharide
The extraction yield of polysaccharide==1.5/100 * 100 %=1.5%
3 conclusions
Petroleum ether degreasing is passed through in this experiment, slough the small-molecule substances such as oligose with 95% ethanol again, then from Common Leafflower Herb, isolate polysaccharide with water extraction and alcohol precipitation method, and employing sevag method deproteinated, the phenolsulfuric acid method is measured its content, its maximum absorption wavelength is 490nm, and polysaccharide extract rate is 1.5%, and sugared content is 28.27%.
1 materials and methods
1.1 material (summary)
1.2. experimental technique
1.2.1 the DEAE-52 post separates
1.2.1.1 DEAE-52 filler pre-treatment
DEAE-52 cellulose wadding distilled water immersion 48h, during 4～5h change water one time, and remove suspended impurity with decantation, then carry out alkali-acid-alkali and process.Be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L first, be washed till neutrality with distilled water behind HCl solution soaking 30 min of 0.5 mol/L again, be washed till neutrality with distilled water behind NaOH solution soaking 30 min of 0.5 mol/L at last, obtain OH
-The fiber type element.(column dimension is 500 * 50mm) to wet method dress post, and distilled water balance 48h avoids bubble and fault-layer-phenomenon in the dress post process.
1.2.1.2 cross the separation and purification of DEAE-52 chromatography column
Take by weighing 640mg Common Leafflower Herb total polysaccharides and be dissolved in (8 mg/mL) in the 80mL redistilled water, excessively loading behind the 0.45 μ m filter membrane.Use successively 0,0.1,0.25,0.5,0.75mol/L NaCl gradient elution, every 10min collects a pipe, flow velocity 4mL/min; The phenolsulfuric acid method is followed the tracks of and detected, and collects each main peak component, and is concentrated, redistilled water dialysis 48h, and lyophilize gets each component of Common Leafflower Herb polysaccharide.
1.2.2. Purity
1.2.2.1 SephadexG-200 dextran gel filtration method
The pre-treatment of SephadexG-200: the SephadexG-200 filler adds an amount of distilled water immersion 24h, during 4～5h change water one time, and remove suspended impurity with decantation, then ultrasonic degas is not until there is bubble to occur in the coagulant liquid.(column dimension is 500 * 25mm) to wet method dress post, and is for subsequent use behind NaCl solution equilibria 48 h with 0.05 mol/L.
The SephadexG-200 column chromatography: each set of dispense of collecting is above made the sample liquid of 25mg/mL, and loading 2mL is with the NaCl wash-out of 0.05 mol/L.Automatically Fraction Collector is collected, and every pipe 5 mL, per 30 min collect a pipe, and the phenolsulfuric acid method is followed the tracks of and detected.
1.2.2 2 HPLC high performance liquid chromatography
Each fraction polysaccharide behind the purifying is mixed with the sample liquid of 1mg/mL, crosses sample introduction behind the filter membrane of 0.45 μ m.
Chromatographic condition: chromatographic column: PolySep-SEC 4000 Part No:00H-3144-K0 Column Size:300 * 7.8mm;
Detector: differential detector; Column temperature: 30 ℃; Moving phase: distilled water; Flow velocity: 0.8 mL/min; Sample size: 20 μ L.
2 results and analysis
2.1 DEAE-52 column chromatography for separation result
Through deproteinated, the smart polysaccharide (PULP) of Common Leafflower Herb after the dialysis treatment is crossed the DEAE-52 ion exchange column, uses successively 0,0.1,0.25,0.5,0.75mol/L NaCl solution gradient wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, and gets as shown in Figure 2 middle elution curve.As seen from Figure 2, PULP can obviously isolate the elution peak of four peak shape symmetries after the DEAE-52 column chromatography for separation, represent respectively four components, is designated as PULP I, PULP II, PULP III and PULP IV.Collecting the larger component PULP II of content is further analyzed.
2.2 SephadexG-200 post and HPLC Purity
2.2.1 SephadexG-200 post result
Get Common Leafflower Herb polysaccharide fraction PULP II and cross the SephadexG-200 gel column, the NaCl wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, make elution curve with absorbance and wash-out pipe number, the elution curve of PULP II on Sephadex G-200 post is symmetrical simple spike, illustrates that PULP II component is the polysaccharide fraction of the relative homogeneous of molecular weight.
