CN104892687A

CN104892687A - Method for separating and purifying monomeric compound from Chinese mahonia leaves through high-speed counter-current chromatography

Info

Publication number: CN104892687A
Application number: CN201510316229.0A
Authority: CN
Inventors: 胡卫成; 王新风; 沈婷; 吴磊
Original assignee: Huaiyin Normal University
Current assignee: Huaiyin Normal University
Priority date: 2015-06-11
Filing date: 2015-06-11
Publication date: 2015-09-09
Anticipated expiration: 2035-06-11
Also published as: CN104892687B

Abstract

The invention discloses a method for separating and purifying monomeric compound from Chinese mahonia leaves through high-speed counter-current chromatography. The method comprises the following steps: drying Chinese mahonia leaves in the shade, smashing the dried dry Chinese mahonia leaves, adding an ethanol solution, carrying out supersonic extraction and filtering, conducting vacuum concentration on the obtained filtrate to obtain a Chinese mahonia leaf extractive, extracting the extractive with solvents of different polarities, separating the ethyl acetate extraction section with a high-speed counter-current chromatographic instrument, collecting corresponding peak components according to a chromatogram, and carrying out vacuum concentration and drying to obtain high-purity caffeotannic acid, quercetin-3-O-beta-D-glucoside, and isorhamnetin-3-O-beta-D-glucoside. The method has the advantages that the defects of complex operation, dead adsorption loss and low yield of the conventional preparation method are overcome; the efficiency is high; the operation is simple; the preparation quantity is large; the comprehensive cost is low; the popularization and use values are high.

Description

The method of monomeric compound in high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia

Technical field

The invention belongs to field of natural medicinal chemistry, be specifically related to a kind of method of fast separating and purifying chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside from Leaf of Chinese Holly Leaf of Leatherleaf Mahonia.

Background technology

Mahonia fortunei leaf system little Ju section plant mahonia bealei ( mahonia bealei(Fort.) Carr.) dry leave, there is clearing heat and detoxicating, anti-inflammatory analgetic, eliminating dampness purging intense heat, the effect of clearing liver and improving vision.On the south the Qinling Mountains, each province is also widely used as Leaf of Chinese Holly.Among the people extensively with it as curing mainly the diseases such as dizziness and tinnitus, soreness of the waist and knees, damp-heat dysentery, toothache due to stomach-fire, icterohepatitis.Modern pharmacological research finds that Mahonia fortunei has antibacterial, anti-inflammatory, antiviral, antitumor, antiproliferative and hypotensive isoreactivity, is a kind of medicinal plant having Development volue.

Focus mostly on its alkaloid to the research of Mahonia fortunei chemical composition, now do not have bibliographical information from Leaf of Leatherleaf Mahonia, isolate chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside compound, the method for existing bibliographical information separation and purification chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside etc. from other herbal medicine often adopts column chromatography method.[the liposoluble ingredient research in Lonicera confusa DC. such as Chai Xingyun, China's natural drug, 6th phase in 2004] use 95 %, 90 %, 85 %, 70 % alcohol refluxs, extraction into ethyl acetate and silicagel column, the method separation and purification that SephadexLH – 20 column purification is separated, identifies its structure according to the physicochemical characteristics of compound and spectroscopic data, from Lonicera confusa DC., is separated to the compounds such as chlorogenic acid.Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside are for being distributed in most of higher plant plant, Wan Chunpeng [east meat fringe straw colour ketone chemical constitution study, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2nd phase in 2009] extract with 70% ethanol room temperature, vinyl acetic monomer solvent extraction and macroporous adsorbent resin, SephadexLH-20, ODS and normal phase silicagel column isochromatic spectrum means are separated, and isolate Quercetin-3-O-β-D-Glucose glycosides.[the lotus leaf chemical constitution study such as Zhao little Liang, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 5th phase in 2013] utilize the separation means such as silicagel column, macroporous adsorptive resins, Sephadex LH-20 post and preparative thin layer chromatography, to be separated from lotus leaf n-butanol fraction with spectroscopic analysis through physicochemical constant and to obtain isorhamnetin-3-O-β-D-glycopranoside.

These compositions adopt column chromatography repeatedly usually, complicated operation, length consuming time, and solvent-oil ratio is large, and sample loss is large, and repeating effect is not ideal enough.

High speed adverse current chromatogram (High-Speed Counter-Current Chromatography, HSCCC) being the liquid-liquid distribution chromatography of a kind of novel, continuous high-efficient of the invention such as Ito, is be based upon a kind of special kinetic balance phenomenon---a kind of separation method on one-way fluid dynamic equilibrium (HDES) system.Compared with conventional solid liquid chromatography separating method, owing to adopting liquid as stationary phase carrier, target compound is separated due to the difference of partition ratio in mutual exclusive two-phase, solve conventional solid support samples extremely to adsorb, sex change, the problems such as peak shape hangover, velocity of separation is not only made greatly to improve with high-speed countercurrent chromatography, and can good repeated collection sample according to uv-absorbing color atlas, there is preparation efficiency high, preparation amount is large, the advantages such as expense is low, thus the concern of people is day by day received in recent years, be widely used in biological medicine, natural product, environmental analysis, the fields such as foods and cosmetics, a kind of effective new separation technology has been considered to especially in natural product industry.

Summary of the invention

The object of the invention is to: overcome the deficiencies in the prior art, a kind of method of easy and simple to handle, comprehensive cost is low, fractional dose is large, product purity is high, sample loss is little efficiently quick separation and purification chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside from Leaf of Leatherleaf Mahonia is provided.

