CN103787885B - A kind of method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts - Google Patents
A kind of method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts Download PDFInfo
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- CN103787885B CN103787885B CN201410079889.7A CN201410079889A CN103787885B CN 103787885 B CN103787885 B CN 103787885B CN 201410079889 A CN201410079889 A CN 201410079889A CN 103787885 B CN103787885 B CN 103787885B
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
The invention discloses a kind of method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts belonging to active ingredient of Chinese herbs extractive technique field.The method is with Flos Lonicerae position on the ground as raw material, high-purity chlorogenic acid is obtained: the first step by following steps, by the 60 80% ethanol solution reflux, extract, twice of pH2 5, or with the sour water of pH2 5 adding cellulase and acid protease extracts twice at 40 60 DEG C；Second step, concentrates by a certain percentage by first step gained extracting solution, and room temperature places 4 12h, and sucking filtration removes the precipitation separated out；3rd step, is concentrated into above filtrate without alcohol taste, adds sour water dilution；It is further purified by macroporous adsorbent resin, purity chlorogenic acid more than 90% can be obtained.The chlorogenic acid purity that the present invention obtains is high, and method is simple, environmental protection, and with low cost, is suitable for industrialized production.
Description
Technical field
The invention belongs to active ingredient of Chinese herbs extractive technique field, be specifically related to a kind of from Flos Lonicerae aerial parts
The method extracting high-purity chlorogenic acid.
Background technology
Flos Lonicerae, formal name used at school Radix Ophiopogonis (Lonicera japonica Thurb), for Caprifoliaceae woodbine, has
The functions such as heat-clearing and toxic substances removing, cool breeze heat radiation, antiviral, hepatic cholagogic, are one of traditional Chinese crude drugs of China.
In Flos Lonicerae, main chemical compositions is chlorogenic acid, isochlorogenic acid, chromocor compound, linalool, Flos Lonicerae alcohol etc..
Recent studies it is generally acknowledged that the biological activity effective ingredient of Flos Lonicerae is chlorogenic acid compound, and often with green
The height of Determination of Chlorogenic Acid evaluates the quality of quality of Flos Lonicerae.
Chlorogenic acid is containing carboxyl and the organic acid of adjacent two phenolic hydroxyl groups, in soluble in water, alcohol, acetone equal solvent,
Molecular weight is 354.3, and its hemihydrate is white or slightly yellow acicular crystal.Chlorogenic acid (Chlorogenic
Acid) it is one of main active in Flos Lonicerae, there is obvious blood pressure lowering, blood fat reducing, antitumor, anti-
The pharmacological actions such as cancer.Chlorogenic acid can not only be used for the additive in food, auxin and some are advanced
The additive etc. of cosmetic, is again the important source material of the industry such as food, medicine, cosmetics.Highly purified green former
Acid is expensive, is the most internationally recognized " plant gold ", therefore, the extraction market prospect of chlorogenic acid
Wide, development potentiality is huge.
At present, Chlorogenic Acid of Flos Lonicerae extracts mainly by solvent extraction method, uses ethanol extraction, ultrasonication
Etc. technique, and extract part is mainly derived from flower, and these extraction process energy consumptions are big, and post processing is polluted more serious.
Also having document or patent to use Enzymatic Extraction, Wang Ruihong etc. uses cellulase enzyme process to extract (document: fine
Dimension element enzyme application in Extracting Process of Honeysuckle, Wang Ruihong etc., Heilungkiang medicine, 2003,16 (4): 286-287),
The compound enzyme of both cellulase and pectase such as Ling Ningsheng carries out the (patent name: a kind of of enzymolysis and extraction
Use the method that combined-enzyme method extracts Chlorogenic Acid of Flos Lonicerae, number of patent application: 201010124495.0).Warp
Overtesting, chlorogenic acid yield and extract purity that above-mentioned Enzymatic Extraction obtains are the highest.Inventor tries respectively
Test employing (1) 60-80% glycolic acid aqueous solution, (2) cellulase, (3) cellulase and pectase two
The compound enzyme of person and (4) cellulase and four kinds of techniques of acid protease compound enzyme, carry out extracting Flos Lonicerae ground
The chlorogenic acid of upper part, the multiplex-enzyme extraction that result display cellulase and acid protease are combined into obtains
Chlorogenic acid yield and extract purity are close with 60-80% glycolic water extraction result.Its principle is probably fibre
Dimension element enzyme can destroy the structure of plant cell wall, is conducive to the effective plant component being wrapped in cell wall molten
Go out, acid protease can under the conditions of lower ph the protein in hydrating solution, be conducive to suppressing water
In extracting solution, protease is on the destruction of chlorogenic acid and the impact on purification by macroporous resin of the subsequent protein class impurity.