2.2.2 HPLC method Purity
When utilizing the HPLC method to detect the purity of Common Leafflower Herb polysaccharide fraction PULP II, the result shows, polysaccharide fraction PULP II after the separation and purification of DEAE-52 cellulose column, relatively more symmetrical in high performance liquid phase collection of illustrative plates superiors type, and calculating its purity through area normalization method reaches more than 95%, figure result matches with Sephadex G-200 gel chromatography, illustrates that purity is higher, can be used for doing Structural Identification.
3 conclusions
Obtain four components after the Common Leafflower Herb polysaccharide process DEAE-52 cellulose column separation and purification behind the water extract-alcohol precipitation deproteinated, be designated as respectively PULP I, PULP II, PULP III and PULP IV.Collect the larger component PULP II of content, carry out Purity with SephadexG-200 gel filtration chromatography method and two kinds of methods of high performance liquid chromatography (HPLC), prove the relative homogeneous of its component, can further do structural analysis.
Physico-chemical property and the structural analysis of embodiment 3 PULP II
1, physico-chemical property
The PULP II is the khaki color powder, easily moisture absorption.Soluble in water, be insoluble to ethanol, acetone and other organic solvent.Molish reaction, phenolsulfuric acid reaction, sulfuric acid-carbazole reaction positive, ninhydrin reaction, IKI reaction, the Fehling reaction, ferric chloride reaction and sulfate reaction are negative.
By physico-chemical property, glucuronic acid content is measured, and ultimate analysis, infrared and ultraviolet, HPLC analysis-by-synthesis show in the PULP II it is that a class does not contain N, S element acidic polysaccharose, and molecular-weight average is 579962.6 relatively.
2, the infrared absorption pattern of PULP II
Infared spectrum shows that the 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 the place.And the PULP II is at 1010～1100 cm
-1Two strong absorption peaks are arranged, illustrated that the furan type glycosidic link exists.
Be comprised of and part acid hydrolysis interpretation of result monose, PULP II monose forms and ratio is Rha:Ara:
Man:Glc is 0.11:0.36:0.08:0.45; Main chain mainly is comprised of Rha, Ara and Glc.
Comprehensive periodate oxidation and Smith degradation results are analyzed, and PULP II glycosidic link is connected to the master with 1 → 6, also has simultaneously 1 → 4 and 1 → 3 mode of connection.
The antioxidation activity in vitro preliminary study of embodiment 4 Common Leafflower Herb polysaccharide PUIP II
1 materials and methods
1.1 material and reagent (summary)
1.2 experimental technique
1.2.1 the mensuration of polysaccharide reducing power
Laboratory reference Oyaizu method is slightly done a little and is changed.Get the polysaccharide soln of 1 mL different concns (1,2,3,4,5mg/mL) in tool plug test tube, then add respectively 2.5 mL 0.2 mol/L pH, 6.6 phosphate buffer solutions and 1% potassium ferricyanide solutions, ice bath cools off rapidly behind 50 ℃ of water-bath 20 min, the trichoroacetic acid(TCA) solution that adds 2.5 mL 10%, shake up, centrifugal (3000 r/min, 10 min).Get supernatant liquor 2.5 mL, add 2.5 mL distilled waters and 0.5 mL, 0.1% FeCl
3, shake up, react 10 min after, measure the absorbancy at 700 nm places.
1.2..2 the external hydroxyl radical free radical (of the polysaccharide ﹒ OH) mensuration of clearance rate
In the Fenton reaction system, H
2O
2With Fe
2+Mix Chan Sheng ﹒ OH.You Yu ﹒ OH reactive behavior is strong, and the survival time is very short, adds Whitfield's ointment, and just Bu Zhuo and is created on the coloring matter that there is strong absorption at 510 nm places Dao ﹒ OH effectively.Simultaneously, if in this system, add with Whitfield's ointment have Competition can Qing Chu ﹒ OH material, then the growing amount of coloring matter tails off, the absorbancy step-down, absorbancy is lower, proves that the ability of this material Qing Chu ﹒ OH is stronger.
The FeSO that in tool plug test tube, adds respectively 2 mL, 9 mmol/L
4The Whitfield's ointment ethanolic soln of solution, 2 mL, 9 mmol/L, the polysaccharide soln of different concns (1,2,3,4,5mg/mL) adds the H of 2 mL 8.8mmol/L at last
2O
2Solution starts reaction, reacts 30 min under the room temperature, measures the absorbancy at 510 nm places, replaces polysaccharide soln to do blank with distilled water.
