CN105622776A - Flos farfarae polysaccharide extraction and purification method - Google Patents
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Abstract
The invention discloses a flos farfarae polysaccharide extraction and purification method. Flos farfarae polysaccharides are extracted by means of the water extraction method under the ultrasonic conditions. Papain and a Sevage reagent are combined to deproteinize the flos farfarae polysaccharides. Pigment is adsorbed through the macroporous resin method for decolorization. Deproteinized and decolorized polysaccharides are subjected to dialysis, impurities in the polysaccharides are further removed, and the flos farfarae polysaccharides high in purity are obtained. The flos farfarae polysaccharide extraction and purification method is high in extraction rate, high in impurity removal rate and good in polysaccharide retention effect.
Description
Technical field
The present invention relates to medicinal plant manufacture field, it is specifically related to the extraction and purification method of Flos Farfarae polysaccharide.
Background technology
Flos Farfarae is the bud of composite family coltsfoot, and property and flavor of peppery and warm, has moistening lung to lower QI, the effect reduced phlegm and only cough. Medical institutions' prescription and folk remedy, folk prescription etc. also adopt Flos Farfarae treatment chronic and acute tracheitis, the disease such as pulmonary tuberculosis. Flos Farfarae polysaccharide has oxidation-resistance, antitumor, antiviral, antiulcer agent, anti-mutation, reducing blood-fat, hypoglycemic, anti-aging, conditioner body immunity function, strengthening immunity etc. act on, Flos Farfarae polysaccharide can being applied to the fields such as food, medicine, healthcare products, the extraction purification of Flos Farfarae polysaccharide has important meaning.
Summary of the invention
It is an object of the present invention to for prior art, it is provided that the extraction and purification method of Flos Farfarae polysaccharide.
For achieving the above object, the present invention takes following technical scheme: the extraction and purification method of Flos Farfarae polysaccharide comprises the following steps:
(1) pulverizing, degreasing pre-treatment: after Flos Farfarae bud is dried 12h in the baking oven of 80 DEG C, pulverized, cross 100 orders sieves, adding 95% ethanolic soln by the solid-liquid ratio of 1g:10ml, backflow degreasing 2 times, each time is 2h, by extracting liquid filtering, the ethanol in filter cake is volatilized;
(2) pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide: pretreated Flos Farfarae polysaccharide is mixed by the solid-liquid ratio of solid-liquid ratio 1g:15��22ml with water, 6h is soaked at 30 DEG C, then at 60��70 DEG C after ultrasonic 10��30min, water extraction 2 times, each 120��160min, collect extracting solution, and centrifugal collection supernatant liquor, merging filtrate after filtering, it is 1/8th of original volume by filtrate vacuum concentration, filtrate after concentrated is added 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, it is drying to obtain Flos Farfarae Crude polysaccharides,
(3) deproteinated: Crude polysaccharides is dissolved in distilled water, it is mixed with the Crude polysaccharides solution of 10mg/ml, the mass concentration adding 1.5��2.5% in Crude polysaccharides solution is 1mg/ml, PH is the papain solution of 7.0, 80��100min at being placed in 55��65 DEG C, add Sevage reagent, 20min is fully mixed through 3000r/min clarifixator, at the centrifugal 10min of 3000r/min, remove precipitation, get supernatant liquor and add Sevage reagent again, repeat aforesaid operations 2 times, supernatant liquor adds 95% ethanol alcohol precipitation of 4 times of volumes, leave standstill 24h, take out filter final vacuum and it is drying to obtain polysaccharide after deproteinated,
(4) decolour: macroporous resin is carried out removal of impurities, acid-alkali treatment, dry pre-treatment, after polysaccharide after deproteinated is configured to the polysaccharide soln of 2mg/ml, macroporous resin and polysaccharide soln are added in constant temperature oscillator in the ratio of 1g:95��100ml, vibration absorption 6h, filters and obtains decolouring polysaccharide;
(5) dialyse: step (4) gained polysaccharide is configured to 10mg/ml solution, with tap water flowing dialysis 48h, use distill water dialysis 24h again, then with 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, namely 50 DEG C of vacuum-dryings obtain Flos Farfarae polysaccharide.