Summary of the invention
It is an object of the invention to provide a kind of method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts,
The method increase the purity of institute's chlorogenic acid extracting, decrease the environmental pollution during extraction, make Flos Lonicerae
The extraction of Content of Chlorogenic Acid is not limited to flower position, improves the utilization rate of Flos Lonicerae herb.
A kind of method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts, is carried out in accordance with the following steps:
(1) dry Flos Lonicerae aerial parts is pulverized, obtain Flos Lonicerae aerial parts coarse powder；
(2) the Flos Lonicerae aerial parts coarse powder obtained in step (1) is added 60-80% ethanol water,
The ratio adding 10-20L ethanol water according to 1kg coarse powder adds, reflux, extract, 1-3h, and reduce pressure sucking filtration,
Repeating this operation, merging filtrate, take 7/8～4/5 concentration of this merging filtrate cumulative volume, concentration ratio is
5: 1～9: 1, this concentrated solution merges with former non-concentrating part solution, and room temperature places 4～12h, has blackish green precipitation
Separating out, sucking filtration removes precipitation, and this filtrate is concentrated into dry powder further；
Or the Flos Lonicerae aerial parts coarse powder that will obtain in step (1) adds the sour water of pH3-5, heat up
To 40-50 DEG C, add cellulase 4000-10000u/kg coarse powder, enzymolysis 2-4h, be subsequently adding acidity
Protease 15000-25000u/kg coarse powder, enzymolysis 2-4h, enzymolysis is warming up to 70-100 DEG C of enzyme denaturing after completing
Processing 10-60min, sucking filtration, the medicinal residues after filtering, with the deionized water backflow 1-3h of 70-100 DEG C, weigh
Operation 1 time, sucking filtration, merge three filtrates, be concentrated into dry powder again；
(3) by the dry powder in step (2) with 1.0-1.5BV (BV, bed volume, resin bed volume)
0.5-1mol/L diluted hydrochloric acid aqueous solution dissolve, solution Content of Chlorogenic Acid is adjusted to 5.0-6.0mg/mL, for
Sample solution, carries out macroporous adsorbent resin column chromatography, with deionized water as eluent, deionized water elution volume
The 20-30% ethanol water eluting of 2-3BV, continuation 7-10BV, merges 20-30% ethanol elution molten
Liquid, concentrates, and concentration ratio is 6: 1-10: 1, after standing 4-12h, has white particle or powder thing to separate out,
For chlorogenic acid finished product.
In described step (1), Flos Lonicerae aerial parts is the leaf of Flos Lonicerae, rattan, flower, fruit, in
One or more combination.
In described step (3), macroporous adsorbent resin is AB-8 resin, HZ-818 resin, X-5 resin or
Diaion HP-20 resin.
Described 60-80% ethanol water 0.5-1mol/L hydrochloric acid adjusts pH2-5.
Beneficial effects of the present invention: 60-80% glycolic acid aqueous solution is extracted by the present invention or cellulase is with acid
Protease composite enzyme extracts the aqueous acid of the Flos Lonicerae overground part extract obtained, and tests employing further
The macroporous resins such as AB-8, HZ-818, X-5, Diaion HP-20, D-101, XDA-1, XDA-1b enter
Row purification, through preferably obtaining the macropore tree of tetra-kinds of models of AB-8, HZ-818, X-5, Diaion HP-20
Fat, has that remove impurity is good, chlorogenic acid eluting is concentrated, use the advantages such as organic solvent is few, resin is reusable.
