CN101168561A - Method for separating and purifying microcystin - Google Patents
Method for separating and purifying microcystin Download PDFInfo
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- CN101168561A CN101168561A CNA2007101906102A CN200710190610A CN101168561A CN 101168561 A CN101168561 A CN 101168561A CN A2007101906102 A CNA2007101906102 A CN A2007101906102A CN 200710190610 A CN200710190610 A CN 200710190610A CN 101168561 A CN101168561 A CN 101168561A
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Abstract
The invention relates to a separating and purifying method for microcystins, and belongs to the technical field of biochemical separation. The invention adopts acetic acid aqueous solution as the extracting agent in toxic cyanophyta water bloom, and adopts economic nacroporous resin D101 as the separating and purifying material, through two times of nacroporous resin chromatography column separating, the elution liquor is alcohol-aqueous solution in different concentration, and the alcohol-aqueous solution with microcystins-RR, microcystins-YR, and microcystins-LR can be gained; the purification of the microcystins-RR, the microcystins-YR, and the microcystins-LR is not lower than 80 percent detected by HPLC-UV/MS. The invention has lower requirement for the equipment, the utilized material during the separating process is safe, the cost is low, and the industrialized production is easy, thus the invention is suitable for the environment experiment and the research of biological degradation, photolysis, and adsorption with large quantity demanded but lower purification.
Description
Technical field
A kind of method of separating and purifying microcystin belongs to technical field of biochemical separation.The method of the present invention's design separation and purification Microcystin from poisonous blue-green algae.Be applicable in the natural blue-green alga bloom thing and remove impurity, separation and purification obtains three kinds of Microcystins.
Background technology
The eutrophication of water body has been a well-known global environmental problem.The blue-green alga bloom pollution had all taken place in numerous lakes such as the Dian Chi of China, Taihu Lake, Chaohu in recent years, and it frequently breaks out the balance of nature that has destroyed the lake, caused water quality extremely to worsen and was on the rise.Constantly producing Microcystin has therebetween caused widely the potential hazard of water body environment and human health and has paid attention to.All kinds of researchs of carrying out around Microcystin comprise the mensuration of its concentration in mechanism that its produces, the environment, effectively remove the full appreciation of measure and toxic effect.Along with extensively carrying out to Microcystin research, scientific research, teaching field are increasing to the demand of the standard substance of Microcystin, and the detection of Microcystin content in water body, the fishery products has been become indispensable index in human drinking-water quality monitoring and the food safety detection.
The standard substance of Microcystin are mainly derived from external chemical reagents corporation, because cost an arm and a leg and the restriction of other non-science factor and economic factors, the acquisition of standard substance becomes the restraining factors that all kinds of correlative studys are carried out in restriction.From Microcystis aeruginosa, extract the purifying microcystin method at present both at home and abroad and mostly adopt the reversed-phase silica gel chromatography filler, obtain the purifying product as elution reagent with methyl alcohol as separating medium.Its weak point is, filler price expensive (being higher than 10000 yuan/kilogram) is used the back deleterious repeatedly, and reagent toxicity is very big, can only obtain very a spot of Microcystin at every turn.
Macroporous adsorbent resin is a kind of novel high polymer polymeric adsorbent with the netted opening structure of polymer, have two kinds of isolating performances of chemisorption and physically screening simultaneously, loading capacity is big, selectivity good and cheap (being lower than 100 yuan/kilogram), successfully separate multiple peptides at present, be widely used in fields such as wastewater treatment, medicine, food, biotechnology.
With macroporous adsorbent resin the method that Microcystin carries out separation and purification is not seen bibliographical information as yet.
Summary of the invention
The object of the present invention is to provide a kind of method of separating and purifying microcystin, on a large scale, safe, cheap from poisonous blue-green alga bloom thing by the technology of two-step chromatography separation and purification simultaneously Microcystin-RR, Microcystin-YR and microcapsule algae toxin, be applicable to the big but not high research experiment of purity requirement of demand.Present method is simple to operate, the solvent safety height, and equipment is simple, and cost is low.
