CN103059069A - Novel technique method for extracting general flavone from wild walking fern - Google Patents
Novel technique method for extracting general flavone from wild walking fern Download PDFInfo
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- CN103059069A CN103059069A CN2012105955382A CN201210595538A CN103059069A CN 103059069 A CN103059069 A CN 103059069A CN 2012105955382 A CN2012105955382 A CN 2012105955382A CN 201210595538 A CN201210595538 A CN 201210595538A CN 103059069 A CN103059069 A CN 103059069A
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Abstract
The invention belongs to the field of natural organic chemistry, and relates to a method for extracting general flavone from Great Khingan wild walking fern, in particular to a novel method for separating and purifying the general flavone through high-speed countercurrent chromatography by using enzyme extraction. The method has the advantages of quickness in extraction, high purity of obtained products and low environmental pollution. 1 The biological enzymolysis technology is applied to fully dissolve out effective components in cells; and 2 the high-speed countercurrent chromatography technology is adopted, so that the separation process is simple, and the solvent consumption is low; the quick separation of the effective components is achieved in a short time; and the purity is high. The biological enzyme method is used for promoting the full dissolution of the effective components; the ethanol is extracted through reflux; the high-speed countercurrent chromatography is adopted to separate and purify; and the purity of the obtained general flavone is improved to 95.3%.
Description
Technical field:
The invention belongs to the natural organic chemistry field, relate to a kind of method of extracting flavones in the wild walking fern in Daxing'an Mountainrange, particularly relate to a kind of Enzymatic Extraction of utilizing, the novel method of high speed adverse current chromatogram separation and purification walking fern total flavones.
Background technology:
Walking fern, plant is up to 20 centimetres.Root stock is short and small, and upright, tip is close by ramentum; The scale lanceolar, chocolate is membranous, full edge.Leafage gives birth to; Basal leaf is sterile, and is less, and the long 1-3 of handle centimetre, the long 1-2 of blade centimetre, wide 5-8 millimeter, ellipse, blunt nosed, the wealthy wedge shape of base portion, slightly downward in petiole; Fertile frond is larger, long 10-15 centimetre in the long 1-5 of handle centimetre blade, wide 5-10 millimeter, lanceolar, full edge or slightly be wavy, base portion wedge shape or circle wedge shape are downward in petiole with narrow wing, and tip is point gradually, and extend into whiplike (long 3-8 centimetre), end is slightly curling, and the capable vegetative propagation of taking root can land.Vein is netted, mays be seen indistinctly above only, has mesh 1-3 capable, and is long and narrow near 1 row mesh of master pulse, parallel with master pulse, its outer capable mesh of 1-2 tiltedly on, the outer scun of mesh separates, not Da Yebian.Sap green after the leaf herbaceous stem is done is without hair.Sorus is linear or oval, respectively form irregular 1-3 in the master pulse both sides capable, usually longer near 1 row of master pulse, be born in mesh to a side of axle, indusium is to the master pulse opening, its outer 1-2 capable when being born in couples in the mesh then indusium split mutually, then indusium is opened to master pulse or limb when being born in individually in the mesh; Indusium is narrow, and similar shape is membranous, greyish-green or light brown.
The shortcomings such as the domestic general extract by solvents total flavones of using exists service temperature high, and yield is on the low side, difference of the present invention is mainly to adopt Enzymatic Extraction, later stage is adopted high-speed countercurrent chromatography separation and purification total flavones, and product not only yield is high, and purity is high.
Summary of the invention:
Advantage of the present invention:
The present invention mainly adopts biological enzymolysis technology, adopts ethanol to cook extraction agent, and the later stage is adopted high-speed countercurrent chromatography (HSCCC) separation and purification total flavones.The inventive method is extracted fast, product purity is high, and yield is high, and the ethanol of selecting food to allow to use extracts for extracting solvent refluxing, without chemical residual.
For achieving the above object, the technical solution used in the present invention is:
A kind of employing Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, its step is as follows:
(1) enzymolysis: the dry herb of walking fern is crushed to the 20-40 order, enzyme concn 0.2-03g/l, hydrolysis temperature 45-50 ℃, enzymolysis time 2-3h;
(2) refluxing extraction: add 60-70% ethanol, adopt the heating and refluxing extraction method, solid-liquid ratio 1: 12-15 extracts 80-90 ℃ of temperature, extracts twice, each extraction time 1-1.5h;
(3) collect: Recycled ethanol, thin-layer chromatography detect to be analyzed, and collects the total flavones part;
(4) filter: filter, merge filtrate twice, get the total flavones crude extract;
(5) phase-splitting: take by weighing certain grams total flavones crude extract, select the HSCCC solvent systems, in proportion all kinds of SOLVENTS is added in the separating funnel, concussion fully mixes solution, and placement is spent the night, and tells upper and lower phase after minute balancing each other;
(6) the total flavones crude extract is dissolved in a certain amount of lower phase solution, concussion makes fully dissolving, and is for subsequent use;
(7) HSCCC separates: with certain flow rate the upper phase (stationary phase) of solvent is pumped into the HSCCC spiral tube with lower mutually (moving phase) with certain proportion, the adjustment rotating speed is 800r/min, flow pump with 2-3ml/min enters moving phase, and UV-detector detects effluent;
(8) collect each chromatographic component, with HSCCC separate to flow point volatilize, get content and be the total flavones more than 90%.