Free radical scavenging activity calculation formula: P=(A
0-A
i)/A
0* 100%
A
0: blank absorbency, A
i: the sample absorbancy
1.2.3 anti peroxidation of lipid ability
Draw 0.4 mL yolk suspension [V(yolk): V(PBS)=1:25], the polysaccharide soln of 1 mL different concns (1,2,3,4,5mg/mL), the FeSO of 0.4 mL, 25 mmol/L
4, in tool plug test tube, PBS solution to the cumulative volume that adds 0.1 mol/LpH7.45 is 4.0 mL, 37 ℃ of constant temperature water baths, 15 min that vibrate.The TCA that adds 1.0 mL 20% after taking out, leave standstill 10 min after, centrifugal (3500 r/min, 10 min).Draw 4.0 mL supernatant liquors, add the thiobarbituricacidα-of 2.0 mL 0.8%, jump a queue, boiling water bath 15 min after the cooling, do reference with PBS, measure the light absorption value at 532 nm places.
Sample represents the ability of the anti-oxidant activity of sample to the inhibiting rate of lipovitellinin lipid peroxidation, that is:
Inhibiting rate I=(A
0-A)/A
0* 100%
A
0The absorbancy of-control tube; The absorbancy of A-sample
2 results and analysis
2.1 the reducing power of PULP II
Experimental result shows that the 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 and strengthens with concentration.
2.2 the external hydroxyl radical free radical clearance rate of PULP II
Experimental result shows that the PULP II polysaccharide soln under each concentration has certain removing ability to external hydroxyl radical free radical, and in the concentration range of 1～5mg/mL, and its clearance rate increases and strengthens with concentration.During 5mg/mL, the clearance rate of PULP II is 35.5%.
2.3 PULP II anti peroxidation of lipid ability
Experimental result shows that the PULP II polysaccharide soln under each concentration all has certain restraining effect to LPO, and has dose-effect relationship in the concentration range of 1～5mg/mL, and its inhibiting rate increases and strengthens with concentration.During 5mg/mL, the inhibiting rate of PULP II is 10.7%.
3 conclusions
Common Leafflower Herb polysaccharide PULP II has certain reducing power, removes external hydroxyl radical free radical ability and anti peroxidation of lipid ability, and strengthens along with the increase of polysaccharide concentration.When 5mg/mL, the reducing power of PULP II is 0.538, and the clearance rate of external hydroxyl free is reached 35.5%, and the inhibiting rate of LPO is respectively 10.7%.This explanation Common Leafflower Herb polysaccharide has certain anti-oxidant activity and its anti-oxidant activity is mainly manifested on the external hydroxyl radical free radical of removing.
The research of embodiment 5 Common Leafflower Herb polysaccharide PUIP II effect on hepatitics B virus in vitros
Present most widely used effect on hepatitics B virus in vitro medicaments sifting model HepG2.2.2.15 cell model is adopted in this test, extract to separate from, Common Leafflower Herb and obtain PUIP II and HepG2.2.2.15 cytosis, get its cell conditioned medium liquid, the titre of measuring respectively its hepatitis B surface antigen (HBsAg), e antigen (HBeAg) changes, judge whether this medicine is effective, and adopt mtt assay to measure the cytotoxicity of each several part.Comprehensive these two indexs, Common Leafflower Herb polysaccharide PUIP II is carried out In Vitro Anti hepatitis B pharmacodynamics test, and select and certifiedly clinically have the acyclovir (ACV) of anti-HBV effect as the positive control medicine, to estimate the effect of each position hepatitis B virus resisting, screen efficient part or effective constituent with this.
1 materials and methods
1.1 material and reagent (summary)
1.2 experimental technique
1.2.1 the recovery of cell
Basic step: allocate 37 ℃-40 ℃ warm water; Take out the HepG2.2.2.15 cell cryopreservation tube from liquid nitrogen container, drop into immediately in 37 ℃-40 ℃ the warm water and rock fast, until frozen storing liquid melts fully, melting process is finished in 1-2min; The cell cryopreservation suspension is moved into centrifuge tube, add about 5mL nutrient solution, blow gently even; With the centrifugal 5min of cell suspension 800r-1000r/min, abandon supernatant liquor; Add complete culture solution to cell precipitation, pressure-vaccum is beaten evenly gently, and cell suspension is moved into culturing bottle, fills up nutrient solution and cultivates, and places 37 ℃, 5%CO
2Cell culture incubator is cultivated.