Wherein, in described step (2), ultrasonic power is 80��90w.
Wherein, described Sevage reagent is chloroform: propyl carbinol is 4:1.
Wherein, in described step (4), the pre-treatment of macroporous resin is: macroporous resin distilled water immersion removes water-soluble impurity agent and suspended substance, first with 95% alcohol immersion 24h, it is washed to distilled water and clarifies without alcohol taste and water liquid, then with 3% dilute hydrochloric acid solution 12h of 3 times of resin volumes, be washed till neutrality with distilled water, soak 12h with 3% Strong oxdiative sodium solution of 3 times of resin volumes again, it is washed till with distilled water neutral rear cool dry.
Wherein, described macroporous resin is XD-5 macroporous resin.
Wherein, in described step (4), the condition of vibration absorption is: temperature 25 DEG C, constant temperature oscillator frequency 120 times/min.
Further, owing to the solvability of protein is similar to polysaccharide, therefore, there is a certain amount of protein in the Crude polysaccharides that step (2) obtains, need to be further purified, and carries out deproteinated process.
Further, the protein in polysaccharide is with the combination of pure sugar chain or sugar chain and peptide chain, and the glycopeptide of formation or glycoprotein, in step (3), deproteinated mainly removes floating preteins. The present invention adopts Papain ferment treatment, the more vegetable-protein of degradable, reduces the loss that polysaccharide precipitates with jello.
Further, containing more pigment in Flos Farfarae polysaccharide extraction liquid, not only affect color and luster and the purity of polysaccharide, also affect the biological activity of polysaccharide, in decoloring method, macroporous adsorbent resin is difficult to absorption glucide, and checks colors and have good adsorptivity, adopts the decolouring of macroporous resin adsorption pigment to have good decolorizing effect.
The invention has the beneficial effects as follows:
1, the present invention adopts pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide, pre-soaking can make solvent fully penetrate into plant cell wall, extraction yield can be improved, ultrasonic cavatition can accelerate cell wall breaking, accelerating release and the dissolution rate of polysaccharide in cell, ultrasonic wave also makes extract and solvent constantly shake, and contributes to the diffusion of polysaccharide, reusable heat water extraction, it is to increase solute dissolution rate and extraction yield.
2, Ben Faming adopts papoid and Sevage agents deproteinated, is removed in precipitation by protein degradation, has higher albumen extrusion rate and polysaccharide retention rate.
3, the present invention adopts Amberlyst process to decolour, and has decolorizing efficiency height, the advantage that polysaccharide retention rate is high.
4, the present invention adopts first with tap water dialysis, then with distill water dialysis, reduces the usage quantity of distilled water, can by further for money polysaccharide impurity and purification.
Embodiment
Below in conjunction with specific embodiment, to further instruction of the present invention.