Embodiment:
Embodiment 1: a kind of employing Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, its step is as follows:
(1) enzymolysis: the dry herb of 500g walking fern is crushed to 20 orders, enzyme concn 0.2g/l, 45 ℃ of hydrolysis temperatures, enzymolysis time 2h;
(2) refluxing extraction: add 60% ethanol, adopt the heating and refluxing extraction method, solid-liquid ratio 1: 12 extracts 80 ℃ of temperature, extracts twice, each extraction time 1.5h;
(3) collect: Recycled ethanol, thin-layer chromatography detect to be analyzed, and collects the total flavones part;
(4) filter: filter, merge filtrate twice, get the total flavones crude extract;
(5) phase-splitting: take by weighing 1g total flavones crude extract, select HSCCC normal hexane-vinyl acetic monomer-methanol-water solvent systems to add in the separating funnel by 30: 70 ratios, concussion fully mixes solution, and placement is spent the night, and divides and tells upper and lower phase after balancing each other;
(6) get in the lower phase solution that 0.5g total flavones crude extract is dissolved in 50ml, concussion makes fully dissolving, and is for subsequent use;
(7) HSCCC separates: with the 10ml/min flow velocity the upper phase (stationary phase) of solvent is pumped into the HSCCC spiral tube with lower mutually (moving phase) with certain proportion, the adjustment rotating speed is 800r/min, flow pump with 2ml/min enters moving phase, and UV-detector detects effluent;
(8) collect each chromatographic component, with HSCCC separate to flow point volatilize, HPLC analyzes, and gets content and be 93.6% total flavones.
Case study on implementation 2: a kind of employing Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, its step is as follows:
(1) enzymolysis: the dry herb of 500g walking fern is crushed to 40 orders, enzyme concn 0.3g/l, 50 ℃ of hydrolysis temperatures, enzymolysis time 2.5h;
(2) refluxing extraction: add 70% ethanol, adopt the heating and refluxing extraction method, solid-liquid ratio 1: 14 extracts 90 ℃ of temperature, extracts twice, each extraction time 1h;
(3) collect: Recycled ethanol, thin-layer chromatography detect to be analyzed, and collects the total flavones part;
(4) filter: filter, merge filtrate twice, get the total flavones crude extract;
(5) phase-splitting: take by weighing 1g total flavones crude extract, select HSCCC chloroform-methanol-water-acetate solvate system to add in the separating funnel by 40: 60 ratios, concussion fully mixes solution, and placement is spent the night, and divides and tells upper and lower phase after balancing each other;
(6) get 0.5 gram total flavones crude extract and be dissolved in the lower phase solution of 50ml, concussion makes fully dissolving, and is for subsequent use;
(7) HSCCC separates: with the 15ml/min flow velocity the upper phase (stationary phase) of solvent is pumped into the HSCCC spiral tube with lower mutually (moving phase) with certain proportion, the adjustment rotating speed is 800r/min, flow pump with 3ml/min enters moving phase, and UV-detector detects effluent;
(8) collect each chromatographic component, with HSCCC separate to flow point volatilize, HPLC analyzes, and gets content and be 95.1% total flavones.
Case study on implementation 3: a kind of employing Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, its step is as follows:
(1) enzymolysis: the dry herb of 500g walking fern is crushed to 40 orders, enzyme concn 0.2mg/ml, 45 ℃ of hydrolysis temperatures, enzymolysis time 3h;
(2) refluxing extraction: add 70% ethanol, adopt the heating and refluxing extraction method, solid-liquid ratio 1: 15 extracts 90 ℃ of temperature, extracts twice, each extraction time 1.5h;
(3) collect: Recycled ethanol, thin-layer chromatography detect to be analyzed, and collects the total flavones part;
(4) filter: filter, merge filtrate twice, get the total flavones crude extract;
(5) phase-splitting: take by weighing 1g total flavones crude extract, select HSCCC normal hexane-vinyl acetic monomer-propyl carbinol-methanol solvate system to add in the separating funnel by 20: 80 ratios, concussion fully mixes solution, and placement is spent the night, and divides and tells upper and lower phase after balancing each other;
(6) get 0.5 gram total flavones crude extract and be dissolved in the lower phase solution of 50ml, concussion makes fully dissolving, and is for subsequent use;
(7) HSCCC separates: with the 20ml/min flow velocity the upper phase (stationary phase) of solvent is pumped into the HSCCC spiral tube with lower mutually (moving phase) with certain proportion, the adjustment rotating speed is 800r/min, flow pump with 2ml/min enters moving phase, and UV-detector detects effluent;
(8) collect each chromatographic component, with HSCCC separate to flow point volatilize, HPLC analyzes, and gets content and be 95.3% total flavones.
