CN102426156A - Determination method for total flavone content in pollen pini supercritical extractant - Google Patents
Determination method for total flavone content in pollen pini supercritical extractant Download PDFInfo
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- CN102426156A CN102426156A CN2011103744293A CN201110374429A CN102426156A CN 102426156 A CN102426156 A CN 102426156A CN 2011103744293 A CN2011103744293 A CN 2011103744293A CN 201110374429 A CN201110374429 A CN 201110374429A CN 102426156 A CN102426156 A CN 102426156A
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Abstract
The invention relates to a determination method for a total flavone content in a pine pollen supercritical extractant. According to the method, rutin is adopted as a standard material; 70% ethanol is adopted to extract the pollen pini supercritical extractant; a polyamide resin chromatographic column is adopted to separate the total flavone; a 5% sodium nitrite solution, a 10% aluminum nitrate solution and a sodium hydroxide solution with the concentration of 1.0 mol.L<-1> are added to carry out a color reaction; the absorbance is detected at the ultraviolet light wave length of 510 nm to detect the total flavone content. According to the present invention, a simple and feasible analysis scheme is established for the total flavone content in the pollen pini extracted by supercritical fluid extraction, the accuracy and the precision are good, and a method basis is provided for quality control of the pollen pini supercritical extractant.
Description
Technical field
The present invention relates to the assay method of general flavone content in a kind of pollen pini supercritical extract.
Background technology
To be pinaceae plant masson pine (Pinus massonina lamb), Chinese pine plants such as (Pinus tabulaefermiscarr) spring spend when just opening pollen pini (Pollen Pini) that male ball is plucked; Dry; Rub pollen down with the hands; Collect, remove the faint yellow thin pollen [referring to ChP (Chinese Pharmacopoeia) .2010.Vol I (an one): 191] of impurity gained.Pollen pini is natural miniature nutrition library.As good health food, the nutritional labeling of pollen pini can be divided into sugar, protein, fat, vitamin, trace element and bioactivator six big classes.The vital movement of its contained plurality of enzymes, hormone, flavones and abundant biological active matter confrontation human bodies such as phosphatide has important regulatory role; Have anti-ageing, antifatigue, adjusting blood fat, hypoglycemic, protect multiple pharmacological effect such as liver, beauty treatment, fat-reducing, be the outstanding person in the pollen product.
The pollen pini active component is complicated, though by " Chinese pharmacopoeia is recorded, and has stipulated method for making, microscopical identification and inspection item, its active content of effective is not made quality control standard.Present document shows: the many forms with glucoside of the flavone compound in the pollen pini exist, and have many-sided physiologically active.Can effectively remove the oxygen radical in the body, it stops the ability of oxidation is more than ten times of vitamin E, can stop degeneration, the aging of cell.The phosphatide that is rich in has multiple pharmacologically actives such as significant reducing blood lipid especially.Biomedical research shows over nearly 20 years: phosphatide has the effect of diseases such as regulating physiological function, enhance immunity power, the control heart, brain, blood vessel.But phosphatide is met thermally labile, brings difficulty for traditional solvent extraction.
Supercritical fluid extraction (supercritical fluid extraction; SFE) be the new and high technology that modern age, separation field occurred; Except that reducing consumption of organic solvent; Also have extraction efficiency with the selectivity height, be applicable to the extraction of determination system of thermal unstable material, environmental pollution is little, operating conditions is easy to characteristics such as change, is a kind of comparatively desirable middle pharmaceutically active substance method for distilling.Pollen pini is carried out supercritical CO
2Extraction can be avoided organic solvent residue, improves productive rate, and the effective active matter in the survivable pollen pini in leaching process simultaneously improves the extraction quantity of active component greatly.[referring to: Shao Xingjun, Ding Dehua, Li Hongyan etc. supercritical CO
2Method for extracting active ingredients of pollen pini. [P] Chinese patent: 200810155138.3,2009-03-18; Feng Wu, Fan Guodong, Liu Jiabao. pinus yunnanensis pollen supercritical CO
2Degreasing research. [J] Yunnan Forestry science and technology .2003, (1): 63.]
The pollen pini supercritical extract is as the Chinese medical extract with wide market outlook; Formulating its criteria of quality evaluation has very important meaning to its development in the Chinese medicine industry, and to hold the quality situation of extract on the whole particularly important thereby particularly set up effective analytical approach to a plurality of typical activity compositions wherein.At present, do not see the assay document and the patent of carrying out flavones ingredient to the pollen pini supercritical extract both at home and abroad as yet.The present invention has set up simple and feasible analytical approach to the general flavone in the supercritical fluid extraction pollen pini first; For the quality standard of formulating the pollen pini supercritical extract provides a reliable foundation, also the quality assessment means are provided simultaneously for further reasonable development and this extract of comprehensive utilization.