1.2.2 the cultivation of going down to posterity of cell
The HepG2.2.2.15 cell places 5%CO
2, 37 ℃ of cultivations.Substratum is DMEM, adds 10% foetal calf serum, 3% L-glutaminate 1%, G418 200ug/mL, penicillin 100u/mL, Streptomycin sulphate 100u/mL.2.2.15 after cell covers with culturing bottle, digest 10-30 second with EDTA first, with 37 ℃ of digestion of 0.25% pancreatin 3-10min, add nutrient solution piping and druming again, 1:3 or 1:4 go down to posterity, and cover with in 8 days, adopt the numeration of cell count plate, are mixed with 10
5Individual/mL inoculating cell culture plate, the every hole 0.1mL of 96 orifice plates; The every hole 1mL of 24 orifice plates, 5%CO
2, cultivate for 37 ℃ and tested in 24 hours.
1.2.3 method for cell count
Generally use blood cell counting plate, count by the white blood cell count(WBC) method.
Getting cell suspension 1mL to be diluted adds physiological saline and does 5 times of dilutions, multiple with 10 * 10 is counted, central authorities use for red blood cell count(RBC), four jiaos of large lattice are that white blood cell count(WBC) is used, only add up complete cell during counting, if the cell that bunches up is counted by a cell, in a grid, if there is cell to be positioned on the line, general meter is reached the standard grade to disregard and is rolled off the production line, and counts left line and disregards right line, and counting error is no more than ± and 5%, need to calculate the cell count in every mL suspension behind the counting, because the area of each grid in the tally is 0.1cm
2, height is 0.01cm, volume is 0.0001cm
3Every mL cell count is calculated by formula 1:
Every mL cell count=(n/4) * 10000 * 5 (1)
In the formula: n is four large lattice total cellular score
1.2.4 the MTT colorimetry is surveyed cell survival rate
The HepG2.2.2.15 cell cultures is in 96 well culture plates, every hole 80 μ l nutrient solutions; Add 20 μ lMTT, continue to cultivate 3-4h; Suck 100 μ l nutrient solutions, add equivalent 0.04-0.1mol/L hydrochloric acid aqueous isopropanol, under the room temperature about 10min(or flat board placed the micro vibrator concussion), crystallisate is dissolved; Measure photoabsorption in microplate reader, the mensuration wavelength is 570nm.
1.2.5 mtt assay is surveyed drug toxicity
The HepG2.2.2.15 cell is pressed 10
5Individual/mL is inoculated in 96 well culture plates, 0.1 mL/ hole, and inferior daily nutrient solution is made into respectively several different concentration with medicine to be measured, adds cell hole, and every hole concentration adds 3 holes, the 0.1mL/ hole, and establish without the contrast of medicine cell and positive control drug.Cultivated 4 days after the dosing, abandon supernatant and dye with MTT, every hole adds the MTT serum-free medium 0.1mL of 1mg/mL, hatches 4 hours, abandons supernatant, and after the adding 0.04mol/L hydrochloric acid Virahol 0.1mL dissolving, with 570nm wavelength colorimetric estimation OD value, experiment repeats 3 times.
1.2.6 survey HBsAg, HBeAg secreting law
With HepG2.2.2.15 cell 10
5/ mL is inoculated in 24 porocyte culture plates, every hole 1.0mL, totally 10 holes, 3rd, collected supernatant 250 μ l in 5,8,11,13 days, nutrient solution was supplied commercial weight to the 13 days, and culture supernatant is frozen in-20 ℃, concentrate at last and press the test kit specification sheets, measure HBsAg, HBeAg with the ELISA method.
1.2.7 medicine is to the HBV inhibition test
The HepG2.2.2.15 cell is pressed 10
5/ mL is inoculated in 24 orifice plates, and every hole 1.0mL abandons nutrient solution next day, carries out doubling dilution take the half toxic concentration of the HepG2.2.2.15 cell of medicine to be measured as initial concentration with nutrient solution, and other establishes without medicine contrast and positive control drug, is incubated at 37 ℃, 5%CO
2In the incubator.Collected supernatant in per 4 days, and change and add the original content liquid and continue to cultivate, the supernatant-20 collected is ℃ frozen, collected in the 12nd day, to concentrate with the ELISA method and measure HBsAg, HBeAg, experiment repeats 3 times.
