CN104892687B - The method that high speed adverse current chromatogram isolates and purifies monomeric compound in Chinese mahonia leaf - Google Patents
The method that high speed adverse current chromatogram isolates and purifies monomeric compound in Chinese mahonia leaf Download PDFInfo
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- CN104892687B CN104892687B CN201510316229.0A CN201510316229A CN104892687B CN 104892687 B CN104892687 B CN 104892687B CN 201510316229 A CN201510316229 A CN 201510316229A CN 104892687 B CN104892687 B CN 104892687B
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- chinese mahonia
- ethyl acetate
- mahonia leaf
- chlorogenic acid
- isorhamnetin
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Abstract
The invention discloses a kind of method of the fast separating and purifying monomeric compound from Chinese mahonia leaf, comprise the following steps：The Chinese mahonia leaf for crushing of drying in the shade is taken, adds ethanol solution ultrasonic extraction, filtering, filtrate decompression is concentrated to give Chinese mahonia leaf extract；Extract is after opposed polarity solvent extraction, ethyl acetate extraction section is separated with high-speed counter-current chromatograph, respective peaks component is collected according to chromatogram, be concentrated under reduced pressure drying, obtain high-purity chlorogenic acid, the O β D glucosides of Quercetin 3, the O β D glucosides of Isorhamnetin 3, overcome traditional preparation methods complex operation, the dead adsorption loss of sample, the shortcomings such as yield is low.This method efficiency high, simple to operate, preparation amount is big, and integrated cost is low, has good value for applications.
Description
Technical field
The invention belongs to field of natural medicinal chemistry, and in particular to one kind quick separating from Ilex Latifolia Thunb Chinese mahonia leaf is pure
Change chlorogenic acid, Quercetin -3-O- β-D-Glucose glycosides, the method for isorhamnetin-3-O-β-D-glycopranoside.
Background technology
Chinese mahonia leaf system little Ju sections plant mahonia bealei (Mahonia bealei(Fort.) Carr.) it is dry
Dry leaf, there is the effect of clearing heat and detoxicating, anti-inflammatory analgetic, eliminating dampness purging intense heat, clear liver and improve vision.Each province is also widely used on the south the Qinling Mountains
For Ilex Latifolia Thunb.It is among the people extensively by the use of it as curing mainly dizziness and tinnitus, soreness and weakness of waist and knees, damp-heat dysentery, gastropyretic toothache, icterepatitis etc.
Disease.Modern pharmacological research finds that Chinese mahonia has antibacterial, anti-inflammatory, antiviral, antitumor, antiproliferative and hypotensive etc. living
Property, it is a kind of medicinal plant for having Development volue.
Research to Chinese mahonia chemical composition focuses mostly on its alkaloid, does not have document report now from Chinese mahonia leaf
In isolate chlorogenic acid, Quercetin -3-O- β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside compound, existing text
Offer report and chlorogenic acid, Quercetin -3-O- β-D-Glucose glycosides, Isorhamnetin -3-O- β-D- are isolated and purified from other Chinese herbal medicines
The method of glucoside etc. is frequently with column chromatography method.Chai Xingyun etc. [study in Honeysuckle flower, Chinese crude drug by liposoluble ingredient
Thing, the 6th phase in 2004] with the % alcohol refluxs of 95 %, 90 %, 85 %, 70, ethyl acetate extraction and silicagel column,
The method of SephadexLH -20 post purifies and separates is isolated and purified, and its knot is identified according to the physicochemical characteristicses of compound and spectroscopic data
Structure, the compounds such as chlorogenic acid are separated to from Honeysuckle flower.Quercetin -3-O- β-D-Glucose glycosides, Isorhamnetin -3-O- β-D- Portugals
Polyglycoside is is distributed in most of higher plant plant, Wan Chunpeng [east meat fringe straw colour ketone chemical constitution studies, in Chinese
Medicine magazine, the 2nd phase in 2009] extracted with 70% ethanol room temperature, ethyl acetate solvent extraction and macroporous absorbent resin,
The chromatogram means such as SephadexLH-20, ODS and normal phase silicagel column are separated, and isolate Quercetin -3-O- β-D-Glucose
Glycosides.Zhao little Liang etc. [lotus leaf chemical constitution study, CHINA JOURNAL OF CHINESE MATERIA MEDICA, the 5th phase in 2013] utilizes silicagel column, macroporous absorption tree
The separation means such as fat post, Sephadex LH-20 posts and preparative thin layer chromatography, through physicochemical constant and spectrum analysis from lotus leaf just
Isolated isorhamnetin-3-O-β-D-glycopranoside in butanol fraction.
These column chromatographies of composition generally use repeatedly, complex operation, time-consuming, and solvent-oil ratio is big, and sample damages
Lose greatly, repeating effect is not ideal enough.
High speed adverse current chromatogram（High-Speed Counter-Current Chromatography, HSCCC）It is Ito etc.