The present invention compares through the test of kinds of processes, it is determined that uses the aqueous acid containing ethanol 60-80% or adopts
Carry out Flos Lonicerae aerial parts chlorogenic acid with the aqueous acid of cellulase and acid protease to extract, extracting solution
Use preferred macroporous resin to be purified further and obtain highly purified chlorogenic acid, after testing chlorogenic acid purity
Up to more than 90%, simple process, it is easy to run in factory, and with low cost.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1
The leaf of extracting honeysuckle, pulverizes, and takes coarse powder 50g, adds 1.0L70% ethanol water (0.5-1mol/L
Hydrochloric acid adjusts pH2.5), reflux, extract, 1h, filtered while hot, medicinal residues repeat to extract 1 time, sucking filtration, twice filter
Liquid merges, and obtains extracting solution 1.85L, takes this extracting solution 1.5L and be concentrated into 300mL, with unconcentrated extraction
Liquid mixes, and stands 8h, and there is obvious blackish green precipitation in solution lower floor, and reduce pressure sucking filtration, obtains filtrate further
Being concentrated into dry powder, dry powder adds deionized water 300-400mL and dissolves and use the aqueous hydrochloric acid solution of 1M to adjust pH
3 (in terms of chlorogenic acid content 5.4mg/mL), are the sample solution of macroporous adsorbent resin column chromatography.Take 500mL
AB-8 macroporous adsorbent resin, first by buy time description by AB-8 macroporous adsorbent resin, (column diameter is
5.5cm, column length is 25cm) carry out acid-base pretreatment, then with this resin column of deionized water balance, take
Stating the dynamic loading of sample solution 300mL configured, elution flow rate is 10mL/min, and sample solution adsorbs completely
After on resin column, continuing and be washed with deionized water de-800mL, 20% ethanol water eluting 2.0L, by water
Eluent (about 1L) discards, and 20% ethanol solution elution fraction (2.0L) merges, and is concentrated into 200mL,
Room temperature (25 DEG C) stands 12h, separates out precipitation, is dried after sucking filtration, and (0.5g receives to obtain chlorogenic acid extract
Rate 1.0%), inverted HPLC detects, and uses area normalization method to detect this extract Content of Chlorogenic Acid pure
Degree ＞ 90%.
Embodiment 2
Extracting honeysuckle aerial parts coarse powder 50g, adds the 70% ethanol/diluted hydrochloric acid aqueous solution (0.5-1 of 1.0L
Mol/L hydrochloric acid adjusts pH4), reflux, extract, 2h, filter, medicinal residues repeat to extract 1 time, and sucking filtration merges twice
Filtrate, obtains total extracting solution 1.8L, takes this extracting solution 1.5L and is concentrated into 200mL, with unconcentrated extracting solution
Mixing, stands 8h, sucking filtration, removes blackish green precipitation, collects filtrate, is concentrated into dry powder, dry powder further
The distilled water adding 300-400mL dissolves, and regulates this solution to pH3.5 with 0.5mol/L dilute hydrochloric acid solution,
This solution Content of Chlorogenic Acid is 4.0mg/mL, then chromatographs through X-5 macroporous adsorbent resin resin column.Take
The X-5 macroporous adsorbent resin of 500mL, first by description when buying, by X-5 macroporous adsorbent resin, (post is straight
Footpath is 5.5cm, and column length is 25cm) carry out acid-base pretreatment, then with this resin column of deionized water balance,
Taking the dynamic loading of sample solution 300mL that above-mentioned configuration is good, elution flow rate is 10mL/min, and sample solution is complete
After being adsorbed on resin column, continuing and use 600mL deionized water eluting, this elution fractions discards, and continues and uses 2L
30% ethanol water eluting, collect merge this elution fractions (2L), this eluent is concentrated into 200
ML, room temperature (15 DEG C) stands 12h, separates out precipitation, will be dried after this precipitation sucking filtration, obtains chlorogenic acid and extracts
Thing, inverted HPLC detects, and uses area normalization method to record chlorogenic acid purity ＞ 90%.
Embodiment 3
Extracting honeysuckle aerial parts coarse powder 50g, adds deionized water 500mL, is warming up to 45 DEG C, with 0.5
Mol/L regulates pH3, adds cellulase 10mg (enzyme activity 300), enzymolysis 2h.After enzymolysis completes
Adding acid protease 20mg (enzyme activity 1000), enzymolysis 2h, enzymolysis is warming up to 80 DEG C and goes out after completing
Ferment treatment 30min.Filtering residue after filtering, with the 800mL deionized water backflow 1.5h of 82 DEG C, repeats
Water extraction 1 time, sucking filtration, merge three filtrates, there are sucking filtration liquid 2.0L, pumping rate liquid is concentrated into dry powder, this
Dry powder deionized water 350mL dissolves, then with 1.0mol/L salt acid for adjusting pH value to 2.5, in this solution
Chlorogenic acid concentration is 6.0mg/mL, and this solution is the most isolated and purified through macroporous adsorbent resin.First, will
HZ-818 macroporous resin 500mL dress post (column diameter is 5.5cm, and column length is 25cm), carries according to producer
The method of confession carries out acid-base pretreatment, takes the dynamic loading of sample solution 400mL that above-mentioned configuration is good, eluting stream
Speed is 10mL/min, after sample solution is adsorbed on resin column completely, continues and uses deionized water 800mL eluting,
Above-mentioned eluent discards, and continues with 30% ethanol water 2.4L eluting and collects, being concentrated into by this elution fractions
200mL, room temperature (20 DEG C) stands 5h, separates out precipitation, sucking filtration, filter residue and drying, obtains chlorogenic acid and extracts
Thing, uses HPLC to analyze, and inverted HPLC detects, and uses area normalization method to record chlorogenic acid pure
Degree ＞ 90%.