Embodiment 1
The extraction and purification method of Flos Farfarae, comprise the following steps: (1) pulverizes, degreasing pre-treatment: after Flos Farfarae bud is dried 12h in the baking oven of 80 DEG C, pulverized, cross 100 order sieves, adding 95% ethanolic soln by the solid-liquid ratio of 1g:10ml, backflow degreasing 2 times, each time is 2 times, by extracting liquid filtering, the ethanol in filter cake is volatilized;
(2) pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide: pretreated Flos Farfarae polysaccharide is mixed by the solid-liquid ratio of solid-liquid ratio 1g:20ml with water, 6h is soaked at 30 DEG C, then at 60 DEG C, ultrasonic power is 70w, after ultrasonic 10min, water extraction 2 times, each 2h, collect extracting solution, and centrifugal collection supernatant liquor, merging filtrate after filtering, it is 1/8th of original volume by filtrate vacuum concentration, filtrate after concentrated is added 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, it is drying to obtain Flos Farfarae Crude polysaccharides,
(3) deproteinated: Crude polysaccharides is dissolved in distilled water, it is mixed with the Crude polysaccharides solution of 10mg/ml, the mass concentration adding 1.5% in Crude polysaccharides solution is 1mg/ml, PH is the papain solution of 7.0, 100min at being placed in 60 DEG C, add chloroform: propyl carbinol is the Sevage reagent of 4:1, 20min is fully mixed through 3000r/min clarifixator, at the centrifugal 10min of 3000r/min, remove precipitation, get supernatant liquor and add chloroform again: propyl carbinol is the Sevage reagent of 4:1, repeat aforesaid operations 2 times, supernatant liquor adds 95% ethanol alcohol precipitation of 4 times of volumes, leave standstill 24h, take out filter final vacuum and it is drying to obtain polysaccharide after deproteinated,
(4) decolour: XD-5 macroporous resin distilled water immersion is removed water-soluble impurity agent and suspended substance, first with 95% alcohol immersion 24h, it is washed to distilled water and clarifies without alcohol taste and water liquid, again with 3% dilute hydrochloric acid solution 12h of 3 times of resin volumes, it is washed till neutrality with distilled water, 12h is soaked again with 3% Strong oxdiative sodium solution of 3 times of resin volumes, it is washed till neutral rear cool dry with distilled water, after polysaccharide after deproteinated is configured to the polysaccharide soln of 2mg/ml, XD-5 macroporous resin and polysaccharide soln are added in constant temperature oscillator in the ratio of 1g:100ml, it it is 25 DEG C in temperature, vibration absorption 6h when constant temperature oscillator frequency is 120 times/min, filter and obtain decolouring polysaccharide,
(5) dialyse: step (4) gained polysaccharide is configured to 10mg/ml solution, with tap water flowing dialysis 48h, use distill water dialysis 24h again, then with 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, namely 50 DEG C of vacuum-dryings obtain Flos Farfarae polysaccharide.
Embodiment 2
The extraction and purification method of Flos Farfarae, comprise the following steps: (1) pulverizes, degreasing pre-treatment: after Flos Farfarae bud is dried 12h in the baking oven of 80 DEG C, pulverized, cross 100 order sieves, adding 95% ethanolic soln by the solid-liquid ratio of 1g:10ml, backflow degreasing 2 times, each time is 2h, by extracting liquid filtering, the ethanol in filter cake is volatilized;
(2) pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide: pretreated Flos Farfarae polysaccharide is mixed by the solid-liquid ratio of solid-liquid ratio 1g:15ml with water, 6h is soaked at 30 DEG C, then at 60 DEG C, ultrasonic power is under 60w, after ultrasonic 20min, water extraction 2 times, each 150min, collect extracting solution, and centrifugal collection supernatant liquor, merging filtrate after filtering, it is 1/8th of original volume by filtrate vacuum concentration, filtrate after concentrated is added 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, it is drying to obtain Flos Farfarae Crude polysaccharides,
(3) deproteinated: Crude polysaccharides is dissolved in distilled water, it is mixed with the Crude polysaccharides solution of 10mg/ml, the mass concentration adding 2.0% in Crude polysaccharides solution is 1mg/ml, PH is the papain solution of 7.0, 90min at being placed in 65 DEG C, add chloroform: propyl carbinol is the Sevage reagent of 4:1, 20min is fully mixed through 3000r/min clarifixator, at the centrifugal 10min of 3000r/min, remove precipitation, get supernatant liquor and add chloroform again: propyl carbinol is the Sevage reagent of 4:1, repeat aforesaid operations 2 times, supernatant liquor adds 95% ethanol alcohol precipitation of 4 times of volumes, leave standstill 24h, take out filter final vacuum and it is drying to obtain polysaccharide after deproteinated,
(4) decolour: XD-5 macroporous resin distilled water immersion is removed water-soluble impurity agent and suspended substance, first with 95% alcohol immersion 24h, it is washed to distilled water and clarifies without alcohol taste and water liquid, again with 3% dilute hydrochloric acid solution 12h of 3 times of resin volumes, it is washed till neutrality with distilled water, 12h is soaked again with 3% Strong oxdiative sodium solution of 3 times of resin volumes, it is washed till neutral rear cool dry with distilled water, after polysaccharide after deproteinated is configured to the polysaccharide soln of 2mg/ml, XD-5 macroporous resin and polysaccharide soln are added in constant temperature oscillator in the ratio of 1g:100ml, it it is 25 DEG C in temperature, vibration absorption 6h when constant temperature oscillator frequency 120 times/min, filter and obtain decolouring polysaccharide,
(5) dialyse: step (4) gained polysaccharide is configured to 10mg/ml solution, with tap water flowing dialysis 48h, use distill water dialysis 24h again, then with 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, namely 50 DEG C of vacuum-dryings obtain Flos Farfarae polysaccharide.