Summary of the invention
The objective of the invention is to design provides a kind of technical scheme of setting up content assaying method to general flavone composition in the supercritical fluid extraction pollen pini.General flavone in extract utilization ultraviolet-visible spectrophotometry is measured; Simple and feasible; Accuracy, precision are good, can hold the quality situation of extract through the assay of active component, can be used as the quality assessment foundation of pollen pini supercritical extract.
Technical scheme of the present invention is following:
The assay method of general flavone content in a kind of pollen pini supercritical extract (SFE-PP), it comprises the steps:
The preparation of step 1. standard reserving solution: it is an amount of that precision takes by weighing control substance of Rutin, adds dissolve with methanol and be settled to 100ml, is made into 150 μ gml
-1The rutin standard reserving solution;
Step 2. supplies the preparation of test agent solution:
1. precision takes by weighing pollen pini supercritical extract 2.0000g, packs tightly with filter paper and places flat bottom flask, adds 100ml 70% ethanolic solution, soaks into the back at 80 ℃ of water-bath refluxed 2h; Crude extract cooling back decompress filter is with a small amount of 25% washing with alcohol flask and filter residue, merging filtrate; Ethanol is wherein removed in decompression distillation, pours out solution in the flask, suction filtration; And with 30ml hot water divide 3 times the washing flask, behind the suction filtration, filtrating is poured in the separating funnel; Divide 3 extraction degreasings with the 75ml methenyl choloride, collect the WS, rotary evaporation is used for following column chromatography application of sample after adding 70% dissolve with ethanol after do;
2. the pretreated polyamide powder wet method of learning from else's experience dress post is used water saturation, and the sample of drawing after the above-mentioned degreasing slowly splashes in the post along chromatographic column; Place certain hour; To the abundant absorption of liquid quilt to be measured, use 70% ethanol elution, collect eluent; Above-mentioned eluent concentrates constant volume must supply test agent solution, is used to measure content of total flavone;
Step 3. assay method: precision is measured above-mentioned confession test agent solution or rutin standard solution in right amount in the 10ml volumetric flask; Add 30% ethanol liquid to 5ml; Adding mass percentage concentration is 5% sodium nitrite solution 0.3ml; Place 5min after the jolting, adding mass percentage concentration is that 10% aluminum nitrate solution 0.3ml shakes up back placement 6min, adds 1.0molL
-1Sodium hydroxide solution 2ml is settled to scale with 30% ethanol, shakes up, and is blank with the reagent corresponding, measures absorbance at the 510nm place;
Step 4. result calculates: with the determination data production standard curve of rutin standard solution, calculate the amount of general flavone in the sample solution for the determination data reference standard opisometer of test agent solution.
Beneficial effect
At present, to the pharmacological action of pollen pini and extract thereof and existing many pieces of bibliographical informations of research of active component.Because the pollen pini functional component is complicated, its quality assessment sporadically appears in the mensuration of vitamin, trace element and single type of active substance of part, and the standardization and the integrality of evaluation content still are left to be desired.The pollen pini supercritical extract is as Chinese medical extract with wide market outlook, and formulating its quality assessment scheme to typical activity composition wherein has very important meaning to its development in the Chinese medicine industry.At present, do not see the document and the patent of carrying out the assay of flavones ingredient to the pollen pini supercritical extract both at home and abroad as yet.The present invention has set up simple and feasible analytical plan to general flavone in the supercritical fluid extraction pollen pini first, and accuracy, precision are good, are the quality controllable method foundation that provides that realizes the pollen pini supercritical extract.
Description of drawings
Fig. 1 is rutin standard items (1) and pollen pini supercritical CO
2Extract supplies the UV scanning figure of test agent (2).
Embodiment
Below through specific embodiment the present invention is described further.
One, experimental section
1. instrument and reagent
UV-2401PC type ultraviolet spectrophotometer (day island proper Tianjin); XT-102A type electronic balance (Switzerland Precisa) etc.
The pollen pini supercritical CO
2Extract (SFE-PP, Jiangsu Nucell Bio-Technology Co., Ltd.); Control substance of Rutin (Nanjing Zelang Pharmaceutical Technology Inc.); All the other reagent analysis pure (Chemical Reagent Co., Ltd., Sinopharm Group).