Claims (1)
1. the method extracting high-purity chlorogenic acid from Flos Lonicerae aerial parts, it is characterised in that according to such as
Lower step is carried out:
(1) dry Flos Lonicerae aerial parts is pulverized, obtain Flos Lonicerae aerial parts coarse powder；
(2) the Flos Lonicerae aerial parts coarse powder that will obtain in step (1) adds the sour water of pH 3-5, rises
Temperature, to 40-50 DEG C, adds cellulase 4 000-10 000u/kg coarse powder, enzymolysis 2-4h, is subsequently adding acid
Property protease 15 000-25 000u/kg coarse powder, enzymolysis 2-4h, enzymolysis is warming up to 70-100 DEG C after completing and goes out
Ferment treatment 10-60min, sucking filtration, the medicinal residues deionized water of 70-100 DEG C after filtering refluxes 1-3h,
Repetitive operation 1 time, sucking filtration, merge three filtrates, be concentrated into dry powder；
(3) the 0.5-1mol/L diluted hydrochloric acid aqueous solution of the dry powder 1.0-1.5BV in step (2) is dissolved,
Solution Content of Chlorogenic Acid is adjusted to 5.0-6.0mg/mL, for sample solution, carries out macroporous adsorbent resin column chromatography,
With deionized water as eluent, deionized water elution volume 2-3BV, the 20-30% of continuation 7-10BV
Ethanol water eluting, merges 20-30% ethanol elution solution, concentrates, and concentration ratio is 6: 1-10: 1, quiet
After putting 4-12h, white particle or powder thing is had to separate out, for chlorogenic acid finished product；
In described step (3), macroporous adsorbent resin is AB-8 resin, HZ-818 resin, X-5 resin or
Diaion HP-20 resin；
In described step (1), Flos Lonicerae aerial parts is the leaf of Flos Lonicerae, rattan, one in fruit or
More than one combination.
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| CN107361204A (en) * | 2017-08-14 | 2017-11-21 | 胡玉涛 | The preparation method and animal feed of a kind of honeysuckle extract feed additive |
| CN109355343A (en) * | 2018-12-28 | 2019-02-19 | 河北肽都生物科技有限公司 | A kind of preparation method of small molecule honeysuckle chelating peptide |
| CN111533658A (en) * | 2020-04-20 | 2020-08-14 | 湖南鸿利药业股份有限公司 | Method for extracting high-purity chlorogenic acid from honeysuckle |
| CN114031498A (en) * | 2021-11-05 | 2022-02-11 | 临沂市农业科学院 | Method for extracting high-purity honeysuckle chlorogenic acid by membrane separation method |
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| CN1616403A (en) * | 2004-09-21 | 2005-05-18 | 济南永曜医药科技有限公司 | Process for preparing chlorogenic acid from honeysuckle |
| CN101985421B (en) * | 2010-10-26 | 2013-01-09 | 西北农林科技大学 | Method for simultaneously preparing chlorogenic acid and luteoloside from honeysuckle flower |
| CN102242100A (en) * | 2011-04-20 | 2011-11-16 | 威海康博尔生物药业有限公司 | Method for using compound biological enzyme in plant extraction process, and conditions thereof |
| CN102911055B (en) * | 2012-11-13 | 2014-07-09 | 西南大学 | Method for extracting chlorogenic acid |
| CN103435486A (en) * | 2013-09-12 | 2013-12-11 | 南京泽朗农业发展有限公司 | Novel method for preparing high-purity chlorogenic acid in honeysuckle |
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