CN110156601A - A method of preparing high-purity chlorogenic acid from STEVIA REBAUDIANA - Google Patents
A method of preparing high-purity chlorogenic acid from STEVIA REBAUDIANA Download PDFInfo
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Abstract
The method that the invention discloses a kind of to prepare high-purity chlorogenic acid from STEVIA REBAUDIANA belongs to the technical field of natural product chemistry.The present invention is to extract medium with water, after leaching liquor is filtered, flocculated using stevia rebaudian leaf as raw material, it is purified with polyamide or class polyamide, using water as eluent, after the concentrated crystallization of eluent, the product that the mass content of chlorogenic acid is 98% or more can be made.Meanwhile during being purified with resin to chlorogenic acid, the other compositions collected can be used for the other stevia rebaudianum polyphenol of separation and Extraction and steviol glycoside.Generally speaking, this method, which is realized, prepares high-purity chlorogenic acid by raw material of stevia rebaudian leaf, and product Content of Chlorogenic Acid mass content can reach 98% or more, and technological operation is simple, hardly uses organic solvent, Environmental Safety.
Description
Technical field
The method that the present invention relates to a kind of to prepare high-purity chlorogenic acid from STEVIA REBAUDIANA, it is especially a kind of with water-borne method from sweet tea
The method that Ye Juzhong extracts high-purity chlorogenic acid, belongs to the technical field of natural product chemistry.
Background technique
Chlorogenic acid also known as caffeotannic acid belong to one kind of chlorogenic acid substance, are by caffeic acid and chinic acid (1- hydroxyl
Hexahydro gallic acid) generate depside, be plant during aerobic respiration through shikimic acid pathway generate a kind of phenylpropyl alcohol
Chlorins compound.
Chlorogenic acid is a kind of important bioactive substance, has very big application value in fields such as medicine, health.It is green
Ortho acid has certain pharmacological action to digestion, blood and reproductive system, has wide antibacterial anti-inflammatory, cholagogue, anastalsis；
Chlorogenic acid has stronger antibiosis and antiviral functions, can effectively inhibit Staphylococcus aureus, hemolytic streptococcus, shigella dysenteriae,
Typhoid bacillus；Chlorogenic acid is a kind of effective phenolic antioxidant, can effectively remove hydroxyl radical free radical and superoxide anion, from
And protect body from the damage of oxidation；In addition, chlorogenic acid also has certain protective effect to angiocarpy, have apparent
Antihypertensive effect.Due to various effects and physiological activity of chlorogenic acid, extraction and purifying to chlorogenic acid also increasingly cause people
Concern.
Chlorogenic acid is widely present in plant, be mainly derived from honeysuckle, Cortex Eucommiae, coffee bean, Acer truncatum leaves, burdock leaf,
Sweet potato leaves etc..STEVIA REBAUDIANA be composite family herbaceos perennial, containing steviol glycoside be STEVIA REBAUDIANA cured leaf 8%-12%, mainly by with
Make to extract natural sweetener steviol glycoside.In addition to steviol glycoside, also contain sweet tea in the alcohol of STEVIA REBAUDIANA, water or alcohol water extract
It chrysanthemum phenol, flavonoids, chinic acid and caffeic acid and derivative, amino acid and fatty acid and its derivative and other needs to remove
The impurity such as tannin, tannin, pigment and pectin.Phenolic compound in stevia rebaudianum mainly includes simple phenolic acid class, chlorogenic acid, Huang
Ketone, flavonols, flavanol compound, osajin etc..For STEVIA REBAUDIANA, research both domestic and external is concentrated mainly on steviol glycoside
On preparative separation, and it is less for polyphenol, the extraction separation report of chlorogenic acid in STEVIA REBAUDIANA.Polyphenol in STEVIA REBAUDIANA or with mentioning
Waste residue is taken to discard together；Or proposed from waste residue, purity is low, and extraction efficiency is poor.