Embodiment 3
The extraction and purification method of Flos Farfarae, comprise the following steps: (1) pulverizes, degreasing pre-treatment: after Flos Farfarae bud is dried 12h in the baking oven of 80 DEG C, pulverized, cross 100 order sieves, adding 95% ethanolic soln by the solid-liquid ratio of 1g:10ml, backflow degreasing 2 times, each time is 2h, by extracting liquid filtering, the ethanol in filter cake is volatilized;
(2) pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide: pretreated Flos Farfarae polysaccharide is mixed by the solid-liquid ratio of solid-liquid ratio 1g:22ml with water, 6h is soaked at 30 DEG C, then at 60 DEG C, ultrasonic power is under 70w, after ultrasonic 30min, water extraction 2 times, each 160min, collect extracting solution, and centrifugal collection supernatant liquor, merging filtrate after filtering, it is 1/8th of original volume by filtrate vacuum concentration, filtrate after concentrated is added 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, it is drying to obtain Flos Farfarae Crude polysaccharides,
(3) deproteinated: Crude polysaccharides is dissolved in distilled water, it is mixed with the Crude polysaccharides solution of 10mg/ml, the mass concentration adding 2.5% in Crude polysaccharides solution is 1mg/ml, PH is the papain solution of 7.0, 80min at being placed in 55 DEG C, add chloroform: propyl carbinol is the Sevage reagent of 4:1, 20min is fully mixed through 3000r/min clarifixator, at the centrifugal 10min of 3000r/min, remove precipitation, get supernatant liquor and add chloroform again: propyl carbinol is the Sevage reagent of 4:1, repeat aforesaid operations 2 times, supernatant liquor adds 95% ethanol alcohol precipitation of 4 times of volumes, leave standstill 24h, take out filter final vacuum and it is drying to obtain polysaccharide after deproteinated,
(4) decolour: XD-5 macroporous resin distilled water immersion is removed water-soluble impurity agent and suspended substance, first with 95% alcohol immersion 24h, it is washed to distilled water and clarifies without alcohol taste and water liquid, again with 3% dilute hydrochloric acid solution 12h of 3 times of resin volumes, it is washed till neutrality with distilled water, 12h is soaked again with 3% Strong oxdiative sodium solution of 3 times of resin volumes, it is washed till neutral rear cool dry with distilled water, after polysaccharide after deproteinated is configured to the polysaccharide soln of 2mg/ml, XD-5 macroporous resin and polysaccharide soln are added in constant temperature oscillator in the ratio of 1g:95ml, it it is 25 DEG C in temperature, vibration absorption 6h when constant temperature oscillator frequency 120 times/min, filter and obtain decolouring polysaccharide,
(5) dialyse: step (4) gained polysaccharide is configured to 10mg/ml solution, with tap water flowing dialysis 48h, use distill water dialysis 24h again, then with 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, namely 50 DEG C of vacuum-dryings obtain Flos Farfarae polysaccharide.