A kind of new, continuous high-efficient liquid-liquid partition chromatography of invention, is built upon a kind of special kinetic balance and shows
As --- one-way fluid dynamic equilibrium（HDES）A kind of separation method on system.With conventional solid liquid chromatography separating method
Compare, due to using liquid as stationary phase carrier, target compound mutual exclusive two-phase due to distribution coefficient not
Separated together, solve the problems such as conventional solid support samples are extremely adsorbed, are denatured, peak shape is trailed, use high speed adverse current chromatogram
Method not only greatly improves separating rate, and according to UV absorption chromatogram can good repeated collection sample, there is preparation efficiency
The high, advantage such as preparation amount is big, expense is low, thus be increasingly subject to the concern of people in recent years, have been widely used for biological medicine,
The fields such as natural products, environmental analysis, foods and cosmetics, especially had been considered as in natural products industry a kind of effective
New separation technology.
The content of the invention
It is an object of the invention to：Overcome the deficiencies in the prior art, there is provided one kind is easy to operate, integrated cost is low, separation
Amount is big, product purity is high, sample loss is small efficient quickly isolates and purifies chlorogenic acid, Quercetin -3-O- from Chinese mahonia leaf
β-D-Glucose glycosides, the method for isorhamnetin-3-O-β-D-glycopranoside.
The present invention technical solution be：Chlorogenic acid, Quercetin -3-O- β-D- Portugals are isolated and purified from Chinese mahonia leaf
Polyglycoside, the method for isorhamnetin-3-O-β-D-glycopranoside comprise the following steps that：
(1) preparation of Chinese mahonia leaf extract：Chinese mahonia leaf dries in the shade, crushed, and straight alcohol extracts for solvent supersonic,
Solid-to-liquid ratio is 1：20, the h of extraction time 2, filtering, filter residue reprocess 2 times；Simultaneously vacuum-concentrcted obtains merging filtrate
Chinese mahonia Leave extract；
(2) crude extract in step 1 is broken up with water, respectively with n-hexane, dichloromethane, ethyl acetate, n-butanol extraction
Take, extract filtering, be concentrated under reduced pressure and respectively obtain n-hexane, dichloromethane, ethyl acetate, n-butanol and water extraction section, 4 °C of ice
Case saves backup；
(3) separation of monomer：N-hexane-methanol-ethyl acetate water is used as dicyandiamide solution, upper phase is stationary phase, under
It is mutually mobile phase, splitter rotating speed is 700-900 rpm, and HSCCC points are carried out to Chinese mahonia leaves ethyl acetate extraction section
From UV-detector is monitored on-line, is collected different fractions respectively and is dried under reduced pressure, obtains chlorogenic acid, Quercetin -3-O- β-D- grapes
Glucosides, isorhamnetin-3-O-β-D-glycopranoside.
Step（3）The volume ratio of described n-hexane-methanol-ethyl acetate-water is 1:1:1:1-1:8:1:8, preferably 1:
5:1:5。
Step（3）Described flow rate of mobile phase is 2 mL/min.
It is an advantage of the invention that：Chinese mahonia leaf extract separation after each cut through HPLC detect purity up to 85% with
On, each cut further purifies available more than 95% sterling chlorogenic acid, Quercetin -3-O- β-D-Glucose glycosides, different sandlwood
Element -3-O- β-D-Glucose glycosides, this method are applied to chlorogenic acid, the quercitrin for walking purifying from Chinese mahonia Ye Zhongyi and preparing high-purity
Element -3-O- β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside.Stability is good, easy to operate.
Brief description of the drawings
Fig. 1 is the chromatogram that high speed adverse current chromatogram isolates and purifies Chinese mahonia leaf；
Fig. 2 is the high-efficient liquid phase chromatogram of chlorogenic acid for isolating and purifying to obtain；
Fig. 3 is the high-efficient liquid phase chromatogram of Quercetin -3-O- β-D-Glucose glycosides for isolating and purifying to obtain；
Fig. 4 is the high-efficient liquid phase chromatogram of isorhamnetin-3-O-β-D-glycopranoside for isolating and purifying to obtain.
Embodiment
The kg of Chinese mahonia leaf 5.0 so to dry in the shade is derived from, is crushed, straight alcohol extracts for solvent supersonic, solid-to-liquid ratio 1：20
, the h of extraction time 2, filtering, filter residue reprocessing 2 times；Merging filtrate and vacuum-concentrcted obtain Chinese mahonia leaf and slightly carried
Thing；Crude extract is broken up with water, dense with n-hexane, dichloromethane, ethyl acetate, extracting n-butyl alcohol, extract filtering, decompression respectively
Contracting respectively obtains n-hexane, dichloromethane, ethyl acetate, n-butanol and extraction section, and 4 °C of refrigerators save backup.Using volume
Than 1:5:1:5 n-hexane-methanol-ethyl acetate water is dicyandiamide solution, mobile phase（Lower phase）Flow velocity is 2 mL/min,
Splitter rotating speed is 900 rpm, and upper phase is stationary phase, and HSCCC separation is carried out to Chinese mahonia leaves ethyl acetate extraction section,
UV-detector is monitored on-line, and Detection wavelength is 254 nm, collects target component, and cut obtains phase through the drying that is concentrated under reduced pressure
High-purity compound is answered, as shown in Figure 1.