Technical solution of the present invention is: from Leaf of Leatherleaf Mahonia, the method for separation and purification chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside comprises following processing step:

(1) preparation of Mahonia fortunei leaf extract: Leaf of Leatherleaf Mahonia dries in the shade, pulverizing, straight alcohol is that solvent supersonic extracts, and solid-to-liquid ratio is 1: 20, extraction time 2 h, filters, filter residue re-treatment 2 times; Merging filtrate vacuum-concentrcted obtains Mahonia fortunei Leave extract;

(2) crude extract water in step 1 is broken up, use normal hexane respectively, methylene dichloride, ethyl acetate, n-butanol extraction, extraction liquid filters, concentrating under reduced pressure obtains normal hexane, methylene dichloride, ethyl acetate, propyl carbinol and water extraction section respectively, and 4 ° of C Refrigerator stores are for subsequent use;

(3) separation of monomer: adopt normal hexane-Jia Chun – ethyl acetate water to be solvent system, upper is stationary phase mutually, lower is moving phase mutually, separator column rotating speed is 700-900 rpm, HSCCC separation is carried out to Leaf of Leatherleaf Mahonia extraction into ethyl acetate section, UV-detector on-line monitoring, collects different fractions respectively and drying under reduced pressure, obtains chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside.

The volume ratio of the normal hexane-methanol-ethyl acetate-water described in step (3) is 1:1:1:1-1:8:1:8, preferred 1:5:1:5.

Flow rate of mobile phase described in step (3) is 2 mL/min.

Advantage of the present invention is: after Mahonia fortunei leaf extract is separated, each cut can reach more than 85% through HPLC detection purity, each cut purify further can obtain more than 95% sterling chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside, the method is applicable to single step purification from Leaf of Leatherleaf Mahonia and prepares highly purified chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside.Good stability, easy and simple to handle.

Accompanying drawing explanation

Fig. 1 is the color atlas of high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia;

Fig. 2 is the high-efficient liquid phase chromatogram of the chlorogenic acid that separation and purification obtains;

Fig. 3 is the high-efficient liquid phase chromatogram of Quercetin-3-O-β-D-Glucose glycosides that separation and purification obtains;

Fig. 4 is the high-efficient liquid phase chromatogram of the isorhamnetin-3-O-β-D-glycopranoside that separation and purification obtains.

Embodiment

Take from Leaf of Leatherleaf Mahonia 5.0 kg so dried in the shade, pulverize, straight alcohol is that solvent supersonic extracts, and solid-to-liquid ratio is 1: 20, extraction time 2 h, filters, filter residue re-treatment 2 times; Merging filtrate vacuum-concentrcted obtains Mahonia fortunei Leave extract; Crude extract water is broken up, and uses normal hexane respectively, methylene dichloride, ethyl acetate, n-butanol extraction, and extraction liquid filters, and concentrating under reduced pressure obtains normal hexane, methylene dichloride, ethyl acetate, propyl carbinol and water extraction section respectively, and 4 ° of C Refrigerator stores are for subsequent use.Normal hexane-Jia Chun – ethyl acetate the water adopting volume ratio 1:5:1:5 is solvent system, moving phase (lower phase) flow velocity is 2 mL/min, separator column rotating speed is 900 rpm, and upper is stationary phase mutually, carries out HSCCC separation to Leaf of Leatherleaf Mahonia extraction into ethyl acetate section, UV-detector on-line monitoring, determined wavelength is 254 nm, collects target component, and cut is dry through concentrating under reduced pressure, obtain corresponding high-purity compound, as shown in Figure 1.

Identify that chemical structure is as follows through 1H-NMR, 13C-NMR,

Namely gained monomeric compound is respectively chlorogenic acid 18.32mg, Quercetin-3-O-β-D-Glucose glycosides 20.46 mg, isorhamnetin-3-O-β-D-glycopranoside 28.36 mg, calculate with chromatographic peak area normalization method, purity all higher than 97%, as shown in figs 2-4.

Claims

1. the method for monomeric compound in high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia, is characterized in that, comprise following processing step:

(2) crude extract water in step (1) is broken up, use normal hexane respectively, methylene dichloride, ethyl acetate, n-butanol extraction, extraction liquid filters, concentrating under reduced pressure obtains normal hexane, methylene dichloride, ethyl acetate, propyl carbinol and water extraction section respectively, and 4 ° of C Refrigerator stores are for subsequent use;

(3) separation of monomer: adopt normal hexane-methanol-ethyl acetate-water to be solvent system, upper is stationary phase mutually, lower is moving phase mutually, separator column rotating speed is 700-900 rpm, HSCCC separation is carried out to Leaf of Leatherleaf Mahonia extraction into ethyl acetate section, UV-detector on-line monitoring, collects different fractions respectively and drying under reduced pressure, obtains chlorogenic acid, Quercetin-3-O-β-D-Glucose glycosides, isorhamnetin-3-O-β-D-glycopranoside.

2. the method for monomeric compound in high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia according to claim 1, it is characterized in that, the volume ratio of the normal hexane-methanol-ethyl acetate-water described in step (3) is 1:1:1:1-1:8:1:8.

3. the method for monomeric compound in high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia according to claim 2, it is characterized in that, the volume ratio of the normal hexane-methanol-ethyl acetate-water described in step (3) is 1:5:1:5.

4. in the high speed adverse current chromatogram separation and purification Leaf of Leatherleaf Mahonia according to any one of claim 1-3, the method for monomeric compound, is characterized in that, the flow rate of mobile phase described in step (3) is 2 mL/min.