Claims (6)
1. the extraction and purification method of Flos Farfarae polysaccharide, it is characterised in that: it comprises the following steps:
(1) pulverizing, degreasing pre-treatment: after Flos Farfarae bud is dried 12h in the baking oven of 80 DEG C, pulverized, cross 100 orders sieves, adding 95% ethanolic soln by the solid-liquid ratio of 1g:10ml, backflow degreasing 2 times, each time is 2h, by extracting liquid filtering, the ethanol in filter cake is volatilized;
(2) pre-soaking-ultrasonic-water extraction combined extracting Flos Farfarae polysaccharide: pretreated Flos Farfarae polysaccharide is mixed by the solid-liquid ratio of solid-liquid ratio 1g:15��22ml with water, 6h is soaked at 30 DEG C, then at 60��70 DEG C after ultrasonic 10��30min, water extraction 2 times, each 120��160min, collect extracting solution, and centrifugal collection supernatant liquor, merging filtrate after filtering, it is 1/8th of original volume by filtrate vacuum concentration, filtrate after concentrated is added 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, it is drying to obtain Flos Farfarae Crude polysaccharides,
(3) deproteinated: Crude polysaccharides is dissolved in distilled water, it is mixed with the Crude polysaccharides solution of 10mg/ml, the mass concentration adding 1.5��2.5% in Crude polysaccharides solution is 1mg/ml, PH is the papain solution of 7.0, 80��100min at being placed in 55��65 DEG C, add Sevage reagent, 20min is fully mixed through 3000r/min clarifixator, at the centrifugal 10min of 3000r/min, remove precipitation, get supernatant liquor and add Sevage reagent again, repeat aforesaid operations 2 times, supernatant liquor adds 95% ethanol alcohol precipitation of 4 times of volumes, leave standstill 24h, take out filter final vacuum and it is drying to obtain polysaccharide after deproteinated,
(4) decolour: macroporous resin is carried out removal of impurities, acid-alkali treatment, dry pre-treatment, after polysaccharide after deproteinated is configured to the polysaccharide soln of 2mg/ml, macroporous resin and polysaccharide soln are added in constant temperature oscillator in the ratio of 1g:95��100ml, vibration absorption 6h, filters and obtains decolouring polysaccharide;
(5) dialyse: step (4) gained polysaccharide is configured to 10mg/ml solution, with tap water flowing dialysis 48h, use distill water dialysis 24h again, then with 95% ethanol alcohol precipitation of 4 times of volumes, filter is taken out after leaving standstill 24h, successively with dehydrated alcohol, acetone, ether drip washing, namely 50 DEG C of vacuum-dryings obtain Flos Farfarae polysaccharide.
2. the extraction and purification method of Flos Farfarae polysaccharide according to claim 1, it is characterised in that, in described step (2), ultrasonic power is 80��90w.
3. the extraction and purification method of Flos Farfarae polysaccharide according to claim 1, it is characterised in that, described Sevage reagent is chloroform: propyl carbinol is 4:1.
4. the extraction and purification method of Flos Farfarae polysaccharide according to claim 1, it is characterized in that, in described step (4), the pre-treatment of macroporous resin is: macroporous resin distilled water immersion removes water-soluble impurity agent and suspended substance, first with 95% alcohol immersion 24h, it is washed to distilled water and clarifies without alcohol taste and water liquid, again with 3% dilute hydrochloric acid solution 12h of 3 times of resin volumes, it is washed till neutrality with distilled water, soak 12h with 3% Strong oxdiative sodium solution of 3 times of resin volumes again, it is washed till with distilled water neutral rear cool dry.
5. the extraction and purification method of Flos Farfarae polysaccharide according to claim 1 or 4, it is characterised in that, described macroporous resin is XD-5 macroporous resin.
6. the extraction and purification method of Flos Farfarae polysaccharide according to claim 1, it is characterised in that, in described step (4), the condition of vibration absorption is: temperature 25 DEG C, constant temperature oscillator frequency 120 times/min.