Technical scheme of the present invention: a kind of method of separating and purifying microcystin, with poisonous blue-green alga bloom dry powder acetic acid aqueous solution is extraction agent, adopt economic macroporous adsorbent resin D101 as the separation and purification material, crossing the macroporous adsorbent resin chromatography column by twice separates, elutriant is Different concentrations of alcohol-aqueous solution, obtains the aqueous ethanolic solution of Microcystin-RR, Microcystin-YR and microcapsule algae toxin; Step is:
A. take by weighing poisonous blue-green alga bloom dry powder, add its weight 20-30 acetic acid solution of 5% doubly, continuously stirring 40-50min with the centrifugal 10min of 6000g, gets supernatant liquor; Precipitation repeats to extract once by the said extracted step, merges the extracted twice supernatant, and with rotary evaporation behind 0.45 micron the membrane filtration, it is standby to get Microcystin acetic acid extraction concentrated solution, and the weight of concentrated solution is 6-10 times of raw material blue-green alga bloom dry powder weight;
B. macroporous adsorbent resin D101 chromatography column, 10 * 100cm, behind the alcohol-pickled resin 24h, respectively soak 3-5h with 4%HCl, 2%HCl successively, deionization is washed neutral, will cross post behind the accent of the extraction concentrated solution in the A step pH2.8-3.2, then successively with being the water that extracts half volume of concentrated solution, 15% ethanol drip washing of half volume, use successively again and extract isopyknic 30% ethanol of concentrated solution, 40% ethanol, 50% ethanol elution, collect elutriant;
C. after the 30% ethanol eluate rotary evaporation that obtains among the step B being removed ethanol, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol drip washing then with the same volume of step B, use 30% ethanol elution again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain containing Microcystin-RR aqueous ethanolic solution;
D. 40% and 50% ethanol eluate that obtains among the step B is merged, after rotary evaporation removes ethanol, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol drip washing with the same volume of step B then, use 40% ethanol elution again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain Microcystin-YR aqueous ethanolic solution and microcapsule algae toxin aqueous ethanolic solution respectively;
E. the purity detecting of Microcystin adopts the HPLC-UV/MS method to measure chromatographic column: Symmetry C18 post, 2.1 * 150mm, 3.0 μ m, 30.0 ℃ of column temperatures; Moving phase: acetonitrile-water-formic acid, volume ratio were followed successively by 30: 70: 0.1; Flow velocity: 0.3ml/min; Ultraviolet-detector: 238nm; Liquid chromatography sampling volume: 10 μ L; The mass spectrum condition: adopt positron ionization mode, spray voltage 3.7KV, the precipitation temperature: 300 ℃, ion source temperature: 120 ℃, ion energy: 1.0v, taper hole voltage: 30-60V, sweep limit: m/z=400-1200; The chromatographic peak area normalization method is calculated Microcystin purity; Judge that by mass signal chromatographic peak separates substantially.
Cross the separation of macroporous adsorbent resin chromatography column for twice and obtain Microcystin-RR, the Microcystin-YR of purity 〉=80% and the aqueous ethanolic solution of microcapsule algae toxin.
Beneficial effect of the present invention: adopt economic macroporous adsorbent resin D101 as the separation and purification material in poisonous Microcystis aeruginosa dry powder, crossing the macroporous adsorbent resin chromatography column by twice separates, elutriant is Different concentrations of alcohol-aqueous solution, obtains the aqueous ethanolic solution of Microcystin-RR, Microcystin-YR and microcapsule algae toxin.The present invention be a kind of can be extensive, safe, cheap from poisonous blue-green alga bloom thing by the two-step chromatography technology of separation and purification Microcystin-RR, Microcystin-YR and microcapsule algae toxin simultaneously, be applicable to the big but not high environmental classes research experiments such as biological degradation, photodissociation and absorption of purity requirement of demand.Present method is simple to operate, the solvent safety height, and equipment is simple, and cost is low.
Description of drawings
The HPLC-UV color atlas of Fig. 1 Microcystin-RR
The HPLC-UV color atlas of Fig. 2 Microcystin-YR
The HPLC-UV color atlas of Fig. 3 microcapsule algae toxin
Embodiment
Embodiment 1:
1, take by weighing poisonous blue-green alga bloom dry powder 20 grams, add 5% acetic acid solution of 500 grams, stir 50min after, with the centrifugal 10min of 6000g, take out supernatant liquor; Precipitation repeats said extracted one time, merges the extracted twice supernatant.
2, with behind 0.45 micron the membrane filtration, rotary evaporation below 50 ℃, it is standby to get Microcystin acetic acid extraction concentrated solution, and the extraction concentrated solution is 150-200mL.
3, macroporous adsorbent resin D101 chromatography column, 10 * 100cm behind the alcohol-pickled resin 24h, soaks 4h with 4%HCl and 2%HCl successively, and deionization is washed neutral.
4, the extraction concentrated solution in 2 steps being regulated its pH value is that macroporous adsorbent resin chromatography column (flow velocity 5mh is crossed in 2.8 backs
-1), each 200mL wash-out of 30%, 40%, 50% ethanol is used in water 100mL, 15% ethanolic soln 100mL drip washing respectively more then, collects elutriant.
5,30% ethanol eluate rotary evaporation is removed ethanol (its vaporization temperature is no more than 50 ℃) after, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol 100mL drip washing then, use 30% ethanol 150-200mL wash-out again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain containing Microcystin-RR aqueous ethanolic solution.