Claims (5)
- One kind adopt Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, its step is as follows:(1) enzymolysis: the dry herb of walking fern is crushed to the 20-40 order, enzyme concn 0.2-0.3mg/ml, hydrolysis temperature 45-50 ℃, enzymolysis time 2-3h;(2) refluxing extraction: add 60-70% ethanol, adopt the heating and refluxing extraction method, solid-liquid ratio 1: 12-15 extracts 8090 ℃ of temperature, extracts twice, each extraction time 1-1.5h;(3) collect: Recycled ethanol, thin-layer chromatography detect to be analyzed, and collects the total flavones part;(4) filter: filter, merge filtrate twice, get the total flavones crude extract;(5) phase-splitting: take by weighing certain grams total flavones crude extract, select the HSCCC solvent systems respectively all kinds of SOLVENTS to be added in the separating funnel in its ratio, concussion fully mixes solution, and placement is spent the night, and tells upper and lower phase after minute balancing each other;(6) get an amount of total flavones crude extract and be dissolved in a certain amount of lower phase solution, concussion makes fully dissolving, and is for subsequent use;(7) HSCCC separates: with certain flow rate the upper phase (stationary phase) of solvent is pumped into the HSCCC spiral tube with lower mutually (moving phase) with certain proportion, the adjustment rotating speed is 800r/min, flow pump with 2-3ml/min enters moving phase, and UV-detector detects effluent;(8) collect each chromatographic component, with HSCCC separate to flow point volatilize, get content and be the total flavones more than 90%.
- According to claim 1 utilize Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, it is characterized in that: in described step (1), the enzyme of employing is cellulase.
- 3. according to claim 2ly utilize Enzymatic Extraction and join with the high speed adverse current chromatogram lotus root, finally obtain the method for high purity walking fern total flavones, it is characterized in that: in described step (5), the HSCCC solvent systems is a kind of of normal hexane-vinyl acetic monomer-methanol-water or chloroform-methanol-water-acetic acid or normal hexane-vinyl acetic monomer-propyl carbinol-methyl alcohol.
- According to claim 3 utilize Enzymatic Extraction and with high speed adverse current chromatogram lotus root connection, finally obtain the method for high purity walking fern total flavones, it is characterized in that: in described step (6), the total flavones crude extract with under the liquor ratio that mixes be 1: 10.
- 5. according to claim 4ly utilize Enzymatic Extraction and join with the high speed adverse current chromatogram lotus root, finally obtain the method for high purity walking fern total flavones, it is characterized in that: in described step (7), the flow velocity of employing 10-20ml/min pumps into the HSCCC spiral tube with the upper and lower phase of solvent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105613595A (en) * | 2015-12-22 | 2016-06-01 | 新乡医学院第一附属医院 | Cleaning disinfectant for CT equipment and preparation method and application thereof |
Citations (2)
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| CN101538296A (en) * | 2008-03-17 | 2009-09-23 | 沈阳药科大学 | Active ingredients of camptosorus sibiricus, and extraction method and use of same |
| EP2428258A1 (en) * | 2010-05-31 | 2012-03-14 | China Agricultural University | Process for extracting plant-derived natural products with polarity or intermediate polarity |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538296A (en) * | 2008-03-17 | 2009-09-23 | 沈阳药科大学 | Active ingredients of camptosorus sibiricus, and extraction method and use of same |
| EP2428258A1 (en) * | 2010-05-31 | 2012-03-14 | China Agricultural University | Process for extracting plant-derived natural products with polarity or intermediate polarity |
Non-Patent Citations (6)
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| 徐绥绪 等: "过山蕨中黄酮类新成分的研究", 《沈阳药学院学报》 * |
| 曹学丽: "《高速逆流色谱分离技术及应用》", 31 March 2005, 化学工业出版社 * |
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| 李宁: "过山蕨和山茶种子的化学成分和药理活性研究", 《沈阳药科大学博士学位论文》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105613595A (en) * | 2015-12-22 | 2016-06-01 | 新乡医学院第一附属医院 | Cleaning disinfectant for CT equipment and preparation method and application thereof |
| CN105613595B (en) * | 2015-12-22 | 2018-03-09 | 新乡医学院第一附属医院 | A kind of cleaning disinfection liquid for CT equipment and its preparation method and application |
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Address after: 165000 Heilongjiang Province, Jiagedaqi Xinyuan District Changhong community road Beilin Gebe AGA group Applicant after: DAXINGANLING LINGOBERRY BOREAL BIOTECH Co.,Ltd. Address before: 165000 Heilongjiang Province, Jiagedaqi Xinyuan District Changhong community road Beilin Gebe AGA group Applicant before: Daxinganling Lingonberry Organic Foodstuffs Co.,Ltd. |
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Free format text: CORRECT: APPLICANT; FROM: GREAT XINGAN MOUNTAINS LINGEBEI ORGANIC FOOD CO., LTD. TO: GREATER KHINGAN MOUNTAIN LINGONBERRY BIOTECHNOLOGY CO., LTD. |
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