Industrialization at present is extracted and the raw material of purifying chlorogenic acid is mainly derived from honeysuckle, folium cortex eucommiae and coffee bean etc..In
State patent CN103288644A describes a kind of preparation method of chlorogenic acid extracting from honeysuckle, and honeysuckle is cleaned and dried
Crushing obtains powder；After multiple water mentions, pH is adjusted, macroporous resin adsorption, then alcohol affords crude extract, through extraction and separation,
Finished product chlorogenic acid is obtained after concentrate drying.The Introduction To Cn Patent of Chinese patent CN1974527A is a kind of prepared from folium cortex eucommiae it is high
The method of purity chlorogenic acid and general flavone is that folium cortex eucommiae will crush after drying, is extracted with ethyl alcohol；Then it is inhaled through macroporous resin column
It is attached, it after silicagel column, is concentrated under reduced pressure, is recrystallized with acetone or water, obtain purity 96%-99% chlorogenic acid fine work.Patent
CN109096110A describes a kind of technique that 99% chlorogenic acid is isolated and purified from coffee bean, and coffee bean pulverizes and sieves to obtain
Coarse powder, ethyl alcohol extract, and stirring centrifugation, after filtering, filtrate is adsorbed through macroporous absorbent resin, desorbed solution through nanofiltration membrane treatment, decoloration,
Filtering after being concentrated under reduced pressure, crystallizes and obtains crude product, 99% chlorogenic acid is recrystallized to give with Diluted Alcohol.Patent CN03134575.1A
A kind of process of chlorogenic acid extracting from Acer truncatum leaves is described, the chlorogenic acid extracting from Acer truncatum leaves is mentioned using low temperature
It taking, control time and pH value, under the conditions of solid-liquid ratio etc., the soluble solids leaching rate of the technique is high, and effect is preferable, and it is easy to operate, it obtains
The chlorogenic acid purity arrived is 40% or so.Patent CN1651398A describes a kind of production work of chlorogenic acid extracting from burdock leaf
Skill, fresh burdock leaf are cleaned impurity elimination, chloroform degreasing, after decoloration, with sour water refluxing extraction, then are extracted with ethyl acetate, are subtracted
It is adsorbed after pressing concentration with AB-8 resin column, through ethanol elution, eluent concentration, recrystallizes and chlorogenic acid pure product is made.Patent
A kind of method that CN200510094347.8 describes chlorogenic acid extracting and purifying from sunflower meal extracts sunflower with ethanol water
The dregs of rice, extract liquor is concentrated go the heavy protein isolate of alcohol, acid after, then be extracted with ethyl acetate, separate and be concentrated to get the green of purity 70%
Ortho acid crude product is finally further purified with D101 macroreticular resin, obtains the chlorogenic acid that content is 92%.
In conclusion at present chlorogenic acid extracting raw material be all mostly the relatively high Chinese herbal medicine of some chlorogenic acid contents and
Resource material not abundant.And using stevia rebaudian leaf as raw material, the relevant report for extracting isolated chlorogenic acid is less, patent
Using stevia rebaudian leaf as raw material in CN102924544A, the method for preparing stevioside and chlorogenic acid step by step, cold water soak extracts sweet tea
Chrysanthemum leaf adds and adjusts pH value to flocculate after divalent iron salt, filters to get filtrate and filter cake, filtrate adsorbed with macroporous absorbent resin,
Stevioside is obtained after parsing, refinement treatment；Filter cake adjusts acid after being dissolved with water, dry through macroporous absorbent resin absorption, parsing, concentration
Chlorogenic acid is obtained after dry, alcohol recrystallization.Patent CN105001281A describes a kind of synchronous production steviol glycoside, flavones and green original
The production method of acid, after stevia rebaudian leaf is mentioned through water, flocculated, is obtained by filtration filter mud and filtrate, filtrate obtains steviol glycoside through purifying resin
Product；Filter mud adsorbs after alcohol solution dissolves through macroporous absorbent resin, and then gradient parses, and low concentration alcohol desorbed solution is prepared into
Chlorogenic acid, the concentration of high concentration alcohol desorbed solution are extracted through organic solvent, obtain flavones.The method achieve steviol glycoside, flavones and
It is extracted while chlorogenic acid, improves the comprehensive utilization value of STEVIA REBAUDIANA, but obtained chlorogenic acid purity is not high.