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| CN107915786A (en) * | 2017-12-29 | 2018-04-17 | 广西汇智生产力促进中心有限公司 | The method for extracting abrus cantoniensis hance polysaccharide |
| CN108017724A (en) * | 2017-12-04 | 2018-05-11 | 浙江大学 | A kind of preparation method of plant-derived chitosan |
| CN108047346A (en) * | 2017-12-26 | 2018-05-18 | 浦江县美泽生物科技有限公司 | The method for removing protein of river snail polysaccharide extraction liquid |
| CN108164619A (en) * | 2017-12-04 | 2018-06-15 | 浙江大学 | A kind of ultrasonic wave added conversion chitin is the method for chitosan |
| CN108472325A (en) * | 2016-06-08 | 2018-08-31 | 自然生物有限公司 | Include the composition for improving memory of petasites folium extract |
| CN110041445A (en) * | 2019-05-17 | 2019-07-23 | 河南科技大学 | A method of by preparing neutral polysaccharide in Syringa pubescens |
| CN110062657A (en) * | 2016-11-24 | 2019-07-26 | 诺瓦塞普工艺公司 | Use the purification process of low granularity resin |
| CN110317279A (en) * | 2019-08-19 | 2019-10-11 | 姜东生 | A kind of Asiatic polysaccharide, the purposes as plant source antiseptic agent and the application in cosmetics |
| CN111097921A (en) * | 2020-01-13 | 2020-05-05 | 山西大学 | Anti-colon cancer silver nanoparticles and preparation method thereof |
| CN112741327A (en) * | 2021-01-08 | 2021-05-04 | 大竹县万康生态农业有限公司 | Cordyceps cicadae polysaccharide health-preserving beverage and preparation method thereof |
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| CN108472325A (en) * | 2016-06-08 | 2018-08-31 | 自然生物有限公司 | Include the composition for improving memory of petasites folium extract |
| CN110062657A (en) * | 2016-11-24 | 2019-07-26 | 诺瓦塞普工艺公司 | Use the purification process of low granularity resin |
| CN108164619A (en) * | 2017-12-04 | 2018-06-15 | 浙江大学 | A kind of ultrasonic wave added conversion chitin is the method for chitosan |
| CN108017724A (en) * | 2017-12-04 | 2018-05-11 | 浙江大学 | A kind of preparation method of plant-derived chitosan |
| CN108017724B (en) * | 2017-12-04 | 2020-03-31 | 浙江大学 | Preparation method of plant-derived chitosan |
| CN108164619B (en) * | 2017-12-04 | 2020-03-31 | 浙江大学 | Method for converting chitin into chitosan under assistance of ultrasonic waves |
| CN108047346A (en) * | 2017-12-26 | 2018-05-18 | 浦江县美泽生物科技有限公司 | The method for removing protein of river snail polysaccharide extraction liquid |
| CN107915786A (en) * | 2017-12-29 | 2018-04-17 | 广西汇智生产力促进中心有限公司 | The method for extracting abrus cantoniensis hance polysaccharide |
| CN110041445A (en) * | 2019-05-17 | 2019-07-23 | 河南科技大学 | A method of by preparing neutral polysaccharide in Syringa pubescens |
| CN110317279A (en) * | 2019-08-19 | 2019-10-11 | 姜东生 | A kind of Asiatic polysaccharide, the purposes as plant source antiseptic agent and the application in cosmetics |
| CN111097921A (en) * | 2020-01-13 | 2020-05-05 | 山西大学 | Anti-colon cancer silver nanoparticles and preparation method thereof |
| CN111097921B (en) * | 2020-01-13 | 2021-05-14 | 山西大学 | Anti-colon cancer silver nanoparticles and preparation method thereof |
| CN112741327A (en) * | 2021-01-08 | 2021-05-04 | 大竹县万康生态农业有限公司 | Cordyceps cicadae polysaccharide health-preserving beverage and preparation method thereof |
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