2. content of total flavone is measured among the experimental technique SFE-PP:
2.1 the preparation of standard reserving solution: it is an amount of that precision takes by weighing control substance of Rutin, adds dissolve with methanol and be settled to 100ml, is made into 150 μ gml
-1Rutin standard inventory solution.
2.2 supply the preparation of test agent solution:
2.2.1 precision takes by weighing pollen pini supercritical extract 2.0000g, packs tightly with filter paper and places flat bottom flask, adds 100ml 70% ethanolic solution, soaks into the back at 80 ℃ of water-bath refluxed 2h.Crude extract cooling back decompress filter is with a small amount of 25% washing with alcohol flask and filter residue, merging filtrate.Ethanol is wherein removed in decompression distillation.Pour out solution in the flask, suction filtration divides the washing flask 3 times with 30ml hot water; Behind the suction filtration, filtrating is poured in the separating funnel, divide 3 extraction degreasings with the 75ml methenyl choloride; After treating complete layering, collect the WS, rotary evaporation is used for application of sample after adding 70% dissolve with ethanol after do.
Pretreated polyamide powder wet method dress post is used water saturation 2.2.2 learn from else's experience.The sample of drawing after the above-mentioned degreasing slowly splashes in the post along chromatographic column, places certain hour, to liquid to be measured by after the abundant absorption; Use 70% ethanol elution; Collect eluent, the concentrated constant volume of above-mentioned eluent promptly gets and supplies test agent solution, to be used to measure content of total flavone.
2.3 assay method: precision is measured above-mentioned need testing solution or rutin standard solution in right amount in the 10ml measuring bottle, adds 30% ethanol to 5ml, adds 5% sodium nitrite solution 0.3ml; Place 5min after the jolting; Add 10% aluminum nitrate solution 0.3ml, shake up the back and place 6min, add 1.0molL
-1Sodium hydroxide solution 2ml is settled to scale with 30% ethanol, shakes up.With the reagent corresponding is blank, measures absorbance at the 510nm place.
2.4 the result calculates: with the determination data production standard curve of rutin standard solution, calculate the amount of general flavone in the sample solution for the determination data reference standard opisometer of test agent solution.
Two, result and discussion
1 determination of total flavonoids optimization of experimental conditions and checking
1.1 measure the selection of wavelength
Precision is measured confession test agent solution under rutin standard solution and 2.2 under 2.1 in right amount in the 10mL measuring bottle; Add reagent according to 2.3 assay methods; With the reagent corresponding is blank, in 200nm～800nm scope, scans respectively, to confirm maximum absorption wavelength.Find that rutin standard items and SFE-PP sample all have the absorption maximum (see figure 1) at the 510nm place, and peak shape is similar, therefore selects 510nm to measure wavelength.
1.2 the drafting of typical curve
Precision is measured rutin standard reserving solution 0.50ml, 1.00ml, 2.00ml, 3.00ml, 4.00ml puts in the 10mL measuring bottle, according to 2.3 assay methods adding reagent, is blank with the reagent corresponding; Measure absorbance at the 510nm place; The gained data are returned, obtain regression equation and be y=0.0099x+0.0289 (r=0.9990, n=5); The result shows that absorbance of rutin and concentration are good linear relationship in the experimental concentration scope.
1.3 precision experiment
Accurate rutin standard inventory solution 1.00ml, 2.00ml, the 4.00ml of drawing gets basic, normal, high three concentration and is respectively 15,30,60 μ gml in the 10ml measuring bottle
-1Standard solution, by 2.3 colour developings.5 parts of each concentration replicate determinations are with the withinday precision of investigation method; More than the standard solution of three concentration, survey once every day, continuous 5 days, with the day to day precision of investigation method.The result sees table 1.
Table 1 general flavone precision experimental result (n=5)
1.4 average recovery experiment
Precision pipettes the sample solution and the reference substance solution of certain volume known content, processes the solution of basic, normal, high 3 concentration, by 2.3 colour developings.3 parts of each concentration replicate determinations, the average recovery of calculating general flavone.The result sees table 2.
Table 2 general flavone average recovery experimental result (n=3)
1.5 content of total flavone is measured in the test sample
Adopt 2.2 to supply the test agent measured in solution down, calculate the general flavone concentration in the need testing solution by typical curve.According to content of total flavone in the computes SFE-PP sample, data are seen table 3 then.