As it can be seen that the technics comparing of chlorogenic acid extracting is mature at present, but there is also some problems.It is that raw material sources are asked first
Topic, chlorogenic acid raw material sources are more single, are some Chinese herbal medicines and resource material not abundant mostly, concentrate on honeysuckle, coffee
On coffee beans, folium cortex eucommiae.Followed by technique extraction step is too many, and used organic reagent is more, increases cost, in addition also deposits
In security risk.Importantly, existing disclosed from the technology for extracting stevia rebaudianum polyphenol in stevia rebaudian leaf, what is mostly obtained is sweet tea
Chrysanthemum polyphenol mixture, and extracted in the filter residue that is obtained by filtration after mentioning, flocculating from stevia rebaudian leaf through water, extraction efficiency and product are pure
It spends all low.In addition, in the prior art, the polyamide used during other plants mention is mostly 30-60 mesh (i.e. 0.25-
Particle 0.5mm) is unfavorable for scale extraction.
Summary of the invention
[technical problem]
Solve the problems, such as the extraction source of chlorogenic acid is single, preparation process is complicated, using a large amount of organic solvents, low output.
[technical solution]
The present invention is to extract medium with water, the chlorogenic acid of high-purity is prepared using stevia rebaudian leaf as raw material.The present invention provides
The technical scheme comprises the following steps:
(1) extraction and flocculation of stevia rebaudian leaf: soaking in water stevia rebaudianum cured leaf piece, and countercurrent leaching filters and collects filtrate to obtain the final product
To STEVIA REBAUDIANA extracting solution, the pH of gained STEVIA REBAUDIANA extracting solution is adjusted to acidity, then while stirring to above-mentioned stevia rebaudian leaf extracting solution
Middle addition chitosan stands flocculation, collects the supernatant after flocculating；
(2) chromatogram purification: by above-mentioned flocculation supernatant through plate compression, gained filtrate is adsorbed with polyamide, outflow
Liquid, which stays, to use it for anything else；Then, it is first collected water lotion with acidification water elution polyamide and is used for chlorogenic acid extracting；Again with volume point
The methanol or ethanol solution of 80% or more number elute polyamide, collect alcohol desorbed solution, and alcohol desorbed solution concentrated spray is dry
After stay and use it for anything else；
(3) it recrystallizes: water lotion obtained by step (2) is adsorbed with polyamide, then with being acidified water elution polyamides
Polyimide resin collects water lotion to get to the water lotion after enriched, after concentration, adjusts pH and temperature, crystallize while stirring, take out
Filter obtains filter cake；It is again recrystallization solvent with water, adjusts pH, recrystallizing to the mass content of chlorogenic acid is 98% or more.
In one embodiment of the invention, in the step (1), when being extracted to stevia rebaudian leaf, stevia rebaudianum cured leaf piece and water
Feed liquid mass ratio be 1:(3-12), extraction water temperature be 20 DEG C -60 DEG C；The pH for adjusting STEVIA REBAUDIANA extracting solution is 3.5-5.0.It is preferred that
Ground, extraction water temperature are 35 DEG C -60 DEG C；The pH for adjusting STEVIA REBAUDIANA extracting solution is 3.5-4.0.
In one embodiment of the invention, in the step (1), the deacetylation of chitosan used is 80%-
90%, the quality for adding chitosan is that stevia rebaudianum cured leaf piece feeds intake the 0.1%-0.2% of quality, and standing the flocculated time is 20min-
1h。
In one embodiment of the invention, it is poly- that the step (2) and step (3), which are enriched with polyamide used,
Amide resin or class polyamide；Including between polyamide 6 (PA6), LX-16, LX-8, polyamide 66 (PA66) or poly hexamethylene adipamide
One of benzene dimethylamine (MXD6).
In one embodiment of the invention, the acidifying water in the step (2) and step (3) is with dilute acid for adjusting pH
The acidic aqueous solution obtained to 3.5-5.0, the amount of acidifying water used are 3-5 times of column volume.
In one embodiment of the invention, in the step (3), adjusting pH is 3.5-5.0, adjusting crystallization temperature is
0 DEG C -25 DEG C, recrystallisation solvent is water.
In one embodiment of the invention, in the step (3), recrystallization solvent is water, and adjusting pH is 3.5-
5.0, crystallization temperature is 0 DEG C -25 DEG C, and recrystallization number is 1-2 times, mixing speed 10-30rpm.
In one embodiment of the invention, the efflux in step (2) and alcohol desorbed solution contain other polyphenol and sweet tea
Other stevia rebaudianum polyphenol and steviol glycoside can be obtained by further separation and Extraction in synanthrin glycosides etc..