6,40% and 50% ethanol eluate is merged, after rotary evaporation removes ethanol (its vaporization temperature is no more than 50 ℃), separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol 100mL drip washing then, use 40% ethanol elution 200mL again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain Microcystin-YR aqueous ethanolic solution and microcapsule algae toxin aqueous ethanolic solution respectively.
7, the purity detecting of Microcystin adopts the HPLC-MS method to judge.Chromatographic column: SymmetryC18 post, 2.1 * 150mm, 3.0 μ m, 30.0 ℃ of column temperatures; Moving phase: acetonitrile-water-formic acid volume ratio 30: 70: 0.1 successively; Flow velocity: 0.3mL/min; Ultraviolet-detector: 238nm; Liquid chromatography sampling volume: 10 μ L.The mass spectrum condition: adopt positron ionization mode, spray voltage 3.7KV, the precipitation temperature: 300 ℃, ion source temperature: 120 ℃, ion energy: 1.0v, taper hole voltage: 35V, sweep limit: m/z=400-1200.Judge Microcystin-RR by mass signal, Microcystin-YR, each peak of microcapsule algae toxin satisfies complete separation requirement, adopts the chromatographic peak area normalization method to calculate Microcystin purity.Obtain Microcystin-RR, the Microcystin-YR of purity 〉=80% and the aqueous ethanolic solution of microcapsule algae toxin.
Embodiment 2:
1, take by weighing poisonous blue-green alga bloom dry powder 50 grams, add 5% acetic acid solution of 1000 grams, stir 40min after, with the centrifugal 10min of 6000g, take out supernatant liquor; Precipitation repeats said extracted one time, merges the extracted twice supernatant.
2, with behind 0.45 micron the membrane filtration, rotary evaporation below 40 ℃, it is standby to get Microcystin acetic acid extraction concentrated solution, and the extraction concentrated solution is 300-400mL.
3, macroporous adsorbent resin D101 chromatography column, 10 * 100cm behind the alcohol-pickled resin 24h, soaks 5h with 4%HCl and 2%HCl respectively, and deionization is washed neutral.
4, the extraction concentrated solution in 2 steps being regulated its pH value is that macroporous adsorbent resin chromatography column (flow velocity 5mh is crossed in 3.2 backs
-1), each 300-400mL wash-out of 30%, 40%, 50% ethanol is used in water 150-200mL, 15% ethanolic soln 150-200mL drip washing respectively more then, collects elutriant.
5,30% ethanol eluate rotary evaporation is removed ethanol (its vaporization temperature is no more than 50 ℃) after, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol 150mL drip washing then, use 30% ethanol 150-200mL wash-out again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain containing Microcystin-RR aqueous ethanolic solution.
6,40% and 50% ethanol eluate is merged, after rotary evaporation removes ethanol (its vaporization temperature is no more than 50 ℃), separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol 150-200mL drip washing then, use 40% ethanol elution 200mL again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain Microcystin-YR aqueous ethanolic solution and microcapsule algae toxin aqueous ethanolic solution respectively.
7, the purity detecting of Microcystin adopts the HPLC-MS method to judge.Chromatographic column: Symmetry C18 post (2.1 * 150mm, 3.0 μ m), 30.0 ℃ of column temperatures; Moving phase: acetonitrile-water-formic acid volume ratio 30: 70: 0.1 successively; Flow velocity: 0.3mL/min; Ultraviolet-detector: 238nm; Liquid chromatography sampling volume: 10 μ L.The mass spectrum condition: adopt positron ionization mode, spray voltage 3.7KV, the precipitation temperature: 300 ℃, ion source temperature: 120 ℃, ion energy: 1.0v, taper hole voltage: 45V, sweep limit: m/z=400-1200.Judge Microcystin-RR by mass signal, Microcystin-YR, each peak of microcapsule algae toxin satisfies complete separation requirement, adopts the chromatographic peak area normalization method to calculate Microcystin purity.Obtain Microcystin-RR, the Microcystin-YR of purity 〉=80% and the aqueous ethanolic solution of microcapsule algae toxin.