1.2.8 the ELISA method is surveyed HBsAg, HBeAg
(1) each component of test kit is taken out from box, balance is to room temperature (18-25 ℃).
(2) fully shake up if any crystal and should fully melt before the concentrated cleaning solution preparation, concentrated cleaning solution uses after pressing the 1:19 dilution with distilled water or deionized water.
(3) the micropore lath is fixed in support, according to the order of sequence numbering.
(4) every hole adds sample 50 μ l to be measured, establishes each 2 hole of yin, yang contrast, and every hole adds each 50 μ l of yin, yang contrast, and establishes blank one hole;
(5) every hole adds enzymic-labelled antibody 50 μ l(except blank), shrouding behind the abundant mixing, place 37 ℃ of environment to hatch 30min;
(6) the manual plate of washing; Discard liquid in the hole, full each hole of washings storage is left standstill and was dried afterwards in 5 seconds, pats dry after repeating 5 times;
(7) every hole adds developer A liquid, each 50 μ l of B liquid, abundant mixing, and shrouding is put in 37 ℃ of environment and is hatched 15min;
(8) every hole adds stop buffer 50 μ l, mixing;
(9) use the microplate reader reading, get wavelength 450nm, use first blank empty school zero.Then read each hole OD value.(negative control OD value is lower than 0.05 as 0.05 calculating, is higher than 0.05 by actual OD value calculating.）
1.3 data analysis
1.3.1 mtt assay can get after surveying drug toxicity:
1.3.2 adopt the ELISA method, the automatic microplate reader of usefulness read OD value after color reaction was finished, the calculating inhibiting rate, with inhibiting rate greater than 50% for restraining effect is arranged.Calculate medicine according to determination data and suppress the antigen percentage; Medicine suppresses antigen medium effective concentration IC
50, the drug level when namely HBsAg and HBeAg inhibiting rate are 50%; Select therapeutic index TI, TI is effectively greater than 1 above person, and the explanation medicine is effective between 1-2, but certain toxicity is arranged; Better greater than 2 effects, toxicity is less; Index is larger, and then curative effect is better, and safety range is larger.Statistical procedures, mean compares between employing t check work group, and data statistic analysis is by the SPSS11.5 software processes, and P＜0.05 expression difference has significance.
2 interpretations of result
2.1 the rule of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Under the described culture condition of test method, the used HepG2.2.2.15 cell of this test began to have measured HBsAg, HBeAg secretion in the 3rd day, peaked at the 8th day, weakened gradually later on, lasted till that the rule of HBsAg, HBeAg secretion as shown in Figure 3 the 13rd day.
2.2 the effect on hepatitics B virus in vitro test-results of positive drug (ACV)
2.2.1 positive drug (ACV) is to the toxicity of HepG2.2.2.15 cell
After different concns ACV process to cultivate, the MTT test-results shows had restraining effect to the HepG2.2.2.15 Growth of Cells, and its effect has certain concentration dependent, and concentration is larger, and restraining effect is more obvious.Cell inhibitory rate when concentration is 0.3125mg/mL is 3.51%, and the cell survival rate under this concentration is 96.49% (〉 95%), the TC of hence one can see that ACV
0Be 0.3125mg/mL.Can get TC according to formula (3)
50Be 4.56 mg/mL.The cytotoxic assay of ACV the results are shown in Table 2-1.
The toxicity of table 2-1 ACV in the HepG2.2.2.15 cell cultures
Show 2-2 ACV restraining effect to HBsAg in the HepG2.2.2.15 cell cultures
2.2.2 positive drug (ACV) suppresses the effect of HepG2.2.2.15 emiocytosis HBsAg, HBeAg
Different concns ACV processes and cultivates collecting cell supernatant liquor after 4 days and 8 days, the ELISA method is surveyed HBsAg, the HBeAg level shows that ACV all has restraining effect to HBsAg, HBeAg, and along with the increase of ACV concentration, its restraining effect strengthens, inhibiting rate increases, and demonstrates certain dose-effect relationship.And along with the passing restraining effect of incubation time also strengthens gradually.Different concns ACV restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures the results are shown in Table 2-2 and 2-3
Show 2-3 ACV restraining effect to HBeAg in the HepG2.2.2.15 cell cultures
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 the time IC
50Be respectively 0.58mg/mL and 0.49mg/mL.Can obtain ACV according to formula (6) the therapeutic index TI of HBsAg, HBeAg is respectively 7.86 and 9.31.Concrete outcome sees Table 2-4.