[beneficial effect]
Method provided by the invention is easy to operate, and obtained chlorogenic acid purity is up to 99%；The preparation of chlorogenic acid is whole
It is water-borne, it is environmentally protective.
Method provided by the invention has expanded the utilization field of STEVIA REBAUDIANA, while preparing the chlorogenic acid of high-purity, no
It influences to extract steviol glycoside and stevia rebaudianum polyphenol, realizes the comprehensive utilization of STEVIA REBAUDIANA.
Detailed description of the invention
The HPLC of 1 gained chlorogenic acid of Fig. 1 embodiment schemes (chlorogenic acid content 99.1%)
The HPLC of 1 gained chlorogenic acid of reference examples schemes (chlorogenic acid content 92.49%) in Fig. 2 embodiment 1
The HPLC of 2 gained intermediate products chlorogenic acid of Fig. 3 embodiment schemes (chlorogenic acid content 63.2%)
The HPLC of 2 gained chlorogenic acid of Fig. 4 embodiment schemes (chlorogenic acid content 98.5%)
The HPLC of 3 gained chlorogenic acid of Fig. 5 embodiment schemes (chlorogenic acid content 99%)
The HPLC of 2 gained chlorogenic acid of reference examples schemes (chlorogenic acid content 80.68%) in Fig. 6 embodiment 3
The HPLC of 4 gained chlorogenic acid of Fig. 7 embodiment schemes (chlorogenic acid content 98.0%)
Fig. 8 reference examples 3 scheme (chlorogenic acid content 60.44%) with the HPLC of the macroreticular resin XDA-8 chlorogenic acid prepared
Specific embodiment
The detection method of content (HPLC) of chlorogenic acid: the assay of chlorogenic acid is referring to Chinese Medicines&Health Produts importers or exporters
Meeting group norms T/CCCMHPIE 1.16-2016, does suitably modified.Using chlorogenic acid as standard items.Chromatographic condition: ultraviolet inspection
Survey device Detection wavelength: 330nm.Chromatographic column: C18 (2),4.6×250mm；Column temperature: 25 DEG C.Mobile phase: acetonitrile-water-
Formic acid gradient elution, flow velocity 1.0mL/min.Sample volume is 10 μ L.Mobile phase A: acetonitrile-water-formic acid (250:750:1, mL/
ML), Mobile phase B: acetonitrile-water-formic acid (100:900:1, mL/mL), gradient service condition such as the following table 1:
Table 1
| Time/min | Mobile phase A % | Mobile phase A % |
| 0-20 | 0 | 100 |
| 20-35 | 100 | 0 |
| 35-37 | 100→0 | 0→100 |
| 37-45 | 0 | 100 |
Unless otherwise indicated, the deacetylation of chitosan used is 80%-90%.Polyamide used and class polyamide
In, polyamide 6 (PA6), trade mark 1013B, density 1.14g/cm3, particle diameter is about 1-3mm, Ube Industries Ltd.'s strain formula
Commercial firm produces；Polyamide 66 (PA66), trade mark EPR27, density 1.14g/cm3, particle diameter is about 1-3mm, table mountain mind
Horse engineering plastics Co., Ltd produces；LX-16 and LX-8, selected particle diameter are about 0.8-1.25mm, and indigo plant dawn science and technology in Xi'an is new
Material limited liability company produces；Poly-meta-xylylene adipamide (MXD6), trade mark S6002, density 1.22g/cm3, particle
Diameter is 1-2.2mm, and Mitsubishi gas Chemical Co., Ltd. is produced.
Below with reference to specific embodiment, the present invention is described further.