Claims (2)
1. the method for a separating and purifying microcystin, it is characterized in that, with poisonous blue-green alga bloom dry powder acetic acid aqueous solution is extraction agent, adopt macroporous adsorbent resin D101 as the separation and purification material, crossing the macroporous adsorbent resin chromatography column by twice separates, elutriant is Different concentrations of alcohol-aqueous solution, obtains the aqueous ethanolic solution of Microcystin-RR, Microcystin-YR and microcapsule algae toxin; Step is:
A. take by weighing poisonous blue-green alga bloom dry powder, add its weight 20-30 acetic acid solution of 5% doubly, continuously stirring 40-50min with the centrifugal 10min of 6000g, gets supernatant liquor; Precipitation repeats to extract once by the said extracted step, merges the extracted twice supernatant, and with rotary evaporation behind 0.45 micron the membrane filtration, it is standby to get Microcystin acetic acid extraction concentrated solution, and the weight of concentrated solution is 6-10 times of raw material blue-green alga bloom dry powder weight;
B. macroporous adsorbent resin D101 chromatography column, 10 * 100cm, behind the alcohol-pickled resin 24h, respectively soak 3-5h with 4%HCl, 2%HCl successively, deionization is washed neutral, will cross post behind the accent of the extraction concentrated solution in the A step pH2.8-3.2, then successively with being the water that extracts half volume of concentrated solution, 15% ethanol drip washing of half volume, use successively again and extract isopyknic 30% ethanol of concentrated solution, 40% ethanol, 50% ethanol elution, collect elutriant;
C. after the 30% ethanol eluate rotary evaporation that obtains among the step B being removed ethanol, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol drip washing then with the same volume of step B, use 30% ethanol elution again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain containing Microcystin-RR aqueous ethanolic solution;
D. 40% and 50% ethanol eluate that obtains among the step B is merged, after rotary evaporation removes ethanol, separate with the new macroporous adsorbent resin of handling of above-mentioned similarity condition again, cross post, use 15% ethanol drip washing with the same volume of step B then, use 40% ethanol elution again, the Fractional Collections elutriant; Measure with the HPLC-UV/MS method, obtain Microcystin-YR aqueous ethanolic solution and microcapsule algae toxin aqueous ethanolic solution respectively;
E. the purity detecting of Microcystin adopts the HPLC-UV/MS method to measure chromatographic column: Symmetry C18 post, 2.1 * 150mm, 3.0 μ m, 30.0 ℃ of column temperatures; Moving phase: acetonitrile-water-formic acid, volume ratio were followed successively by 30: 70: 0.1; Flow velocity: 0.3ml/min; Ultraviolet-detector: 238nm; Liquid chromatography sampling volume: 10 μ L; The mass spectrum condition: adopt positron ionization mode, spray voltage 3.7KV, the precipitation temperature: 300 ℃, ion source temperature: 120 ℃, ion energy: 1.0v, taper hole voltage: 30-60V, sweep limit: m/z=400-1200; The chromatographic peak area normalization method is calculated Microcystin purity; Judge that by mass signal chromatographic peak separates substantially.
2. the method for a kind of separating and purifying microcystin according to claim 1, it is characterized in that twice mistake macroporous adsorbent resin chromatography column separates Microcystin-RR, Microcystin-YR and the microcapsule algae toxin aqueous ethanolic solution that obtains purity 〉=80%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN102388924A (en) * | 2011-08-28 | 2012-03-28 | 鞠志国 | Biological insecticide and preparation method thereof |
| CN101750460B (en) * | 2008-12-03 | 2012-09-19 | 北京林业大学 | Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography |
| CN104017059A (en) * | 2014-06-19 | 2014-09-03 | 南京麦思德餐饮管理有限公司 | Method for extracting microcystic toxins |
| CN106841371A (en) * | 2017-04-12 | 2017-06-13 | 东南大学 | A kind of method that utilization flight time mass spectrum differentiates Cells of Blue-green Algae |
| CN110606876A (en) * | 2019-08-01 | 2019-12-24 | 青岛普瑞邦生物工程有限公司 | N-15 labeled microcystin and production method thereof |
| CN115343378A (en) * | 2021-09-06 | 2022-11-15 | 国家海洋环境监测中心 | Method for separating and purifying okadaic acid and finotoxin homologues in prorocentrum lima |
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2007
- 2007-11-27 CN CNB2007101906102A patent/CN100558743C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750460B (en) * | 2008-12-03 | 2012-09-19 | 北京林业大学 | Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography |
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN102388924A (en) * | 2011-08-28 | 2012-03-28 | 鞠志国 | Biological insecticide and preparation method thereof |
| CN102388924B (en) * | 2011-08-28 | 2014-04-16 | 鞠志国 | Biological insecticide and preparation method thereof |
| CN104017059A (en) * | 2014-06-19 | 2014-09-03 | 南京麦思德餐饮管理有限公司 | Method for extracting microcystic toxins |
| CN106841371A (en) * | 2017-04-12 | 2017-06-13 | 东南大学 | A kind of method that utilization flight time mass spectrum differentiates Cells of Blue-green Algae |
| CN110606876A (en) * | 2019-08-01 | 2019-12-24 | 青岛普瑞邦生物工程有限公司 | N-15 labeled microcystin and production method thereof |
| CN110606876B (en) * | 2019-08-01 | 2021-10-12 | 青岛普瑞邦生物工程有限公司 | N-15 labeled microcystin and production method thereof |
| CN115343378A (en) * | 2021-09-06 | 2022-11-15 | 国家海洋环境监测中心 | Method for separating and purifying okadaic acid and finotoxin homologues in prorocentrum lima |
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