Show 2-4 ACV IC to HBsAg and HBeAg in the HepG2.2.2.15 cell cultures
50And TI
2.3 the effect on hepatitics B virus in vitro effect of Common Leafflower Herb polysaccharide PUIP II
2.3.1 Common Leafflower Herb polysaccharide PUIP II is to the toxicity of HepG2.2.2.15 cell
After different concns Common Leafflower Herb polysaccharide PUIP II treatment group was cultivated, the MTT test-results shows had restraining effect to the HepG2.2.2.15 Growth of Cells, and its effect has certain concentration dependent, and concentration is larger, and restraining effect is more obvious.Cell inhibitory rate when concentration is 0.156mg/mL is 5.76%, and the cell survival rate under this concentration is 94.24% (≈ 95%), the TC of Common Leafflower Herb polysaccharide PUIP II that hence one can see that
0＜0.156mg/mL.Maximal percentage inhibition in the experimental concentration scope＜50% is so can only calculate TC
5010mg/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 the 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 is processed and is cultivated collecting cell supernatant liquor after 4 days and 8 days, the ELISA method is surveyed HBsAg, the HBeAg level shows that Common Leafflower Herb polysaccharide PUIP II all has restraining effect to HBsAg, HBeAg, and the increase along with 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 restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures the results are shown in Table 2-6 and 2-7.
Show 2-6 Common Leafflower Herb polysaccharide PUIP II restraining effect to HBsAg in the HepG2.2.2.15 cell cultures
Show 2-7 Common Leafflower Herb polysaccharide PUIP II restraining effect to HBeAg in the HepG2.2.2.15 cell cultures
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 the time IC
50Be respectively 1.59mg/mL and 0.49mg/mL.Can obtain Common Leafflower Herb polysaccharide PUIP III to the therapeutic index TI of HBsAg, HBeAg respectively according to formula (6)〉6.29 for and 20.41.Concrete outcome sees Table 2-8.
Show 2-8 Common Leafflower Herb polysaccharide PUIP II IC to HBsAg and HBeAg in the HepG2.2.2.15 cell cultures
50And TI
3 discuss
This test has been adopted mtt assay to carry out cytotoxicity and has been detected, and the result shows the maximal non-toxic concentration (TC of Common Leafflower Herb polysaccharide PUIP 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 restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures, the result shows in 7 concentration that Common Leafflower Herb polysaccharide PUIP II sets under non-toxic concn, except minimum concentration, other each concentration group is compared with the cell control group and is all shown obvious restraining effect, and the increase along with Common Leafflower Herb polysaccharide PUIP II concentration, its restraining effect strengthens, and inhibiting rate increases, and demonstrates certain dose-effect relationship.The result for the treatment of index that is used for clinically at present estimating medicine is therapeutic index (TI), is that medicine is invalid when TI＜1, and the medicine poor efficiency is poisonous when 1＜TI＜2, as TI〉the effective low toxicity of medicine 2 time.Hence one can see that, and Common Leafflower Herb polysaccharide PUIP II is to the effective low toxicity of therapeutic index of HBsAg, HBeAg (TI be respectively 〉=6.29 and 20.41); Positive control medicine acyclovir is to the effective low toxicity of therapeutic index (TI is respectively 7.86 and 9.31) of HBsAg, HBeAg.
In sum, the Common Leafflower Herb polysaccharide II all has certain restraining effect to HBsAg, HBeAg in the HepG2.2.2.15 cell cultures.
The above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (10)
1. the extraction and separation method of a Common Leafflower Herb polysaccharide component PUIP II comprises the steps:
1) the Common Leafflower Herb herb is pulverized after with the distilled water cleaning, drying, uses petroleum ether degreasing, after ethanol takes off small molecular sugar, and dregs of a decoction flooding, the vat liquor centrifugation is got clear liquid and is added the ethanol alcohol precipitation after concentrated, and pure hypostasis is thick total polysaccharides; Thick total polysaccharides is through 2) except albumen, 3) alcohol precipitation, washing and 4) dialyse, be drying to obtain the smart total polysaccharides of Common Leafflower Herb; 5) each component separation and purification of polysaccharide: the smart total polysaccharides of Common Leafflower Herb is crossed ion exchange column, with NaCl solution gradient wash-out, the phenolsulfuric acid method is followed the tracks of and is detected, and draws elution curve, four peaks appear on the elution curve successively, represent respectively four polysaccharide fractions, be designated as respectively PULP I, PULP II, PULP III and PULP IV according to the order that goes out the peak, collect the 2nd main peak component, concentrated, the redistilled water dialysis, lyophilize gets Common Leafflower Herb polysaccharide PULP II pure component, i.e. Common Leafflower Herb polysaccharide component PUIP II.