Embodiment 1:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 35 DEG C with the water of 3 × 4L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 3.5.The 1wt% chitosan solution of 120mL is added to extracting solution while stirring, stands
50min flocculation purification.Supernatant plate-frame filtering after flocculation purification, after filtered clear liquid passes through PA6 resin adsorption, stream
Liquid, which stays, out uses it for anything else；Deresination is washed with 3 times of column volumes again, water elution is collected and is used as chlorogenic acid extracting.Then, dense with volume
Degree parses PA6 resin for 80% methanol aqueous solution, collects alcohol desorbed solution, and alcohol desorbed solution gives over to it after being further concentrated and dried
With.Using the chlorogenic acid in PA6 resin adsorption enrichment water elution, then, then with 3 times of column volumes deresinations are washed, collection is simultaneously
It is concentrated water lotion, and with 0.1mol/L hydrochloric acid adjusting efflux pH to 3.5,20 DEG C, crystallize 12h under 10rpm, is filtered
Cake；It is again recrystallization solvent with water, crystallizes 18h at 20 DEG C, pH 3.5,10rpm again.It filters, drying filter residue obtains content
For 99.1% chlorogenic acid (HPLC map is shown in Fig. 1).
Reference examples 1: it is extracted at pH 7.0, remaining condition is the same as embodiment 1.
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 35 DEG C with the water of 3 × 4L, gained STEVIA REBAUDIANA extracting solution
PH to 7.0 is adjusted with 0.05mol/L sodium bicarbonate.The 1wt% chitosan solution of 120mL is added to extracting solution while stirring, it is quiet
Set 50min flocculation purification.Supernatant plate-frame filtering after flocculation purification, after filtered clear liquid passes through PA6 resin adsorption, stream
Liquid, which stays, out uses it for anything else；Deresination is washed with 3 times of column volumes again, water elution is collected and is used as chlorogenic acid extracting.Then, dense with volume
Degree parses PA6 resin for 80% methanol aqueous solution, collects alcohol desorbed solution, and alcohol desorbed solution gives over to it after being further concentrated and dried
With.Using the chlorogenic acid in PA6 resin adsorption enrichment water elution, then, then with 3 times of column volumes deresinations are washed, collection is simultaneously
It is concentrated water lotion, and with 0.05mol/L sodium bicarbonate adjusting efflux pH to 7.0,20 DEG C, crystallize 12h under 10rpm；Exist again
20 DEG C, pH 7.0, crystallize 18h under 10rpm.Suction filtration, drying filter residue obtain the chlorogenic acid that content is 92.49%, and (HPLC map is shown in
Fig. 2).After comparison diagram 1, Fig. 2 can be seen that adjustment pH to neutrality, the purity of resulting chlorogenic acid drops to 92.4%.
Embodiment 2:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 60 DEG C with the water of 3 × 2.5L, gained STEVIA REBAUDIANA is extracted
Liquid 0.1mol/L vinegar acid for adjusting pH to 4.5.1% chitosan solution of 100mL is added under stirring to extracting solution, stands 20min
Flocculation purification.Supernatant plate-frame filtering after going flocculation purification, after filtered clear liquid passes through PA66 resin adsorption, efflux
It stays and uses it for anything else；Deresination is washed with 4 times of column volumes again, water elution is used as chlorogenic acid extracting.Then, it is parsed with 80% ethyl alcohol
PA66 resin, alcohol desorbed solution are stayed after being further concentrated and dried and are used it for anything else.It is enriched with using PA66 resin adsorption green in water elution
Then ortho acid, then with 4 times of column volumes washes deresinations, the chlorogenic acid content collected is that 63.2% (HPLC map is shown in figure
3)；After concentration, its pH to 4.5 is adjusted with 0.1mol/L hydrochloric acid, then crystallizes 12h at 0 DEG C, 10rpm；Again in 0 DEG C, pH
4.5,12h is crystallized under 10rpm.It filters, drying filter residue obtains the chlorogenic acid that content is 98.5% (HPLC map is shown in Fig. 4).
Embodiment 3:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 50 DEG C with the water of 3 × 3L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 4.0.1% chitosan solution of 120mL is added under stirring to extracting solution, stands 50min wadding
Solidifying removal of impurities.Supernatant plate-frame filtering after going flocculation purification, after filtered clear liquid passes through LX-16 resin adsorption, efflux
It stays and uses it for anything else；Deresination is washed with 5 times of column volumes again, eluent is used as chlorogenic acid extracting.Then LX-16 is parsed with 85% methanol
Resin, alcohol desorbed solution are stayed after being further concentrated and dried and are used it for anything else.Utilize the green original in LX-16 resin adsorption enrichment water elution
Then acid, then with 5 times of column volumes washes deresinations, collect and be concentrated water lotion, and with 0.1mol/L hydrochloric acid adjusting efflux pH
12h is crystallized under to 4.0,20 DEG C, 10rpm；16h is crystallized at 20 DEG C, pH 4.0,10rpm again.It filters, drying filter residue obtains
The chlorogenic acid that content is 99.0% (HPLC map is shown in Fig. 5).