In the formula: X---content of total flavone in the sample, μ gg
-1
C---according to the bent concentration that calculates general flavone in the test solution of mark, μ gml
-1
V
2---it is long-pending to measure the consumption bottle, ml
V---appearance liquid cumulative volume, ml
V
1---measure sample volume, ml
M---the quality of SFE-PP sample, g
Table 3 general flavone is measured the result
Claims (1)
1. the assay method of general flavone content in the pollen pini supercritical extract is characterized in that it comprises the steps:
The preparation of step 1. standard reserving solution: it is an amount of that precision takes by weighing control substance of Rutin, adds dissolve with methanol and be settled to 100 ml, is made into 150 μ g ml
-1The rutin standard reserving solution;
Step 2. supplies the preparation of test agent solution:
Precision takes by weighing pollen pini supercritical extract 2.0 g, packs tightly with filter paper and places flat bottom flask, adds 100 ml, 70% ethanolic solution, soaks into the back at 80 ℃ of water-bath refluxed 2 h; Crude extract cooling back decompress filter is with a small amount of 25% washing with alcohol flask and filter residue, merging filtrate; Ethanol is wherein removed in decompression distillation, pours out solution in the flask, suction filtration; And with 30 ml hot water divide 3 times the washing flask, behind the suction filtration, filtrating is poured in the separating funnel; Divide 3 extraction degreasings with 75 ml methenyl cholorides, collect the WS, rotary evaporation is used for following column chromatography application of sample after adding 70% dissolve with ethanol after do;
The pretreated polyamide powder wet method of learning from else's experience dress post is used water saturation, and the sample of drawing after the above-mentioned degreasing slowly splashes in the post along chromatographic column; Place certain hour; To the abundant absorption of liquid quilt to be measured, use 70% ethanol elution, collect eluent; Above-mentioned eluent concentrates constant volume must supply test agent solution, is used to measure content of total flavone;
Step 3. assay method: precision is measured above-mentioned confession test agent solution or rutin standard solution in right amount in 10 ml measuring bottles; Add 30% ethanol liquid to 5 ml; Adding mass percentage concentration is 5% sodium nitrite solution, 0.3 ml; Place 5 min after the jolting, adding mass percentage concentration is that 10% aluminum nitrate solution, 0.3 ml shakes up back placement 6 min, adds 1.0 mol L
-1Sodium hydroxide solution 2 ml are settled to scale with 30% ethanol, shake up, and are blank with the reagent corresponding, measure absorbance at 510 nm places;
Step 4. result calculates: with the determination data production standard curve of rutin standard solution, calculate the amount of general flavone in the sample solution for the determination data reference standard opisometer of test agent solution.
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| CN104215494A (en) * | 2014-09-26 | 2014-12-17 | 武汉大学 | Sample preprocessing method with pollen as solid phase extractant |
| CN107860733A (en) * | 2017-10-30 | 2018-03-30 | 广西中医药大学 | The strong floor Determination Method of Flavone Content of medicine nine |
| CN108324751A (en) * | 2018-05-14 | 2018-07-27 | 浙江医药高等专科学校 | A kind of elegant jessamine alcohol extracts from the leaves and its purposes in relieving alcoholism and protecting liver |
| CN111912801A (en) * | 2020-08-21 | 2020-11-10 | 平顶山神马工程塑料有限责任公司 | Method for measuring copper ion content in polyamide slice |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267738A (en) * | 2013-05-15 | 2013-08-28 | 浙江大学 | Method for measuring content of flavonoid of silkworm larva body |
| CN104215494A (en) * | 2014-09-26 | 2014-12-17 | 武汉大学 | Sample preprocessing method with pollen as solid phase extractant |
| CN107860733A (en) * | 2017-10-30 | 2018-03-30 | 广西中医药大学 | The strong floor Determination Method of Flavone Content of medicine nine |
| CN108324751A (en) * | 2018-05-14 | 2018-07-27 | 浙江医药高等专科学校 | A kind of elegant jessamine alcohol extracts from the leaves and its purposes in relieving alcoholism and protecting liver |
| CN111912801A (en) * | 2020-08-21 | 2020-11-10 | 平顶山神马工程塑料有限责任公司 | Method for measuring copper ion content in polyamide slice |
| CN111912801B (en) * | 2020-08-21 | 2023-02-24 | 平顶山神马工程塑料有限责任公司 | Method for measuring copper ion content in polyamide slice |
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Application publication date: 20120425 |