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, with sherwood oil 70～90 ℃ of lower refluxing extraction 2～4 times, each 1～3 hour, discard phegma, the dregs of a decoction continue with 50～100% ethanol 80～95 ℃ of lower refluxing extraction 2～4 times, each 1～3 hour, discard phegma, the dregs of a decoction that obtain are used 60～95 ℃ of floodings 3 times again, each 1～3 hour, merge 3 vat liquors and carry out centrifugation, centrifugal speed 2000～6000r/min, get clear liquid and be concentrated into original volume 1/4, add 90～100% ethanol of 4 times of volumes, leave standstill 24h, centrifugal, get pure 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) describedly adopt Sevag method deproteinated except albumen: the pure hypostasis that step 1) is obtained redissolves to get Crude polysaccharides solution with distilled water, in Crude polysaccharides solution, add chloroform and propyl carbinol mixed solution, Crude polysaccharides solution: chloroform and propyl carbinol mixeding liquid volume are 1～6:1～3, chloroform in described chloroform and the propyl carbinol mixed solution: propyl carbinol volume ratio=1～4:1～4, stir, the sufficient standing layering, discard precipitation, repeat 1～10 time after until the interface without white precipitate.
4. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that the described alcohol precipitation of step 3), washing: 75～100% ethanol that in except the polysaccharide soln behind the albumen, add 3～6 times of volumes, leave standstill 12～36h, centrifugal, precipitation is cleaned with dehydrated alcohol, acetone, ether successively, repeats 2～5 times.
5. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that the described dialysis of step 4), drying: the polysaccharide of getting after alcohol precipitation, the washing adds distilled water, use distill water dialysis after fully redissolving, dialysis is carried out under magnetic agitation, sample liquid and distilled water volume ratio are 1:10～30,4～8h changes water one time, dialysis 12～120h, dialyse complete after with sugar soln 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 the ion exchange column of step 5) adopts DEAE-52 anionite-exchange resin.
7. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, the concentration gradient scope that it is characterized in that the described NaCl solution of step 5) is 0～3mol/L.
8. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 7, the concentration gradient that it is characterized in that described NaCl solution are respectively 0,0.1,0.25,0.5,0.75mol/L.
9. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 1, it is characterized in that step 5) uses different concns NaCl gradient elution successively, every kind of concentration NaCl eluant solution 8 hours, flow velocity 4mL/min, collect elutriant with pipe, every 10min collects a pipe, draws elution curve.
10. the extraction and separation method of Common Leafflower Herb polysaccharide component PUIP II according to claim 9 is characterized in that the Guan Xuwei X-coordinate of described elution curve to collect, and the absorbance of solution is that the ordinate zou drafting forms.
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| CN109234335A (en) * | 2018-11-14 | 2019-01-18 | 浙江海洋大学 | A kind of preparation method of polysaccharide in tabasheer rich in galactofuranose |
| CN111647095A (en) * | 2020-06-30 | 2020-09-11 | 华润三九医药股份有限公司 | Polysaccharide of fraxinus chinensis, preparation method and application thereof |
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| CN1695647A (en) * | 2004-05-12 | 2005-11-16 | 四川省中药研究所 | Method for preparing general extractive from effective part of blattbulume |
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| 周宇等: "正交试验优选叶下珠中多糖的提取工艺", 《化工时刊》 * |
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| CN109234335A (en) * | 2018-11-14 | 2019-01-18 | 浙江海洋大学 | A kind of preparation method of polysaccharide in tabasheer rich in galactofuranose |
| CN109234335B (en) * | 2018-11-14 | 2021-08-20 | 浙江海洋大学 | Preparation method of polysaccharide rich in galactofuranose in tabasheer |
| CN111647095A (en) * | 2020-06-30 | 2020-09-11 | 华润三九医药股份有限公司 | Polysaccharide of fraxinus chinensis, preparation method and application thereof |
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