Reference examples 2: reducing the deacetylation of chitosan, remaining condition is the same as embodiment 3.
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 50 DEG C with the water of 3 × 3L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 4.0.1% chitosan (deacetylation 70%) of 120mL is added under stirring to extracting solution
Solution stands 50min flocculation purification.Supernatant plate-frame filtering after going flocculation purification, filtered clear liquid pass through LX-16
After resin adsorption, efflux, which stays, to use it for anything else；Deresination is washed with 5 times of column volumes again, eluent is used as chlorogenic acid extracting.Then it uses
85% methanol parses LX-16 resin, and alcohol desorbed solution is stayed after being further concentrated and dried and used it for anything else.It is enriched with using LX-16 resin adsorption
Then chlorogenic acid in water elution, then with 5 times of column volumes washes deresinations, collects and be concentrated water lotion, and use 0.1mol/
L hydrochloric acid adjusts efflux pH to 4.0, and 20 DEG C, crystallize 12h under 10rpm；16h is crystallized at 20 DEG C, pH 4.0,10rpm again.
It filters, drying filter residue obtains the chlorogenic acid that content is 80.68% (HPLC map is shown in Fig. 6).
Embodiment 4:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 60 DEG C with the water of 3 × 2L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 4.0.1% chitosan solution of 100mL is added under stirring to extracting solution, stands 40min
Flocculation purification.Supernatant plate-frame filtering after going flocculation purification, after filtered clear liquid passes through LX-8 resin adsorption, efflux
It stays and uses it for anything else；It is washed with water deresination, eluent is used as chlorogenic acid extracting.Then LX-8 resin, alcoholysis are parsed with 90% ethyl alcohol
Analysis liquid is stayed after being further concentrated and dried and is used it for anything else.Water elution is concentrated after resin concentration through LX-8 resin concentration chlorogenic acid
Efflux, and adjust efflux pH to 4.0 with 0.1mol/L hydrochloric acid, 10 DEG C, crystallize 12h under 30rpm；Again in 10 DEG C, pH
4.0,16h is crystallized under 30rpm.It filters, drying filter residue obtains the chlorogenic acid that content is 98.0% (HPLC map is shown in Fig. 7).
Reference examples 3:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 50 DEG C with the water of 3 × 3L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 3.5.1% chitosan solution of 100mL is added under stirring to extracting solution, stands 30min wadding
Solidifying removal of impurities.Supernatant plate-frame filtering after going flocculation purification, after filtered clear liquid passes through XDA-8 resin adsorption, efflux
It stays and uses it for anything else；It is washed with water deresination, eluent is used as chlorogenic acid extracting.Then XDA-8 resin, alcoholysis are parsed with 85% methanol
Analysis liquid is stayed after being further concentrated and dried and is used it for anything else.Water elution is concentrated after resin concentration through XDA-8 resin concentration chlorogenic acid
Efflux, and adjust efflux pH to 3.5 with 0.1mol/L hydrochloric acid, 5 DEG C, crystallize 12h under 10rpm；Again in 5 DEG C, pH
3.5,12h is crystallized under 10rpm.It filters, drying filter residue obtains the chlorogenic acid that content is 60.44% (HPLC map is shown in Fig. 8).
Embodiment 5:
1kg STEVIA REBAUDIANA cured leaf piece is placed in storage tank, is extracted at 40 DEG C with the water of 3 × 3L, gained STEVIA REBAUDIANA extracting solution
With 0.1mol/L salt acid for adjusting pH to 4.0.1% chitosan solution of 120mL is added under stirring to extracting solution, stands 50min wadding
Solidifying removal of impurities.Supernatant plate-frame filtering after going flocculation purification, after filtered clear liquid passes through MXD6 resin adsorption, efflux is stayed
It uses it for anything else；Again with the washing deresination of 3 times of column volumes, eluent is as chlorogenic acid extracting.Then MXD6 is parsed with 85% methanol
Resin, alcohol desorbed solution are stayed after being further concentrated and dried and are used it for anything else.It is enriched with the chlorogenic acid in water elution using MXD6 resin adsorption,
Then, deresination then with 3 times of column volumes is washed, collects and be concentrated the water elution of outflow, and adjusted and flowed with 0.1mol/L hydrochloric acid
Liquid pH to 4.0 out, 20 DEG C, crystallize 12h under 10rpm；16h is crystallized at 20 DEG C, pH 4.0,10rpm again.It filters, drying filter
Slag obtains the chlorogenic acid that content is 98.9%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
Subject to people's claims of art are defined, without departing from the spirit and scope of the present invention, can all do various change with
Modification, therefore protection scope of the present invention should subject to the definition of the claims.