It is as follows through 1H-NMR, 13C-NMR identification chemical constitution,
I.e. gained monomeric compound be respectively chlorogenic acid 18.32mg, Quercetin -3-O- β-mg of D-Glucose glycosides 20.46,
The mg of isorhamnetin-3-O-β-D-glycopranoside 28.36, calculated with chromatographic peak area normalization method, purity is above 97%, such as schemes
Shown in 2- Fig. 4.
Claims (4)
1. high speed adverse current chromatogram isolates and purifies Chinese mahonia leaf Content of Chlorogenic Acid, Quercetin -3-O- β-D-Glucose glycosides, different sandlwood
The method of element -3-O- β-D-Glucose glycosides, it is characterised in that comprise the following steps that：
(1) preparation of Chinese mahonia leaf extract：Chinese mahonia leaf dries in the shade, crushed, and straight alcohol extracts for solvent supersonic, solid-liquid
Than for 1：20, the h of extraction time 2, filtering, filter residue reprocess 2 times；Simultaneously vacuum-concentrcted obtains Chinese mahonia to merging filtrate
Leave extract；
(2) by step（1）Middle crude extract is broken up with water, respectively with n-hexane, dichloromethane, and ethyl acetate, extracting n-butyl alcohol,
Extract filters, and is concentrated under reduced pressure and respectively obtains n-hexane, dichloromethane, ethyl acetate, n-butanol and water extraction section, 4 °C of refrigerators
Save backup；
(3) separation of monomer：N-hexane-methanol-ethyl acetate-water is used as dicyandiamide solution, upper phase is stationary phase, and lower phase is
Mobile phase, splitter rotating speed are 700-900 rpm, and HSCCC separation is carried out to Chinese mahonia leaves ethyl acetate extraction section, purple
External detector is monitored on-line, collects different fractions and be dried under reduced pressure respectively, obtain chlorogenic acid, Quercetin -3-O- β-D-Glucose glycosides,
Isorhamnetin-3-O-β-D-glycopranoside.
2. high speed adverse current chromatogram according to claim 1 isolate and purify Chinese mahonia leaf Content of Chlorogenic Acid, Quercetin -3-O- β -
The method of D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside, it is characterised in that step（3）Described n-hexane-first
The volume ratio of alcohol-ethyl acetate-water is 1:1:1:1- 1:8:1: 8.
3. high speed adverse current chromatogram according to claim 2 isolate and purify Chinese mahonia leaf Content of Chlorogenic Acid, Quercetin -3-O- β -
The method of D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside, it is characterised in that step（3）Described n-hexane-first
The volume ratio of alcohol-ethyl acetate-water is 1:5:1:5.
4. the high speed adverse current chromatogram according to claim any one of 1-3 isolates and purifies Chinese mahonia leaf Content of Chlorogenic Acid, quercitrin
Element -3-O- β-D-Glucose glycosides, the method for isorhamnetin-3-O-β-D-glycopranoside, it is characterised in that step（3）Described
Flow rate of mobile phase is 2 mL/min.
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| CN105732611B (en) * | 2016-03-09 | 2017-05-03 | 山东省分析测试中心 | Method for separating alkaloids compound in caulis mahoniae by high-speed countercurrent chromatography |
| CN105994386B (en) * | 2016-06-02 | 2018-09-07 | 湖南省农业生物资源利用研究所 | A kind of mahonia bealei extract and preparation method thereof and the application in resisting tobacco black shank |
| CN107903176B (en) * | 2017-11-07 | 2021-02-05 | 中国科学院西北高原生物研究所 | Preparation method of three chemical reference substances in dracocephalum tanguticum |
| CN109293509B (en) * | 2018-11-30 | 2021-08-03 | 浙江科技学院 | Method for preparing high-purity chlorogenic acid from bamboo leaf extract |
| CN109776474A (en) * | 2019-03-27 | 2019-05-21 | 淮阴师范学院 | A method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum |
| CN110613739A (en) * | 2019-07-31 | 2019-12-27 | 湖州耕香生物科技有限公司 | Method for separating flavonoid compounds in cotton rose based on high-speed countercurrent chromatography |
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| KR20100022883A (en) * | 2008-08-20 | 2010-03-03 | 서울대학교산학협력단 | A method for isolating and producing the saponin- rich fractions and further highly purified platycodin d from the root extract of platycodon grandiflorum using by high-speed count-current chromatography |
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| CN103641867A (en) * | 2013-12-05 | 2014-03-19 | 湖南农业大学 | High-speed counter-current chromatography separation method and application of phenolic acid glycoside compounds in traditional Chinese medicine lily |
| CN104031013A (en) * | 2014-06-17 | 2014-09-10 | 浙江工业大学 | Method for preparing salvianolic acid B and rosmarinic acid by adopting high-speed counter-current chromatography separation and purification process |
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