Claims (10)
1. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA, which is characterized in that with water be extract medium, comprising the following steps:
(1) extraction and flocculation of stevia rebaudian leaf: soaking in water stevia rebaudianum cured leaf piece, countercurrent leaching, filters and collects filtrate and obtains sweet tea
Leaf chrysanthemum extracting solution adjusts the pH of gained STEVIA REBAUDIANA extracting solution to acidity, then adds while stirring into above-mentioned stevia rebaudian leaf extracting solution
Enter chitosan and stand flocculation, collects the supernatant after flocculating；
(2) chromatogram purification: by above-mentioned flocculation supernatant through plate compression, gained filtrate is adsorbed with polyamide, and efflux stays
It uses it for anything else；Then, it is first collected water lotion with acidification water elution polyamide and is used for chlorogenic acid extracting；Volume fraction is used again
80% or more methanol or ethanol solution elutes polyamide, collects alcohol desorbed solution, after the drying of alcohol desorbed solution concentrated spray
It stays and uses it for anything else；
(3) it recrystallizes: water lotion obtained by step (2) is adsorbed with polyamide, then with being acidified water elution polyamide resin
Rouge collects water lotion to get to the water lotion after enriched, after concentration, adjusts pH and temperature, crystallize, filter while stirring
To filter cake；It is again recrystallization solvent with water, adjusts pH, recrystallizing to the mass content of chlorogenic acid is 98% or more.
2. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1, which is characterized in that the step
(1) in, when extracting to stevia rebaudian leaf, the feed liquid mass ratio of stevia rebaudianum cured leaf piece and water is 1:(3-12), extraction water temperature is 20 DEG C -60
℃；The pH for adjusting STEVIA REBAUDIANA extracting solution is 3.5-5.0.
3. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1 or 2, which is characterized in that the step
Suddenly in (1), the deacetylation of chitosan used is 80%-90%, and the quality for adding chitosan is that stevia rebaudianum cured leaf piece feeds intake quality
0.1%-0.2%, stand the flocculated time be 20min-1h.
4. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1, which is characterized in that the step
(2) and polyamide used in step (3) enrichment is polyamide or class polyamide.
5. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 4, which is characterized in that including polyamide
One of 6 (PA6), LX-16, LX-8, polyamide 66 or poly-meta-xylylene adipamide.
6. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1 or 4, which is characterized in that the step
Suddenly the acidifying water in (2) and step (3) is the acidic aqueous solution obtained with dilute acid for adjusting pH to 3.5-5.0, acidifying water used
Amount is 3-5 times of column volume.
7. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1, which is characterized in that the step
(3) in, adjusting pH is 3.5-5.0, adjusting crystallization temperature is 0 DEG C -25 DEG C, and recrystallisation solvent is water.
8. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1 or claim 7, which is characterized in that the step
Suddenly in (3), recrystallization solvent is water, and adjusting pH is 3.5-5.0, crystallization temperature is 0 DEG C -25 DEG C, and recrystallization number is 1-2 times,
Mixing speed is 10-30rpm.
9. a kind of method for preparing chlorogenic acid from STEVIA REBAUDIANA according to claim 1, which is characterized in that in step (2)
Efflux and alcohol desorbed solution contain other polyphenol and steviol glycoside etc., other stevia rebaudianums can be obtained by further separation and Extraction
Polyphenol and steviol glycoside.
10. the chlorogenic acid that -9 any the methods are prepared according to claim 1.
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| CN118063319A (en) * | 2024-04-17 | 2024-05-24 | 诸城市浩天生物科技有限公司 | Method for preparing high-purity chlorogenic acid by using stevia rebaudiana extract |
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